

Th1 / Th2 type cytokines in hepatitis B patients treated with interferon-alpha . 
OBJECTIVE : To investigate the relationship between the expression of Th1 / Th2 type cytokines and the effect of interferon-alpha therapy . 
METHODS : Th1 / Th2 type cytokines were assayed by enzyme-linked immunosorbent assay ( ELISA ) and reverse transcription polymerase chain reaction ( RT-PCR ) on 23 patients with chronic hepatitis B who were treated with interferon-alpha . 
RESULTS : Levels of IFN-gamma in the supernatant of peripheral blood mononuclear cells ( PBMC ) cultures from the patients with hepatitis B were slightly lower than those of controls ( P = 0.07 ) . 
However , the levels of IL-4 were higher than those of controls ( P = 0.01 ) . 
Cytokines measurements during IFN-alpha treatment showed a trend to decreasing levels of IL-4 at 4 , 12 , and 24 weeks . 
Levels of IFN-gamma were slightly increased following IFN-alpha treatment ( P = 0.09 ) . 
In patients with a complete response to IFN-alpha , the levels of IFN-gamma were higher at 24 weeks following IFN-alpha treatment than that of pre-treatment ( P = 0.04 ) , and the levels of IL-4 decreased markedly at 12 and 24 weeks ( P = 0.02 , 0.03 , respectively ) . 
mRNA expression positively correlated with the level of Th1 / Th2 type cytokines in the supernatant . 
CONCLUSION : The expression of Th2 type cytokines is predominant in patients with chronic hepatitis B . 
Interferon-alpha therapy can modulate the balance of Th1 / Th2 type cytokines , and this is related to its clinical effect . 
Levels of Th1 / Th2 type cytokines could be a predictor of clinical response during Interferon-alpha treatment . 


Involvement of BMP-2 signaling in a cartilage cap in osteochondroma . 
This study describes the distributions of bone morphogenetic protein ( BMP )-2 as well as mRNAs for BMP receptor type IB ( BMPRIB ) . collagen types II ( Col II ) and III ( Col III ) in a growing " cartilage cap " of osteochondroma . 
In situ hybridization and immunohistochemical study were performed using histological sections obtained during surgery . 
BMP-2 was detected in mesenchymal cells in the outer fibrous layer and chondrocytes in the inner cartilaginous matrix , positive for Col III and Col II , respectively . 
BMPRIB mRNA was distributed in chondrocytes . 
This is the first study to provide observational evidence of the involvement of BMP-2 signaling in the pathogenesis of cartilage cap of osteochondroma. and suggests the role of BMP-2 in the growth of cartilage cap in osteochondroma . 


The molar ratio of serum retinol-binding protein ( RBP ) to transthyretin ( TTR ) is not useful to assess vitamin A status during infection in hospitalised children . 
OBJECTIVE : To assess the usefulness of the molar ratio of serum retinol-binding protein ( RBP ) to transthyretin ( TTR ) to determine vitamin A ( VA ) status during infection . 
DESIGN : We took advantage of previously collected data during a randomised double-blind , placebo-controlled clinical trial to conduct a secondary analysis of the RBP / TTR ratio and its relationship to infection and VA status . 
In this clinical trial , children were randomly assigned to one of three groups and received either one single oral high dose of VA ( 200 000 IU ) on the day of admission and subsequently a placebo daily until discharge or daily oral low doses of VA ( 5000 IU ) from admission until discharge or a placebo daily from admission until discharge . 
SETTING : Lwiro pediatric hospital , Province of South Kivu , Democratic Republic of Congo . 
SUBJECTS : A total of 900 children aged 0-72 months hospitalised consecutively between March 1994 and March 1996 . 
MAIN OUTCOME MEASURES : RBP / TTR molar ratio after 7 days hospitalisation . 
RESULTS : After 7 days hospitalisation , molar RBP : TTR ratio ( mean+ /-s.d. ) of infected children ( C-reactive proteins > 10 mg / l ) was 0.67+ /-0.31 in the high-dose group ( n = 81 ) , 0.74+ /-0.44 in the low dose group ( n = 71 ) and 0.73+ /-0.39 in the placebo group ( n = 81 ) . 
These values did not differ significantly ( one-way ANOVA P = 0.472 ) . In patients with baseline serum retinol concentrations < 0.70 micromol / l , changes in RBP : TTR ratio between admission and day 7 were not statistically different in the three groups ( one-way ANOVA P = 0.548 ) . 
CONCLUSIONS : In this population of malnourished hospitalised children , molar RBP : TTR ratio does not appear to be useful to assess VA status during infection. 
SPONSORSHIP : Our research was partially supported by a grant from the Fonds de la Recherche Scientifique et Medicale ( contract 3.4505.94 ) and the David and Alice Van Buuren Foundation. 


Role of cyclin-dependent kinase inhibitors in the growth arrest at senescence in human prostate epithelial and uroepithelial cells . 
Cellular senescence has been proposed to be an in vitro and in vivo block that cells must overcome in order to immortalize and become tumorigenic . 
To characterize these pathways , we focused on changes in the cyclin-dependent kinase inhibitors and their binding partners that underlie the cell cycle arrest at senescence . 
As a model , we utilized normal human prostate epithelial cell ( HPEC ) and human uroepithelial cell ( HUC ) cultures . 
After 30-40 population doublings cells became growth-arrested in G0 / 1 with a threefold decrease in Cdk2-associated activity , a point defined as pre-senescence . 
Temporally following this growth arrest , the cells develop a senescence morphology and express senescence-associated beta-galactosidase ( SA-beta-gal ) . 
Levels of p16 ( INK4a ) and p57 ( KIP2 ) rise in HUCs during progressive passages , whereas only p16 increases in HPEC cultures . 
The induced expression of p57 , similar to p16 , produces a senescent-like phenotype . 
pRB , cyclin D , p19 ( INK4d ) and p27 ( KIP1 ) decrease in both cell types . 
We find that p53 , p21 ( CIP1 ) and p15 ( INK4b ) are transiently elevated in HPECs and HUCs at the pre-senescent growth arrest , then return to low proliferating levels at terminal senescence . 
Analysis of p53 , p21 ( CIP1 ) , p15 ( INK4b ) , p16 ( INK4a ) , and p57 ( KIP2 ) reveals altered expression in immortalized , non-tumorigenic HPV16 E6 and E7 prostate lines and in tumorigenic prostate cancer cells . 
These results indicate : ( i ) the existence of a subset of growth inhibiting genes elevated at the onset of the senescence , ( ii ) a distinct class of genes involved in the maintenance of senescence , and ( iii ) the frequent inactivation of these pathways during immortalization . 


Positive inotropic responses mediated by endothelin ET ( A ) and ET ( B ) receptors in human myocardial trabeculae . 
The aim of the present study was to determine possible inotropic effects mediated by endothelin ET ( A ) and ET ( B ) receptors in human myocardial trabeculae from the right atrium and the left ventricle . 
Isolated trabeculae from human hearts were paced at 1.0 Hz in tissue baths , and changes in isometric contractile force upon exposure to agonist were studied . 
Endothelin-1 ( ET-1 ) and ET-3 had a strong positive inotropic effect in all trabeculae . 
ET-1 was significantly more potent than ET-3 in both atrial ( P < 0.001 ) and ventricular ( P < 0.05 ) trabeculae . 
Preincubation with the ET ( A ) receptor antagonist FR139317 ( 1 microM ) decreased significantly ( P < 0.005 ) the potency of ET-I in both atrial and ventricular trabeculae , without any significant changes in Emax ( maximum effect obtained with an agonist ) . 
The ET ( B ) receptor agonist IRL 1620 had a positive inotropic effect only in some trabeculae , and the ET ( B ) receptor antagonist BQ 788 ( 1 microM ) almost completely blocked this effect . 
These results suggest that both ET ( A ) and ET ( B ) receptors mediate positive inotropic responses at both the atrial and ventricular level in the human heart . 


Antiapoptotic proteins . 
The bcl-2 and inhibitor of apoptosis protein families . 
The balance between pro- and antiapoptotic proteins can determine cellular fate . 
In this regard , the Bcl-2 and IAP protein families have evolved as highly conserved regulators of cell death . 
A further testament to their critical roles in maintaining balance between cell life and death may be the increasing implication of Bcl-2 and TAP proteins in the pathologies of human diseases . 
Although much has been learned about these families of proteins , future studies of the Bcl-2 and IAP families are sure to hold more exciting discoveries and will continue to reveal new strategies for combating human diseases . 


Apoptotic proteins . 
p53 and c-myc related pathways . 
c-Myc and p53 are two proteins that have critical roles in the regulation of apoptosis and the cell cycle . 
The authors review how these two proteins are thought to control the opposing events of proliferation and apoptosis and examine whether their well-documented biological roles in tumorigenesis can be applied to the vascular system . 


A comparative study of E-cadherin and stromelysin-3 expression in de novo and ex adenoma carcinoma of the colorectum . 
An apparent exception to the colorectal adenoma-carcinoma carcinogenetic pathway is the so-called " de novo " carcinoma which has no evidence of adenoma in its vicinity . 
Despite the fact that they are often quite small , these lesions appear to be more aggressive ( i.e. , greater likelihood of lymph-node metastases ) than carcinomas that clearly arise from surrounding adenomas exadenoma carcinoma . 
The purpose of the present comparative immunohistochemical study was to compare rates of cell adhesion molecule ( E-cadherin ) and protease [ stromelysin-3 ( ST-3 ) ] expression in groups of de novo ( n = 64 ) and ex adenoma ( n = 42 ) lesions in order to see whether their more aggressive behavior is associated with decreased cell adhesion and increased protease expression . 
The rates of extensive ST-3 expression and decreased E-cadherin expression were significantly higher in the de novo group ( P = 0.014 and 0.005 , respectively ) . 
Histopathologically , the de novo group also had a significantly higher percentage of cases with an infiltrative invasion pattern . 
These differences highlight the more aggressive phenotype of the de novo colorectal carcinoma and fit with their greater invasive potential . 


Genetic profile of 22 pancreatic carcinoma cell lines . 
Analysis of K-ras , p53 , p16 and DPC4 / Smad4 . 
The K-ras , p53 , p16 and DPC4 genes are among those most frequently altered in pancreatic ductal carcinoma . 
We analyzed 22 cell lines for alterations in these genes by direct sequence analysis and methylation-specific polymerase chain reaction . 
These cell lines showed mutations in K-ras and p53 at frequencies of 91 % and 95 % , respectively . 
Alterations in p16INK4a were found in all cases and included nine homozygous deletions , seven mutations and promoter methylation in six cases . 
Eight cell lines ( 36 % ) had an alteration of DPC4 , including one mutation and seven homozygous deletions . 
The most typical mutational profile involved K-ras , p53 , and p16INK4a , concurrently aberrated in 20 cases ( 91 % ) . 
Eight cell lines had alterations in all four genes . 
Inactivation of DPC4 was always accompanied by alteration of all of the other three genes . 
This comprehensive data regarding the cumulative genetic alterations in pancreatic carcinoma cell lines will be of great value for studies involving drug sensitivity or resistance that may be associated with inactivation of a particular gene or molecular pathway . 


Human gastrinoma cells express calcium-sensing receptor . 
The intravenous calcium injection test has been reported to be useful for the diagnosis of gastrinoma . 
However , the mechanism underlying calcium-evoked gastrin release is not fully understood . 
We investigated the mechanism of calcium-stimulated gastrin release from gastrinoma cells in vitro with a particular focus on the calcium-sensing receptor ( CaR ) . 
Human gastrinoma cells were taken from mechanically minced gastrinoma tissues obtained at surgery . 
In the perifusion system , high [ Ca2+ ] o induced gastrin release from gastrinoma cells . 
As [ Ca2+ ] o increased , [ Ca2+ ] i rapidly increased , as monitored by fluorometry . 
The response was not inhibited by nifedipine , a blocker of the voltage-dependent calcium channel . 
Reverse transcriptase-polymerase chain reaction and subsequent Southern blot hybridization revealed the presence of the CaR gene in human gastrinoma tissues . 
Moreover , the expression of CaR in gastrinoma tissues was confirmed by immunohistochemistry . 
Our results demonstrated that CaR was expressed in human gastrinoma cells and could be involved in the mechanism of calcium-evoked gastrin release . 


BMP-4 and BMP-6 involvement in the osteogenic properties of the HeLa cell line . 
The heterotopically induced ossicles are used in our research on bone tissue . 
The ossicles are formed in the thigh muscle of BALB / c mice under the influence of injected suspension of 3 x 10 ( 6 ) HeLa cells . 
We postulate that the mechanism of bone induction is based on the secretion of bone morphogenetic proteins BMP-4 and BMP-6 by the grafted HeLa cells . 
This was proved by the use of specific immunohistochemical reaction and Western blots of conditioned culture medium . 
It seems that HeLa cells secrete BMPs continuously into the culture medium , even without contact with the mice muscle tissue , were induction of bone tissue is observed . 


Immunohistochemical analysis of epidermal growth factor receptor family members in stage I non-small cell lung cancer . 
BACKGROUND : To elucidate the relationship between the expression of epidermal growth factor receptor family members ( ErbB-1 , neu / ErbB-2 , ErbB-3 , and ErbB-4 ) and tumor recurrence . 
METHODS : We used immunohistochemistry to examine the expression of four epidermal growth factor receptor family members in 73 patients with stage I non-small cell lung cancer . 
RESULTS : Using Cox univariate analysis , we determined that angiolymphatic tumor emboli and non-well-differentiated tumor cells were two significant conventional pathologic predictors of tumor recurrence , and that ErbB-1 and ErbB-3 were also significant predictors . 
Co-expression of ErbB-1 + ,-3+ , or expression of three or more epidermal growth factor receptor family members had a significant effect on lung cancer recurrence . 
A stepwise multivariate Cox proportional hazards regression analysis provided a predictive model for tumor recurrence . 
CONCLUSIONS : The present study shows that in patients with a non-well-differentiated tumor , overexpression of ErbB-3 is a useful marker for predicting tumor recurrence . 
The present study also confirmed that ErbB-1 expression increased in proportion to the loss of tumor differentiation . 
The correlation between ErbB-3 and distant metastasis was good . 


Soluble interleukin 2 receptor and interleukin 1alpha in toxic epidermal necrolysis : a comparative analysis of serum and blister fluid samples . 
BACKGROUND : Toxic epidermal necrolysis ( TEN ) is a rare but severe adverse drug disease , characterized by extensive skin and mucosal detachment with participation of different immunoinflammatory pathways , in particular with early participation of activated CD8 + T lymphocytes . 
OBJECTIVE : To further study the potential role of T lymphocytes in the early phase of keratinocyte necrosis . 
DESIGN : Prospective study . 
SETTING : University hospitals . 
PATIENTS : Thirteen patients with clinical and histopathologic criteria of TEN and 6 patients with second-degree burns . 
MAIN OUTCOME MEASURES : Measurement of soluble interleukin ( IL ) 2 receptor ( sIL-2R ) and IL-1alpha in serum samples and fluid of recent blisters . 
RESULTS : In the blister fluid of patients with TEN , we found significantly higher levels of sIL-2R than in patients with burns , whereas IL-1alpha levels were higher in the blister fluid of burned patients . 
No significant differences were found in serum samples of patients with TEN and burns , in either sIL-2R or IL-1alpha . 
In TEN we also found significantly higher levels of sIL-2R in the blister fluid compared with serum samples , pointing to a predominantly local production contrasting with the low concentration of sIL-2R in the blister fluid of burned patients . 
CONCLUSIONS : Our findings of elevated sIL-2R levels in blister fluid of patients with TEN are probably related to a local down-regulation of an immunologically mediated cytotoxic reaction and further support the involvement of activated T lymphocytes in the early blisters of TEN . 


Potential cellular signatures of viral infections in human hematopoietic cells . 
Expression profiling of cellular genes was performed using a 10 , 000 cDNA human gene array in order to identify expression changes following chronic infection of human hematopoietic cells with Kaposi 's Sarcoma-associated Virus ( KSHV ) also known as Human Herpesvirus 8 ( HHV8 ) and Human T cell leukemia virus-1 ( HTLV-1 ) . 
We performed cell-free in vitro infection of primary bone marrow derived CD34 + cells using semi-purified HHV8 and a mature IL-2 dependent T cell line , KIT 225 , using highly concentrated viral stocks prepared from an infectious molecular clone of HTLV-1 . 
Thirty days post infection , mRNA was isolated from infected cultures and uninfected controls and submitted for microarray analysis . 
More than 400 genes were differentially expressed more than two-fold following HHV8 infection of primary bone marrow derived CD34 + cells . 
Of these 400 , interferon regulatory factor 4 ( IRF4 ) , cyclin B2 , TBP-associated factor , eukaryotic elongation factor and pim 2 were up-regulated more than 3.5 fold . 
In contrast , less than 100 genes were differentially expressed more than two-fold following chronic infection of a mature T cell line with HTLV-1 . 
Of these , only cdc7 was up-regulated more than 3.5 fold . 
These data may provide insight into cellular signatures of infection useful for diagnosis of infection as well as potential targets for therapeutic intervention . 


Biological data and clinical symptoms as predictors of astrogliosis and neurodegeneration in patients with second-stage Trypanosoma brucei gambiense sleeping sickness . 
Concentrations of glial fibrillary acidic protein ( GFAp ) and light subunit neurofilament protein ( NFL ) in cerebrospinal fluid ( CSF ) were measured in patients with second-stage Trypanosoma brucei gambiense sleeping sickness . 
Correlations between GFAp and NFL in CSF as markers for astrogliosis and neurodegeneration , and clinical and biological data were investigated . 
Abnormal levels of GFAp and NFL were significantly associated with increasing CSF cell number and protein concentration , and with the absence of lymph nodes or the absence of trypanosomes in lymph node aspirate . 
A significant association was found between abnormal NFL and presence of trypanosomes in CSF , abnormal limb movements , difficulties in gait and coordination , and low Karnofsky index . 
By multivariate analysis , it was shown that increasing CSF cell number , increasing CSF protein concentration , and the absence of lymph nodes or the absence of trypanosomes in the lymph node aspirate were the best predictors for astrogliosis and neurodegeneration among the variables tested . 
These results demonstrate the importance of CSF cell count and protein determination in assessment of the severity of central nervous system involvement and reinforces the importance of laboratory diagnosis to assess the stage of the disease . 
The clinical symptoms studied were less useful in predicting astrogliosis or neurodegeneration . 


Interleukin-10 levels in seminal plasma : implications for chronic prostatitis-chronic pelvic pain syndrome . 
PURPOSE : Chronic prostatitis-chronic pelvic pain syndrome is a disease of unknown pathogenesis . 
We investigated whether the chronic inflammation and pain experienced by patients may be caused by an imbalance of proinflammatory versus anti-inflammatory cytokines within the prostate , namely interleukin ( IL )-8 , interferon-gamma and IL-2 versus IL-10 . 
We measured these inflammatory cytokines in seminal plasma as a reflection of the prostate environment . 
MATERIALS AND METHODS : We measured levels of IL-8 , interferon-gamma , IL-2 and IL-10 in the seminal plasma of 31 patients with chronic prostatitis-chronic pelvic pain syndrome and 14 controls using enzyme-linked immunosorbent assay . 
Results were correlated with health related quality of life , as measured by the Multidimensional Pain Inventory , McGill pain questionnaire and International Prostate Symptom Score . 
RESULTS : The cytokines analyzed were detectable in the seminal plasma of patients with chronic prostatitis-chronic pelvic pain syndrome and controls . 
Interferon-gamma , IL-2 and IL-10 levels were statistically greater in patients . 
Furthermore , IL-10 levels correlated directly with measures of life interference ( r = 0.52 , p < 0.01 ) and pain severity ( r = 0.53 , p < 
CONCLUSIONS : Our study suggests that IL-10 may have a role in the pain symptoms experienced by patients with chronic prostatitis-chronic pelvic pain syndrome . 


Adeno-associated virus vector-mediated triple gene transfer of dopamine synthetic enzymes . 
OBJECTIVE : To explore triple gene transfer of dopamine synthetic enzymes with separate adeno-associated virus ( AAV ) vectors . 
METHODS : The genes for dopamine synthetic enzymes , tyrosine hydroxylase ( TH ) , aromatic L-amino acid decarboxylase ( AADC ) , and GTP cyclohydrolase I ( GCH , an enzyme critical for tetrahydrobiopterin synthesis ) were contransduced into 293 cells with separate AAV vectors . 
Expressions of TH , AADC and GCH were detected by Western blot analysis . 
Intracellular dopamine level was assayed by high-performance liquid chromatography . 
RESULTS : TH , AADC and GCH were effectively coexpressed in transduced cells with three separate AAV vectors , AAV- TH , AAV- AADC and AAV- GCH . 
Furthermore , the coexpression resulted in an effectively spontaneous dopamine production in cotransduced cells . 
CONCLUSION : The triple transduction of TH , AADC and GCH genes with separate AAV vectors is effective , which might be important to gene therapy for Parkinson 's disease . 


Genetic studies on the role of proteinases and growth factors in atherosclerosis and aneurysm formation . 
Expression analysis and epidemiologic studies have provided indirect evidence that proteinases and growth factors play a role in the development of atherosclerosis and complications such as aneurysm formation and plaque rupture . 
Studies using genetically altered mice have proven to be an elegant tool to study the causal involvement of these factors in atherogenesis and to gain insight into the underlying mechanisms . 
Recently , proteinases of the plasminogen and matrix metalloproteinase ( MMP ) systems as well as their inhibitors have received much attention , and these studies together have emphasized the complexity of their role in vascular disease . 
This overview summarizes the current knowledge on plasminogen activator inhibitor-1 ( PAI-1 ) in the progression of atherosclerosis and the influence of MMPs in aneurysm formation . 
In addition , a possible role for Gas6 , the product of growth arrest-specific gene 6 , in atherosclerotic lesion development is put into perspective . 


Chromatin image analysis provides new evidence of the relation of lymphocytes to cytokines and sCAM in the inflammatory nature of atherosclerosis . 
Comparative analysis of cytokines and sCAM secretion within the lymphocyte chromatin state are possible evidence of inflammatory reactions in atherosclerosis . 
Two types of response were studied : coagulation and fibrinolysis ( incubation of blood clot within 6 hours at 37 degrees C ) and standardized viscosimetric flow using a rotational viscometer ( shear rate 100 l / s , 60 seconds at 37 degrees C , and incubation within 6 hours at 37 degrees C ) . 
Cytokines IL-1alpha , IL-1beta , IL-6 , IL-8 , IL-10 ( Immunotech , France ) , endothelin-1 , and soluble cell adhesion molecules ( sCAM ) sP- and sE-selectin , sICAM-1 , and sVCAM-1 ( R AMPPPP D , UK ) have been determined using ELISA kits ( photometer , Biomek-1000 , Beckman , USA ) . 
The chromatin of lymphocyte nuclei was studied using the computer TV morphodensitometry system DiaMorph ( Russia ) and smears dyed specifically for DNA . 
Correlational changes in morphodensitometric ( MDM ) parameters and cytokine and sCAM levels in two tests were compared to initial levels . 
After rheologic testing , lymphocyte nuclei as a whole had not changed , but chromatin activity had decreased . 
Reorganization of nuclei after the coagulation test was observed . 
Endothelin-1 and sP- and sE-selectin levels were not related to function of lymphocytes ( by MDM data ) as seen in both tests ; it is probable that another cell-cell communication mechanism had been switched on . 
We established a strong correlation between chromatin activity of lymphocytes and the serum concentration of IL-1beta , IL-6 , and IL-10 , which are the active participants in the pro- and anti-inflammatory program in atherogenesis . 
Results are evidence of the role of lymphocytes in pro- and anti-inflammatory cytokine reactions and cytokine-like sCAM activity in atherosclerosis . 


The genetics of host resistance and susceptibility to tuberculosis . 
The study of human genomics has the potential to aid our understanding of the interindividual and interpopulation differences in susceptibility to tuberculosis . 
Resistance to infection is affected by the ability of macrophages to phagocytose and destroy the bacilli . 
Several genes are involved in this process , and two have been the focus of recent interest : the natural resistance-associated protein ( NRAMP1 ) gene and the genes coding for the vitamin D receptor . 
Susceptibility genes have also been discovered -- for example , one on the X chromosome that may explain the increased susceptibility of males to tuberculosis . 
Studies have also focused on the variations in virulence of the bacillus in both its drug-susceptible and drug-resistant forms . 
These mechanisms must be understood in order to prevent , or combat , the emergence of a virulent , multidrug-resistant form of the bacillus that would be uncontrollable by means of today 's treatment strategies . 


Inflammatory gene polymorphisms and ischaemic heart disease : review of population association studies . 
Inflammation and genetics are both prominent mechanisms in the pathogenesis of atherosclerosis and arterial thrombosis . 
Accordingly , a number of population studies have explored the association of ischaemic heart disease with gene polymorphisms of the inflammatory molecules tumour necrosis factors ( TNF ) alpha and beta , transforming growth factors ( TGF ) beta1 and 2 , interleukin ( IL ) 1 and its receptor antagonist ( IL 1ra ) , CD14 ( the receptor for lipopolysaccharide ) , P and E selectins , and platelet endothelial cell adhesion molecule ( PECAM ) 1 . 
Although they are very preliminary and partly conflicting , the data provide some evidence that alterations in the genetics of the inflammatory system may modify the risk of ischaemic heart disease . 


Regulated trafficking of neurotransmitter transporters : common notes but different melodies . 
The activity of biogenic amine and amino acid neurotransmitters is limited by presynaptic and astrocytic Na ( + ) -dependent transport systems . 
Their functional importance is underscored by the observation that these transporters are the targets of broad classes of psychotherapeutic agents , including antidepressants and stimulants . 
Early studies suggested that the activity of these transporters can be fine tuned by a number of different signaling pathways . 
In the past five years , several groups have provided compelling evidence that changing the cell surface availability of these transporters contributes to this fine tuning . 
This regulated trafficking can result in rapid ( within minutes ) increases or decreases in the plasma membrane expression of these transporters and is independent of transcriptional or translational control mechanisms . 
Many of the same signaling molecules , including protein kinase C ( PKC ) , tyrosine kinase , phosphatidylinositol 3-kinase ( P13-K ) , and protein phosphatase , regulate the transporters for different neurotransmitters . 
In addition to these classical receptor activated pathways , transporter substrates also regulate activity and cell surface expression of these transporters . 
In fact , some of the transporters form complexes with signaling molecules . 
Given the functional and genetic similarities of these transporters , it is not surprising that the same signaling molecules regulate their trafficking , but except for the molecules , the actual effects on individual transporters are remarkably different . 
It is as if the same musical notes have been rearranged into several different melodies . 


Present data concerning the renin- angiotensin system of extrarenal origin . 
After a brief historical incursion regarding RAS of renal origin , we present the main extrarenal angiotensin-forming enzymes , starting with isorenin , tonin , D and G cathepsin and ending with the conversion enzyme and chymase . 
This is followed by a short presentation of data concerning tissue RAS in the heart and vessels , brain and neurocranial accessory glands , cortico-suprarenal and gonadal glandular territory . 
In the end we refer to the relations between circulating RAS of renal origin and the extrarenal RAS specific to tissue underlying the fact that the two RAS components cooperate and constitute a unitary hormonal system , regulating the major functions of the organism and maintaining the main homeostatic equilibria . 
While the circulating RAS of renal origin is acutely activated to contribute to the accomplishment of short term homeostatic reactions , tissue RAS , in paracrine or autocrine ways , exerts local-regional adaptive actions of long duration . 


Expression of glucocorticoid receptors and cyclooxygenase-2 in nasal polyps from nonallergic patients . 
Glucocorticoid treatment has been widely used to suppress inflammatory and immune responses . 
However , from a clinical point of view , its efficacy in the treatment of nasal polyposis seems to vary individually from patient to patient . 
In the present study , we examined the presence of glucocorticoid receptors ( GRs ) and cyclooxygenase-2 ( COX-2 ) in the nasal polyps of nonallergic patients as compared with normal controls . 
Reverse transcription-polymerase chain reaction analyses revealed the presence of both GR messenger RNA and COX-2 messenger RNA expression in nasal polyps from nonallergic patients , as well as in normal nasal mucosa from controls . 
Consistent with this finding , immunohistochemical analysis demonstrated that GRs and COX-2 were labeled in both tissues . 
In nasal polyps , GR labeling was associated with the cytoplasm and nucleus of surface mucosa , submucosal glands , endothelial cells , and inflammatory cells . 
Importantly , COX-2 labeling was seen in the cytoplasm of surface mucosa , submucosal glands , and the vascular wall in nasal polyps . 
In contrast , in normal nasal tissues , COX-2 labeling was only found in the vascular wall , and the expression was weaker -- a finding demonstrating that COX-2 is upregulated in nasal polyps . 
Therefore , 1 ) the presence of GRs and COX-2 in nasal polyps from nonallergic patients , as well as upregulation of COX-2 expression , suggests that inflammation may play an important role in the pathophysiology of nasal polyps ; and 2 ) glucocorticoid could be a potential treatment method for suppressing inflammatory processes. 


Instability of extracellular matrix gene expression in primary cell culture of fibroblasts from human vocal fold lamina propria and tracheal AB-Primary fibroblast cell cultures were established from lamina propria of human vocal fold and tracheal scar . 
There exists a crucial need to provide new tools for studying voice biology , and one of the first steps is the development of a human primary laryngeal cell culture bank . 
Because cell lines can lose their differentiated phenotype in culture across passages , documentation of gene expression must be determined for passage populations , for us to have knowledge of cell behavior in vitro . 
Comparison of messenger RNA gene expression of extracellular matrix proteins ( procollagen I , collagenase , elastin , hyaluronic acid synthase 2 , hyaluronidase , fibronectin , cd44 , fibromodulin , and decorin ) across cell passages ( 3 , 4 , 5 , 6 , 10 , and 12 fornormal laminapropria and 3 , 4 , 5 , 6 , and 10 for tracheal scar ) revealed varied growth patterns . 
Cytogenetic analysis demonstrated relative stability of the karyotypes across passages for the tracheal scar cell cultures , whereas the karyotypes of the normal lamina propria fibroblasts showed instability in in vitro cultures . 
Recommendations for use of primary cell cultures for further studies of gene expression are made . 


Assaying of tumor necrosis factor alpha , complement factors , and alpha-1-antitrypsin in the diagnosis of malignant serous effusions . 
The objective of this study was to measure the concentrations of tumor necrosis factor-alpha ( TNFalpha ) in pleural and peritoneal effusions of different causes and to verify whether TNFalpha , alpha-1-antitrypsin ( alpha1AT ) , and complement factors C3 and C4 can be used in the differential diagnosis of serous effusion . 
One hundred forty-five serous effusions of various origins were analyzed . 
TNFalpha , alpha1AT , and complement factors C3 and C4 concentrations were measured simultaneously in blood and serous effusion using commercially available methods . 
Serous effusions were classified as follows : 102 exudates and 43 transudates . 
All variables were found to have good diagnostic value in the differential diagnosis of serous effusion . 
In the stepwise discriminant analysis , four variables were selected , producing a significant discriminant function ( p < 0.001 ) . 
Their order of selection was alpha1AT effusion , C4 serum , TNFalpha-effusion , and C3 effusion . 
Combined use of these variables increased remarkably the diagnostic value ( in diagnosing exudates versus transudates ) giving sensitivity = 93.14 % ; specificity = 90.70 % ; positive predictive value = 95.96 % ; negative predictive value = 84.78 % . 
Determination of TNFalpha , complement factors C3 and C4 , and alpha1AT may be a significant parameter in the differential diagnosis of serous effusions , particularly in those patients with malignant disease . 
Moreover , the combination of them significantly increased their diagnostic power . 


Differential expression of granulysin and perforin by NK cells in cancer patients and correlation of impaired granulysin expression with^M progression of cancer. 
Granulysin has been identified as an effector molecule co-localized with perforin in the cytotoxic granules of cytotoxic T lymphocytes and natural killer ( NK ) cells , and has been reported to kill intracellular pathogens in infected cells in the presence of perforin and to induce a cytotoxic effect against tumor cells . 
The aim of the present study was to elucidate whether intracellular expression of granulysin and perforin by NK cells might be associated with progression of cancer . 
Flow cytometric analysis demonstrated high levels of perforin and granulysin expression by CD3 (-) CD16 ( + ) cells in healthy controls . 
In contrast , cancer patients exhibited significantly decreased levels of granulysin expression ( P < 0.005 ) , despite having equally high levels of perforin expression in comparison with healthy controls . 
The tumor-free patients expressed granulysin at levels similar to healthy controls , while the progressive tumor-bearing patients expressed remarkably lower levels of granulysin compared to healthy controls ( P < 0.0001 ) . 
Similarly , patients with an advanced performance status had significantly fewer granulysin-positive NK cells than healthy controls . 
Meanwhile , a considerable number of the tumor-bearing patients showed a decrease in the number of circulating NK cells , and a correlation between impaired granulysin expression and reduced circulating NK cells was observed . 
These findings suggest that the tumor-bearing patients with impaired granulysin expression were in an immunosuppressive state . 
In conclusion , impaired expression of granulysin by NK cells correlates with progression of cancer , and determination of granulysin expression might prove informative for assessing the immunological condition of cancer patients . 


Regulation of cell proliferation , apoptosis , and carcinogenesis by activin AB-The aim of this review is to provide insight into the molecular mechanisms by which activin A modulates cell proliferation , apoptosis , and carcinogenesis in vitro and in vivo . 
Activin A , a member of the TGFbeta superfamily , has various effects on diverse biological systems , including cell growth inhibition in many cell types . 
However , the mechanism ( s ) by which activin exerts its inhibitory effects are not yet understood . 
This review highlights activin 's effects on activin receptors and signaling pathway , modulation of activin signaling , and regulation of cell proliferation and apoptosis by activin . 
Based on the experiences of all the authors , we emphasized cell cycle inhibitors such as p16 and p21 and regulators of apoptosis such as p53 and members of the bcl-2 family . 
Aside from activin 's inhibition of cell proliferation and enhancement of apoptosis , other newly developed methods for molecular studies of apoptosis by activin were briefly presented that support the role of activin as an inhibitor of carcinogenesis and cancer progression . 
These methods include subtractive hybridization based on covalent bonding , a simple and accurate means to determine molecular profile of as few as 20 cells based on an RNA-PCR approach , and a messenger RNA-antisense DNA interference phenomenon ( D-RNAi ) , resulting in a long-term gene knockout effects . 


Identification of a new member of the tumor necrosis factor family and its receptor , a human ortholog of mouse GITR . 
The tumor necrosis factor ( TNF ) and TNF receptor ( TNFR ) gene superfamilies regulate diverse biological functions , including cell proliferation , differentiation , and survival . 
We have identified a new TNF-related ligand , designated human GITR ligand ( hGITRL ) , and its human receptor ( hGITR ) , an ortholog of the recently discovered murine glucocorticoid-induced TNFR-related ( mGITR ) protein . 
The hGITRL gene mapped to chromosome 1q23 , near the gene for the TNF homolog Fas / CD95 ligand . 
The hGITR gene mapped to chromosome 1p36 , near a cluster of five genes encoding TNFR homologs . 
We found hGITRL mRNA in several peripheral tissues , and detected hGITRL protein on cultured vascular endothelial cells . 
The levels of hGITR mRNA in tissues were generally low ; in peripheral blood T cells , however , antigen-receptor stimulation led to a substantial induction of hGITR transcripts . 
Cotransfection of hGITRL and hGITR in embryonic kidney 293 cells activated the anti-apoptotic transcription factor NF-kappaB , via a pathway that appeared to involve TNFR-associated factor 2 ( TRAF2 ) and NF-kappaB-inducing kinase ( NIK ) . 
Cotransfection of hGITRL and hGITR in Jurkat T leukemia cells inhibited antigen-receptor-induced cell death . 
Thus , hGITRL and hGITR may modulate T lymphocyte survival in peripheral tissues . 


Differential mechanisms of recognition and activation of interleukin-8 receptor subtypes . 
We have probed an epitope sequence ( His18-Pro19-Lys20-Phe21 ) in interleukin-8 ( IL-8 ) by site-directed mutagenesis . 
This work shows that single and double Ala substitutions of His18 and Phe21 in IL-8 reduced up to 77-fold the binding affinity to IL-8 receptor subtypes A ( CXCR1 ) and B ( CXCR2 ) and to the Duffy antigen . 
These Ala mutants triggered neutrophil degranulation and induced calcium responses mediated by CXCR1 and CXCR2 . 
Single Asp or Ser substitutions , H18D , F21D , F21S , and double substitutions , H18A / F21D , H18A / F21S , and H18D / F21D , reduced up to 431-fold the binding affinity to CXCR1 , CXCR2 , and the Duffy antigen . 
Interestingly , double mutants with charged residue substitutions failed to trigger degranulation or to induce wild-type calcium responses mediated by CXCR1 . 
Except for the H18A and F21A mutants , all other IL-8 mutants failed to induce superoxide production in neutrophils . 
This study demonstrates that IL-8 recognizes and activates CXCR1 , CXCR2 , and the Duffy antigen by distinct mechanisms . 


Isolation of human delta-catenin and its binding specificity with presenilin 1 . 
We screened proteins for interaction with presenilin ( PS ) 1 , and cloned the full-length cDNA of human delta-catenin , which encoded 1225 amino acids . 
Yeast two-hybrid assay , GST binding assay and immunoprecipitation demonstrated that delta-catenin interacted with a hydrophilic loop region in the endoproteolytic C-terminal fragment of PS1 , but not with that of PS-2 . 
These results suggest that PS1 and PS2 partly differ in function . 
PS1 loop fragment containing the pathogenic mutation retained the binding ability . 
We also found another armadillo-protein , p0071 , interacted with PS1 . 


A newly identified member of tumor necrosis factor receptor superfamily ( TR6 ) suppresses LIGHT-mediated apoptosis . 
TR6 ( decoy receptor 3 ( DcR3 ) ) is a new member of the tumor necrosis factor receptor ( TNFR ) family . 
TR6 mRNA is expressed in lung tissues and colon adenocarcinoma , SW480 . 
In addition , the expression of TR6 mRNA was shown in the endothelial cell line and induced by phorbol 12-myristate 13-acetate / ionomycin in Jurkat T leukemia cells . 
The open reading frame of TR6 encodes 300 amino acids with a 29-residue signal sequence but no transmembrane region . 
Using histidine-tagged recombinant TR6 , we screened soluble forms of TNF-ligand proteins with immunoprecipitation . 
Here , we demonstrate that TR6 specifically binds two cellular ligands , LIGHT ( herpes virus entry mediator ( HVEM )-L ) and Fas ligand ( FasL / CD95L ) . 
These bindings were confirmed with HEK 293 EBNA cells transfected with LIGHT cDNA by flow cytometry . 
TR6 inhibited LIGHT-induced cytotoxicity in HT29 cells . 
It has been shown that LIGHT triggers apoptosis of various tumor cells including HT29 cells that express both lymphotoxin beta receptor ( LTbetaR ) and HVEM / TR2 receptors . 
Our data suggest that TR6 inhibits the interactions of LIGHT with HVEM / TR2 and LTbetaR , thereby suppressing LIGHT-mediated HT29 cell death . 
Thus , TR6 may play a regulatory role for suppressing in FasL- and LIGHT-mediated cell death . 


Shared and unique determinants of the erythropoietin ( EPO ) receptor are important for binding EPO and EPO mimetic peptide . 
We have shown previously that Phe93 in the extracellular domain of the erythropoietin (EPO ) receptor ( EPOR ) is crucial for binding EPO . 
Substitution of Phe93 with alanine resulted in a dramatic decrease in EPO binding to the Escherichia coli-expressed extracellular domain of the EPOR ( EPO-binding protein or EBP ) and no detectable binding to full-length mutant receptor expressed in COS cells . 
Remarkably , Phe93 forms extensive contacts with a peptide ligand in the crystal structure of the EBP bound to an EPO-mimetic peptide ( EMP1 ) , suggesting that Phe93 is also important for EMP1 binding . 
We used alanine substitution of EBP residues that contact EMP1 in the crystal structure to investigate the function of these residues in both EMP1 and EPO binding . 
The three largest hydrophobic contacts at Phe93 , Met150 , and Phe205 and a hydrogen bonding interaction at Thr151 were examined . 
Our results indicate that Phe93 and Phe205 are important for both EPO and EMP1 binding , Met150 is not important for EPO binding but is critical for EMP1 binding , and Thr151 is not important for binding either ligand . 
Thus , Phe93 and Phe205 are important binding determinants for both EPO and EMP1 , even though these ligands share no sequence or structural homology , suggesting that these residues may represent a minimum epitope on the EPOR for productive ligand binding . 


Association of terminal complement proteins in solution and modulation by suramin . 
The association of terminal complement proteins was investigated by analytical ultracentrifugation and multi-angle laser light scattering . 
Native C8 and C9 formed a heterodimer in solution of physiological ionic strength with a free-energy change DeltaG degrees of-8.3 kcal / mol and a dissociation constant Kd of 0.6 microM ( at 20 degrees C ) that was ionic strength- and temperature-dependent . 
A va n't Hoff plot of the change in Kd was linear between 10 and 37 degrees C and yielded values of DeltaH degrees =-12.9 kcal / mol and DeltaS degrees =-15.9 cal mol-1 deg-1 , suggesting that electrostatic forces play a prominent role in the interaction of C8 with C9 . 
Native C8 also formed a heterodimer with C5 and low concentrations of polyionic ligands such as protamine and suramin inhibited the interaction . 
Suramin induced high-affinity trimerization of C8 ( Kd = 0.10 microM at 20 degrees C ) and dimerization of C9 ( Kd = 0.86 microM at 20 degrees C ) . 
Suramin-induced C8 oligomerization may be the primary reason for the drug 's ability to prevent complement-mediated hemolysis . 
Analysis of sedimentation equilibria and also of the fluorescence enhancement of suramin when bound to protein provided evidence for two suramin-binding sites on each C9 and three on each C8 in the oligomers . 
Oligomerization could be reversed by high suramin concentrations , but 8-aminonaphthalene-1 , 3 , 6-trisulfonate ( ANTS2-) , which mimics half a suramin molecule , could not compete with suramin binding and oligomerization suggesting that the drug also binds nonionically to the proteins . 


Presenilins interact with Rab11 , a small GTPase involved in the regulation of vesicular transport . 
Presenilin 1 ( PS1 ) mutations account for the majority of early-onset dominant cases of familial Alzheimer 's disease . 
Presenilins ( PSs ) are located in many intra-cellular compartments such as the endoplasmic reticulum , Golgi apparatus , nuclear region and vesicular structures . 
These proteins include from seven to nine putative transmembrane domains , with the N- and C-terminal ends and a large hydrophilic loop orientated towards the cytoplasm . 
We report an interaction between the human PS1 or PS2 hydrophilic loop and Rab11 , a small GTPase belonging to the Ras-related superfamily . 
Interaction domains were mapped to codons 374-400 for PS1 and to codons 106-179 for Rab11 , a region including the fourth GTP-binding domain . 
Considering the implication of Rab proteins in vesicular transport pathways , the PS- Rab11 interaction suggests that PSs might be involved in amyloid precursor protein vesicular routing . 


Endogenous presenilin 1 redistributes to the surface of lamellipodia upon adhesion of Jurkat cells to a collagen matrix . 
Most familial early-onset Alzheimer 's disease cases are caused by mutations in the presenilin 1 ( PS1 ) gene . 
Subcellular localization of the endogenous PS1 is essential for understanding its function , interactions with proteins , and role in Alzheimer 's disease . 
Although numerous studies revealed predominant localization of PS1 to endoplasmic reticulum and Golgi , there are conflicting reports on the localization of PS1 to the cell surface . 
We found that endogenous PS1 is highly expressed in T lymphocytes ( Jurkat cells ) . 
Using a variety of methods , we present evidence that endogenous PS1 is localized to the cell surface in addition to intracellular membrane compartments . 
Moreover , PS1 appeared in high levels on the surface of lamellipodia upon adhesion of the cells to a collagen matrix . 
The redistribution of PS1 in adhered cells was strikingly similar to that of the well characterized adhesion protein CD44 . 
Cell surface PS1 formed complexes in vivo with actin-binding protein filamin ( ABP-280 ) , which is known to form bridges between cell surface receptors and cytoskeleton and mediate cell adhesion and cell motility . 
Taken together , our results suggest a role of PS1 in cell adhesion and / or cell-matrix interaction . 


Molecular determinants of the interaction between the C-terminal domain of Alzheimer 's beta-amyloid peptide and apolipoprotein E alpha-helices . 
In a previous work , we predicted and demonstrated that the 29-42-residue fragment of beta-amyloid peptide ( Abeta peptide ) has in vitro capacities close to those of the tilted fragment of viral fusion proteins . 
We further demonstrated that apolipoprotein E2 and E3 but not apolipoprotein E4 can decrease the fusogenic activity of Abeta ( 29-42 ) via a direct interaction . 
Therefore , we suggested that this fragment is implicated in the neurotoxicity of Abeta and in the protective effects of apolipoprotein E in Alzheimer 's disease . 
Because structurally related apolipoproteins do not interact with the Abeta C-terminal domain but inhibit viral fusion , we suggested that interactions existing between fusogenic peptides and apolipoproteins are selective and responsible for the inhibition of fusion . 
In this study , we simulated interactions of all amphipathic helices of apolipoproteins E and A-I with Abeta and simian immunodeficiency virus ( SIV ) fusogenic fragments by molecular modeling . 
We further calculated cross-interactions that do not inhibit fusion in vitro . 
The results suggest that interactions of hydrophobic residues are the major event to inhibit the fusogenic capacities of Abeta ( 29-42 ) and SIV peptides . 
Selectivity of those interactions is due to the steric complementarity between bulky hydrophobic residues in the fusogenic fragments and hydrophobic residues in the apolipoprotein C-terminal amphipathic helices . 


CCR5 binds multiple CC-chemokines : MCP-3 acts as a natural antagonist . 
CCR5 was first characterized as a receptor for MIP-1alpha , MIP-1beta , and RANTES , and was rapidly shown to be the main coreceptor for M-tropic human immunodeficiency virus ( HIV ) -1 strains and simian immunodeficiency virus ( SIV ) . 
Chemokines constitute a rapidly growing family of proteins and receptor-chemokine interactions are known to be promiscuous and redundant . 
We have therefore tested whether other CC-chemokines could bind to and activate CCR5 . 
All CC-chemokines currently available were tested for their ability to compete with [ ( 125 ) I ]- MIP-1beta binding on a stable cell line expressing recombinant CCR5 , and / or to induce a functional response in these cells . 
We found that in addition to MIP-1beta , MIP-1alpha , and RANTES , five other CC-chemokines could compete for [ ( 125 ) I ]-MIP-1beta binding : MCP-2 , MCP-3 , MCP-4 , MCP-1 , and eotaxin binding was characterized by IC ( 50 ) values of 0.22 , 2.14 , 5.89 , 29.9 , and 21.7 nmol / L , respectively . 
Among these ligands , MCP-3 had the remarkable property of binding CCR5 with high affinity without eliciting a functional response , MCP-3 could also inhibit the activation of CCR5 by MIP-1beta and may therefore be considered as a natural antagonist for CCR5 . 
It was unable to induce significant endocytosis of the receptor . 
Chemokines that could compete with high affinity for MIP-1beta binding could also compete for monomeric gp120 binding , although with variable potencies ; maximal gp120 binding inhibition was 80 % for MCP-2 , but only 30 % for MIP-1beta . 
MCP-3 could compete efficiently for gp120 binding but was , however , found to be a weak inhibitor of HIV infection , probably as a consequence of its inability to downregulate the receptor . 


Molecular cloning of a novel human CC chemokine ( Eotaxin-3 ) that is a functional ligand of CC chemokine receptor 3 . 
Previously , we mapped the novel CC chemokine myeloid progenitor inhibitory factor 2 ( MPIF-2 ) / eotaxin-2 to chromosome 7q11.23 ( Nomiyama , H. , Osborne , L. R. , Imai , T. , Kusuda , J. , Miura , R. , Tsui , L.-C. , and Yoshie , O. ( 1998 ) Genomics 49 , 339-340 ) . 
Since chemokine genes tend to be clustered , unknown chemokines may be present in the vicinity of those mapped to new chromosomal loci . 
Prompted by this hypothesis , we analyzed the genomic region containing the gene for MPIF-2 / eotaxin-2 ( SCYA24 ) and have identified a novel CC chemokine termed eotaxin-3 . 
The genes for MPIF-2 / eotaxin-2 ( SCYA24 ) and eotaxin-3 ( SCYA26 ) are localized within a region of approximately 40 kilobases . 
By Northern blot analysis , eotaxin-3 mRNA was constitutively expressed in the heart and ovary . 
We have generated recombinant eotaxin-3 in a baculovirus expression system . 
Eotaxin-3 induced transient calcium mobilization specifically in CC chemokine receptor 3 ( CCR3 ) -expressing L1.2 cells with an EC ( 50 ) of 3 nM . 
Eotaxin-3 competed the binding of ( 125 ) I-eotaxin to CCR3-expressing L1.2 cells with an IC ( 50 ) of 13 nM . 
Eotaxin-3 was chemotactic for normal peripheral blood eosinophils and basophils at high concentrations . 
Collectively , eotaxin-3 is yet another functional ligand for CCR3 . 
The potency of eotaxin-3 as a CCR3 ligand seems , however , to be approximately 10-fold less than that of eotaxin . 
Identification of eotaxin-3 will further promote our understanding of the control of eosinophil trafficking and other CCR3-mediated biological phenomena . 
The strategy used in this study may also be applicable to identification of other unknown chemokine genes . 


Presenilin 1 suppresses the function of c-Jun homodimers via interaction with QM / Jif-1 . 
Presenilin 1 ( PS1 ) is the causative gene for an autosomal dominant familial Alzheimer 's disease ( AD ) mapped to chromosome 14 . 
Here we show that QM / Jun-interacting factor ( Jif )-1 , a negative regulator of c-Jun , is a candidate to mediate the function of PS1 in the cell . 
We screened for proteins that bind to PS1 from a human embryonic brain cDNA library using the two-hybrid method and isolated one clone encoding the QM / Jif-1 gene . 
The binding of QM / Jif-1 to full-length PS1 was confirmed in vitro by pull-down assay , and in vivo by immunoprecipitation assays with human samples , including AD brains . 
Immunoelectronmicroscopic analysis showed that QM / Jif-1 and PS1 are colocalized at the endoplasmic reticulum , and the nuclear matrix in human brain neurons . 
Chloramphenicol acetyltransferase assays in F9 cells showed that PS1 suppresses transactivation by c-Jun / c-Jun but not by c-Jun / c-Fos heterodimers , consistent with the reported function of QM / Jif-1 . 
By monitoring fluorescent recombinant protein and by gel mobility shift assays , PS1 was shown to accelerate the translocation of QM from the cytoplasm to the nucleus and to thereby suppress the binding of c-Jun homodimer to 12-O-tetradecanoylphorbol-13-acetate ( TPA ) -responsive element ( TRE ) . 
PS1 suppressed c-jun-associated apoptosis by retinoic acid in F9 embryonic carcinoma cells , whereas this suppression of apoptosis is attenuated by mutation in PS1 . 
Collectively , the novel function of PS1 via QM / Jif-1 influences c-jun-mediated transcription and apoptosis . 


Structural interactions of fibroblast growth factor receptor with its ligands . 
Fibroblast growth factors ( FGFs ) effect cellular responses by binding to FGF receptors ( FGFRs ) . 
FGF bound to extracellular domains on the FGFR in the presence of heparin activates the cytoplasmic receptor tyrosine kinase through autophosphorylation . 
We have crystallized a complex between human FGF1 and a two-domain extracellular fragment of human FGFR2 . 
The crystal structure , determined by multiwavelength anomalous diffraction analysis of the selenomethionyl protein , is a dimeric assemblage of 1 : 1 ligand : receptor complexes . 
FGF is bound at the junction between the two domains of one FGFR , and two such units are associated through receptor : receptor and secondary ligand : receptor interfaces . 
Sulfate ion positions appear to mark the course of heparin binding between FGF molecules through a basic region on receptor D2 domains . 
This dimeric assemblage provides a structural mechanism for FGF signal transduction . 


Crystal structure of the hereditary haemochromatosis protein HFE complexed with transferrin receptor . 
HFE is related to major histocompatibility complex ( MHC ) class I proteins and is mutated in the iron-overload disease hereditary haemochromatosis . 
HFE binds to the transferrin receptor ( TfR ) , a receptor by which cells acquire iron-loaded transferrin . 
The 2.8 A crystal structure of a complex between the extracellular portions of HFE and TfR shows two HFE molecules which grasp each side of a twofold symmetric TfR dimer . 
On a cell membrane containing both proteins , HFE would ' lie down ' parallel to the membrane , such that the HFE helices that delineate the counterpart of the MHC peptide-binding groove make extensive contacts with helices in the TfR dimerization domain . 
The structures of TfR alone and complexed with HFE differ in their domain arrangement and dimer interfaces , providing a mechanism for communicating binding events between TfR chains . 
The HFE-TfR complex suggests a binding site for transferrin on TfR and sheds light upon the function of HFE in regulating iron homeostasis . 


Bone morphogenetic protein receptor complexes on the surface of live cells : a new oligomerization mode for serine / threonine kinase receptors . 
The bone morphogenetic proteins ( BMPs ) play important roles in embryogenesis and normal cell growth . 
The BMP receptors belong to the family of serine / threonine kinase receptors , whose activation has been investigated intensively for the transforming growth factor-beta ( TGF-beta ) receptor subfamily . 
However , the interactions between the BMP receptors , the composition of the active receptor complex , and the role of the ligand in its formation have not yet been investigated and were usually assumed to follow the same pattern as the TGF-beta receptors . 
Here we demonstrate that the oligomerization pattern of the BMP receptors is different and is more flexible and susceptible to modulation by ligand . 
Using several complementary approaches , we investigated the formation of homomeric and heteromeric complexes between the two known BMP type I receptors ( BR-Ia and BR-Ib ) and the BMP type II receptor ( BR-II ) . 
Coimmunoprecipitation studies detected the formation of heteromeric and homomeric complexes among all the BMP receptor types even in the absence of ligand . 
These complexes were also detected at the cell surface after BMP-2 binding and cross-linking . 
Using antibody-mediated immunofluorescence copatching of epitope-tagged receptors , we provide evidence in live cells for preexisting heteromeric ( BR-II / BR-Ia and BR-II / BR-Ib ) and homomeric ( BR-II / BR-II , BR-Ia / BR-Ia , BR-Ib / BR-Ib , and also BR-Ia / BR-Ib ) oligomers in the absence of ligand . 
BMP-2 binding significantly increased hetero- and homo-oligomerization ( except for the BR-II homo-oligomer , which binds ligand poorly in the absence of BR-I ) . 
In contrast to previous observations on TGF-beta receptors , which were found to be fully homodimeric in the absence of ligand , the BMP receptors show a much more flexible oligomerization pattern . 
This novel feature in the oligomerization mode of the BMP receptors allows higher variety and flexibility in their responses to various ligands as compared with the TGF-beta receptors . 


TrkA amino acids controlling specificity for nerve growth factor . 
Neurotrophins are important for the development and maintenance of the vertebrate nervous system , mediating their signal into the cell by specific interaction with tyrosine kinase receptors of the Trk family . 
The extracellular portion of the Trk receptors has been previously proposed to consist of a cysteine-rich motif , a leucine-rich motif , a second cysteine-rich motif followed by two immunoglobulin-like domains . 
Earlier studies have shown that a major neurotrophin-binding site in the Trk receptors resides in the second immunoglobulin-like domain . 
Although the individual amino acids in TrkA involved in binding to nerve growth factor ( NGF ) and those in TrkC involved in binding to neurotrophin-3 have been mapped in this domain , the Trk amino acids that provide specificity remained unclear . 
In this study , a minimum set of residues in the human TrkC second immunoglobulin-like domain , which does not bind nerve growth factor ( NGF ) , were substituted with those from human TrkA . 
The resulting Trk variant recruited binding of NGF equivalent to TrkA , maintained neurotrophin-3 binding equivalent to TrkC , and also bound brain-derived neurotrophin , although with lower affinity compared with TrkB . 
This implies that the amino acids in the second immunoglobulin-like domain that determine Trk specificity are distinct for each Trk . 


Characterization of the CD30L binding domain on the human CD30 molecule using anti- CD30 antibodies . 
CD30 and its counter-receptor CD30 ligand ( CD30L ) are members of the TNF-receptor / TNFalpha superfamily and function to regulate lymphocyte survival and differentiation . 
Several monoclonal antibodies ( MAbs ) have been developed against CD30 and , based on mutual inhibition assays , are grouped into three nonoverlapping serologic clusters . 
However , the relationship between the epitopes recognized by the antibodies comprising each cluster and the binding domain for CD30L is not known . 
Using a soluble CD30L / CD8alpha chimeric protein , we assessed the ability of anti- CD30 MAb to inhibit the binding of CD30L to CD30 expressed by the CD30+ Karpas 299 cell line . 
CD30L binding by CD30 is blocked by MAb that recognize epitopes belonging to cluster Group A ( like Ber-H2 , Ber-H8 , and HRS-4 ) as well as cluster Group C ( like HeFi-1 and M44 ) . 
Cluster Group B antibodies , including M67 and Ki-1 , do not affect CD30L binding to CD30 . 
The pattern of CD30L binding inhibition shows only limited correspondence to the functional capacity of some anti- CD30 MAb to trigger CD30 signaling . 
Finally , we demonstrate that the anti- CD30L MAb M81 also completely inhibits CD30 / CD30L interaction . 
This information is useful for applying these MAbs in functional studies to further investigate the CD30 / CD30L system and for designing assays for soluble CD30L . 


Design , characterization , and structure of a biologically active single-chain mutant of human IFN-gamma . 
A mutant form of human interferon-gamma ( IFN-gamma SC1 ) that binds one IFN-gamma receptor alpha chain ( IFN-gamma R alpha ) has been designed and characterized . 
IFN-gamma SC1 was derived by linking the two peptide chains of the IFN-gamma dimer by a seven-residue linker and changing His111 in the first chain to an aspartic acid residue . 
Isothermal titration calorimetry shows that IFN-gamma SC1 forms a 1 : 1 complex with its high-affinity receptor ( IFN-gamma R alpha ) with an affinity of 27 ( + /-9 ) nM . 
The crystal structure of IFN-gamma SC1 has been determined at 2.9 A resolution from crystals grown in 1.4 M citrate solutions at pH 7.6 . 
Comparison of the wild-type receptor-binding domain and the Asp111-containing domain of IFN-gamma SC1 show that they are structurally equivalent but have very different electrostatic surface potentials . 
As a result , surface charge rather than structural changes is likely responsible for the inability of the His111 --> Asp domain of to bind IFN-gamma R alpha . 
The AB loops of IFN-gamma SC1 adopt conformations similar to the ordered loops of IFN-gamma observed in the crystal structure of the IFN-gamma / IFN-gamma R alpha complex . 
Thus , IFN-gamma R alpha binding does not result in a large conformational change in the AB loop as previously suggested . 
The structure also reveals the final six C-terminal amino acid residues of IFN-gamma SC1 ( residues 253-258 ) that have not been observed in any other reported IFN-gamma structures . 
Despite binding to only one IFN-gamma R alpha , IFN-gamma SC1 is biologically active in cell proliferation , MHC class I induction , and anti-viral assays . 
This suggests that one domain of IFN-gamma is sufficient to recruit IFN-gamma R alpha and IFN-gamma R beta into a complex competent for eliciting biological activity . 
The current data are consistent with the main role of the IFN-gamma dimer being to decrease the dissociation constant of IFN-gamma for its cellular receptors . 


BMP-2 antagonists emerge from alterations in the low-affinity binding epitope for receptor BMPR-II . 
Bone morphogenetic protein-2 ( BMP-2 ) induces bone formation and regeneration in adult vertebrates and regulates important developmental processes in all animals . 
BMP-2 is a homodimeric cysteine knot protein that , as a member of the transforming growth factor-beta ( TGF-beta ) superfamily , signals by oligomerizing type I and type II receptor serine-kinases in the cell membrane . 
The binding epitopes of BMP-2 for BMPR-IA ( type I ) and BMPR-II or ActR-II ( type II ) were characterized using BMP-2 mutant proteins for analysis of interactions with receptor ectodomains . 
A large epitope 1 for high-affinity BMPR-IA binding was detected spanning the interface of the BMP-2 dimer . 
A smaller epitope 2 for the low-affinity binding of BMPR-II was found to be assembled by determinants of a single monomer . 
Symmetry-related pairs of the two juxtaposed epitopes occur near the BMP-2 poles . 
Mutations in both epitopes yielded variants with reduced biological activity in C2C12 cells ; however , only epitope 2 variants behaved as antagonists partially or completely inhibiting BMP-2 activity . 
These findings provide a framework for the molecular description of receptor recognition and activation in the BMP / TGF-beta superfamily . 


Crystal structure of the BMP-2-BRIA ectodomain complex . 
Bone morphogenetic proteins ( BMPs ) belong to the large transforming growth factor-beta ( TGF-beta ) superfamily of multifunctional cytokines . 
BMP-2 can induce ectopic bone and cartilage formation in adult vertebrates and is involved in central steps in early embryonal development in animals . 
Signaling by these cytokines requires binding of two types of transmembrane serine / threonine receptor kinase chains classified as type I and type II . 
Here we report the crystal structure of human dimeric BMP-2 in complex with two high affinity BMP receptor IA extracellular domain ( BRIAec ) . 
The receptor chains bind to the ' wrist ' epitopes of the BMP-2 dimer and contact both BMP-2 monomers . 
No contacts exist between the receptor domains . 
The model reveals the structural basis for discrimination between type I and type II receptors and the variability of receptor-ligand interactions that is seen in BMP- TGF-beta systems . 


LEC induces chemotaxis and adhesion by interacting with CCR1 and CCR8 . 
Liver-expressed chemokine ( LEC ) is an unusually large CC chemokine , which is also known as LMC , HCC-4 , NCC-4 , and CCL16 . 
Previously , LEC was shown to induce leukocyte migration but the responsible signaling receptors were not characterized . 
We report chemotaxis and competitive binding studies that show LEC binds to and activates CCR1 and CCR8 transfected HEK-293 cells . 
LEC induced maximal migration of CCR1 and CCR8 transfected cells at 89.3 nmol / L and cell adhesion at 5.6 nmol / L . 
The molar concentration of LEC required to induce maximum cell migration is 20-to 200-fold greater than that required for RANTES or I309 , respectively . 
All 3 chemokines induced maximal static adhesion at 5 to 7 nmol / L . 
A neutralizing polyclonal antibody to LEC was developed to demonstrate that the unusually high concentration of LEC required to induce chemotaxis was a property of LEC and not as a result of an irrelevant protein contamination . 
This study suggests that LEC may be a more effective inducer of cell adhesion than cell migration . 


Identification of residues important both for primary receptor binding and specificity in fibroblast growth factor-7 . 
Fibroblast growth factors ( FGFs ) mediate a multitude of physiological and pathological processes by activating a family of tyrosine kinase receptors ( FGFRs ) . 
Each FGFR binds to a unique subset of FGFs and ligand binding specificity is essential in regulating FGF activity . 
FGF-7 recognizes one FGFR isoform known as the FGFR2 IIIb isoform or keratinocyte growth factor receptor ( KGFR ) , whereas FGF-2 binds well to FGFR1 , FGFR2 , and FGFR4 but interacts poorly with KGFR . 
Previously , mutations in FGF-2 identified a set of residues that are important for high affinity receptor binding , known as the primary receptor-binding site . 
FGF-7 contains this primary site as well as a region that restricts interaction with FGFR1 . 
The sequences that confer on FGF-7 its specific binding to KGFR have not been identified . 
By utilizing domain swapping and site-directed mutagenesis we have found that the loop connecting the beta4-beta5 strands of FGF-7 contributes to high affinity receptor binding and is critical for KGFR recognition . 
Replacement of this loop with the homologous loop from FGF-2 dramatically reduced both the affinity of FGF7 for KGFR and its biological potency but did not result in the ability to bind FGFR1 . 
Point mutations in residues comprising this loop of FGF-7 reduced both binding affinity and biological potency . 
The reciprocal loop replacement mutant ( FGF2-L4 / 7 ) retained FGF-2 like affinity for FGFR1 and for KGFR . 
Our results show that topologically similar regions in these two FGFs have different roles in regulating receptor binding specificity and suggest that specificity may require the concerted action of distinct regions of an FGF . 


New structural and functional aspects of the type I interferon-receptor interaction revealed by comprehensive mutational analysis of the binding interface . 
Type I interferons bind to two cell surface receptors , ifnar1 and ifnar2 , as the first step in the activation of several signal transduction pathways that elicit an anti-viral state and an anti-proliferative response . 
Here , we quantitatively mapped the complete binding region of ifnar2 on interferon ( IFN ) alpha2 by 35 individual mutations to alanine and isosteric residues . 
Of the six " hot-spot " residues identified ( Leu-30 , Arg-33 , Arg-144 , Ala-145 , Met-148 , and Arg-149 ) , four are located on the E-helix , which is located at the center of the binding site flanked by residues on the A-helix and the AB-loop . 
The contribution of residues of the D-helix , which have been previously implicated in binding , proved to be marginal for the interaction with the extracellular domain of ifnar2 . 
Interestingly , the ifnar2 binding site overlaps the largest continuous hydrophobic patch on IFNalpha2 . 
Thus , hydrophobic interactions seem to play a significant role stabilizing this interaction , with the charged residues contributing toward the rapid association of the complex . 
Relating the anti-viral and anti-proliferative activity of the various interferon mutants with their affinity toward ifnar2 results in linear function over the whole range of affinities investigated , suggesting that ifnar2 binding is the rate-determining step in cellular activation . 
Dose-time analysis of the anti-viral response revealed that shortening the incubation time of low-level activation cannot be compensated by higher IFN doses . 
Considering the strict dependence of the cellular response on affinity , these results suggest that for maintaining transcription of IFN-responsive genes over a longer time period , low but continuous signaling through the IFN receptor is essential . 


Crystal structure of fibroblast growth factor receptor ectodomain bound to ligand and heparin . 
Fibroblast growth factors ( FGFs ) are a large family of structurally related proteins with a wide range of physiological and pathological activities . 
Signal transduction requires association of FGF with its receptor tyrosine kinase ( FGFR ) and heparan sulphate proteoglycan in a specific complex on the cell surface . 
Direct involvement of the heparan sulphate glycosaminoglycan polysaccharide in the molecular association between FGF and its receptor is essential for biological activity . 
Although crystal structures of binary complexes of FGF-heparin and FGF-FGFR have been described , the molecular architecture of the FGF signalling complex has not been elucidated . 
Here we report the crystal structure of the FGFR2 ectodomain in a dimeric form that is induced by simultaneous binding to FGF1 and a heparin decasaccharide . 
The complex is assembled around a central heparin molecule linking two FGF1 ligands into a dimer that bridges between two receptor chains . 
The asymmetric heparin binding involves contacts with both FGF1 molecules but only one receptor chain . 
The structure of the FGF1-FGFR2-heparin ternary complex provides a structural basis for the essential role of heparan sulphate in FGF signalling . 


Loss of fibroblast growth factor receptor 2 ligand-binding specificity in Apert syndrome . 
Craniosynostosis syndromes are autosomal dominant human skeletal diseases that result from various mutations in fibroblast growth factor receptor genes ( Fgfrs ) . 
Apert syndrome ( AS ) is one of the most severe craniosynostosis syndromes and is associated with severe syndactyly of the hands and feet and with central nervous system malformations . 
AS is caused by specific missense mutations in one of two adjacent amino acid residues ( S252W or P253R ) in the highly conserved region linking Ig-like domains II and III of FGFR2 . 
Here we demonstrate that these mutations break one of the cardinal rules governing ligand specificity of FGFR2 . 
We show that the S252W mutation allows the mesenchymal splice form of FGFR2 ( FGFR2c ) to bind and to be activated by the mesenchymally expressed ligands FGF7 or FGF10 and the epithelial splice form of FGFR2 ( FGFR2b ) to be activated by FGF2 , FGF6 , and FGF9 . 
These data demonstrate loss of ligand specificity of FGFR2 with retained ligand dependence for receptor activation . 
These data suggest that the severe phenotypes of AS likely result from ectopic ligand-dependent activation of FGFR2 . 


Structure of an extracellular gp130 cytokine receptor signaling complex . 
The activation of gp130 , a shared signal-transducing receptor for a family of cytokines , is initiated by recognition of ligand followed by oligomerization into a higher order signaling complex . 
Kaposi 's sarcoma-associated herpesvirus encodes a functional homolog of human interleukin-6 ( IL-6 ) that activates human gp130 . 
In the 2.4 angstrom crystal structure of the extracellular signaling assembly between viral IL-6 and human gp130 , two complexes are cross-linked into a tetramer through direct interactions between the immunoglobulin domain of gp130 and site III of viral IL-6 , which is necessary for receptor activation . 
Unlike human IL-6 ( which uses many hydrophilic residues ) , the viral cytokine largely uses hydrophobic amino acids to contact gp130 , which enhances the complementarity of the viral IL-6-gp130 binding interfaces . 
The cross-reactivity of gp130 is apparently due to a chemical plasticity evident in the amphipathic gp130 cytokine-binding sites . 


Platelet-derived growth factor C ( PDGF-C ) , a novel growth factor that binds to PDGF alpha and beta receptor . 
We have characterized platelet-derived growth factor ( PDGF ) C , a novel growth factor belonging to the PDGF family . 
PDGF-C is a multidomain protein with the N-terminal region homologous to the extracellular CUB domain of neuropilin-1 , and the C-terminal region consists of a growth factor domain ( GFD ) with homology to vascular endothelial growth factor ( 25 % ) and PDGF A-chain ( 23 % ) . 
A serum-sensitive cleavage site between the two domains allows release of the GFD from the CUB domain . 
Competition binding and immunoprecipitation studies on cells bearing both PDGF alpha and beta receptors reveal a high affinity binding of recombinant GFD ( PDGF-CC ) to PDGF receptor-alpha homodimers and PDGF receptor-alpha / beta heterodimers . 
PDGF-CC exhibits greater mitogenic potency than PDGF-AA and comparable or greater mitogenic activity than PDGF-AB and PDGF-BB on several mesenchymal cell types . 
Analysis of PDGF-CC in vivo in a diabetic mouse model of delayed wound healing showed that PDGF-CC significantly enhanced repair of a full-thickness skin excision . 
Together , these studies describe a third member of the PDGF family ( PDGF-C ) as a potent mitogen for cells of mesenchymal origin in in vitro and in vivo systems with a binding pattern similar to PDGF-AB . 


Human CC chemokine liver-expressed chemokine / CCL16 is a functional ligand for CCR1 , CCR2 and CCR5 , and constitutively expressed by hepatocytes . 
Liver-expressed chemokine ( LEC ) / CCL16 is a human CC chemokine selectively expressed in the liver . 
Here , we investigated its receptor usage by calcium mobilization and chemotactic assays using mouse L1.2 pre-B cell lines stably expressing a panel of 12 human chemokine receptors . 
At relatively high concentrations , LEC induced calcium mobilization and chemotaxis via CCR1 and CCR2 . 
LEC also induced calcium mobilization , but marginal chemotaxis via CCR5 . 
Consistently , LEC was found to bind to CCR1 , CCR2 and CCR5 with relatively low affinities . 
The binding of LEC to CCR8 was much less significant . 
In spite of its binding to CCR5 , LEC was unable to inhibit infection of an R5-type HIV-1 to activated human peripheral blood mononuclear cells even at high concentrations . 
In human liver sections , hepatocytes were strongly stained by anti-LEC antibody . 
HepG2 , a human hepatocarcinoma cell line , was found to constitutively express LEC . 
LEC was also present in the plasma samples from healthy adult donors at relatively high concentrations ( 0.3 -- 4 nM ) . 
Taken together , LEC is a new low-affinity functional ligand for CCR1 , CCR2 and CCR5 , and is constitutively expressed by liver parenchymal cells . 
The presence of LEC in normal plasma at relatively high concentrations may modulate inflammatory responses . 


Cloning of TRAP , a ligand for CD40 on human T cells . 
A cDNA clone , designated TRAP ( TNF-related activation protein ) was isolated from a collection of T cell activation genes . 
The polypeptide encoded by a mRNA of approx . 
2.3 kb is 261 amino acids long with a calculated M ( r ) of 29.3 kDa . 
The structural features predict a type II transmembrane protein , but are also compatible with a secreted form . 
TRAP is highly similar to an identified murine CD40 ligand both at the cDNA ( 82.8 % identity ) and the protein ( 77.4 % identity ) levels , and related to tumor necrosis factor / lymphotoxin . 
Expressed in a murine myeloma , TRAP was identified as a ligand for CD40 by binding to a soluble CD40 construct . 
TRAP mRNA is expressed in a T cell-specific fashion with a maximum at 8 h after stimulation . 
The TRAP gene is located in the q26.3-q27.1 region of the X chromosome . 


Reconstitution of the multiprotein complex of pp60src , hsp90 , and p50 in a cell-free system . 
A rabbit reticulocyte lysate system that has been used to reconstitute functional complexes between steroid receptors and the 90-kDa heat shock protein ( hsp90 ) has been used here to form complexes between the pp60src tyrosine kinase and hsp90 . 
Reticulocyte lysate forms complexes between hsp90 and a temperature-sensitive mutant of Rous sarcoma virus pp60v-src , which is normally present in cytosol virtually entirely in the multiprotein complex form . 
In addition , hsp90 in the lysate complexes with wild-type pp60v-src , of which only a small portion is normally recovered in cytosol in the native multiprotein complex , and with the cellular homolog , pp60c-src , which has never been recovered in cytosol in the form of a native multiprotein complex with hsp90 . 
Moreover , the reticulocyte lysate-reconstituted complex also contains the 50-kDa phosphoprotein component of the native pp60v-src multiprotein complex . 
The native and reconstituted pp60src-hsp90 complexes have similar thermal stability and , like steroid receptor heterocomplexes , they are stabilized by molybdate . 
As previously shown with reticulocyte lysate-reconstituted steroid receptor heteroprotein complexes , the reconstituted pp60src multiprotein complex contains hsp70 , which is a major candidate for providing the protein unfoldase activity required for hsp90 association . 


The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling . 
A cDNA clone encoding a novel , widely expressed protein ( called growth factor receptor-bound protein 2 or GRB2 ) containing one src homology 2 ( SH2 ) domain and two SH3 domains was isolated . 
Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors ( EGFRs ) and platelet-derived growth factor receptors ( PDGFRs ) via its SH2 domain . 
Interestingly , GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5 . 
It has been shown that sem-5 and two other genes called let-23 ( EGFR like ) and let-60 ( ras like ) lie along the same signal transduction pathway controlling C. elegans vulval induction . 
To examine whether GRB2 is also a component of ras signaling in mammalian cells , microinjection studies were performed . 
While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect , microinjection of GRB2 together with H-ras protein stimulated DNA synthesis . 
These results suggest that GRB2 / sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling . 


Analysis of the oligomerization of myogenin and E2A products in vivo using a two-hybrid assay system . 
Members of the helix-loop-helix ( HLH ) family of proteins bind DNA and activate transcription as homo- and heterodimers . 
Myogenin is a muscle-specific HLH protein that binds DNA in vitro as a heterodimer with several widely expressed HLH proteins , such as the E2A gene products E12 and E47 . 
We describe a method for detection of protein-protein interactions among HLH proteins in vivo in which dimerization through the HLH motif reconstructs a hybrid transcription factor containing the DNA-binding domain of yeast GAL4 linked to one HLH motif and the activation domain of VP-16 linked to another . 
We have used this assay to investiagate whether myogenin forms homomeric or heteromeric complexes in vivo and to determine whether growth factors and oncogenes that inhibit myogenesis influence myogenin 's ability to dimerize . 
The results show that myogenin heterodimerizes with E12 and E47 in vivo , but it does not homodimerize to a measurable extent . 
Peptide growth factors , as well as the immediate early gene products c-Jun , v-Fos , and c-Myc , inhibit the activity of myogenin through a mechanism independent of its association with E2A products . 


Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro . 
Mitogen-activated protein ( MAP ) kinases are 42- and 44-kD serine-threonine protein kinases that are activated by tyrosine and threonine phosphorylation in cells stimulated with mitogens and growth factors . 
MAP kinase and the protein kinase that activates it ( MAP kinase kinase ) were constitutively activated in NIH 3T3 cells infected with viruses containing either of two oncogenic forms ( p35EC12 , p3722W ) of the c-Raf-1 protein kinase . 
The v-Raf proteins purified from cells infected with EC12 or 22W viruses activated MAP kinase kinase from skeletal muscle in vitro . 
Furthermore , a bacterially expressed v-Raf fusion protein ( glutathione S-transferase-p3722W ) also activated MAP kinase kinase in vitro . 
These findings suggest that one function of c-Raf-1 in mitogenic signaling is to phosphorylate and activate MAP kinase kinase . 


Biochemical properties of the 75-kDa tumor necrosis factor receptor . 
Characterization of ligand binding , internalization , and receptor phosphorylation . 
An expression plasmid encoding the human 75-kDa tumor necrosis factor ( TNF ) type 2 receptor ( TNF-R2 ) was constructed and used to generate a stable human cell line ( 293 / TNF-R2 ) overexpressing TNF-R2 . 
Ligand binding analysis revealed high affinity binding ( Kd = 0.2 nM ) with approximately 94 , 000 + /-7 , 500 sites / cell for 125I- TNF-alpha and approximately 5-fold lower affinity for TNF-beta ( Kd = 1.1 nM ) with 264 , 000 + /-2 , 000 sites / cell . 
Cross-linking of 125I- TNF-alpha and 125I- TNF-beta to 293 / TNF-R2 cells yielded predominant complexes with apparent molecular weights of 211 , 000 for TNF-alpha and 205 , 000 and 244 , 000 for TNF-beta , suggesting these complexes contain two or three TNF-R2 molecules . 
Immunoprecipitation of TNF-R2 from 32P-labeled 293 / TNF-R2 cells demonstrated that the receptor is phosphorylated . 
The majority ( 97 % ) of 32Pi incorporation was found in serine residues with a very low level of incorporation ( 3 % ) in threonine residues . 
TNF-alpha treatment of 293 / TNF-R2 cells did not significantly affect the degree or pattern of phosphorylation . 
Cell surface-bound 125I-TNF-alpha was slowly internalized by the 293 / TNF-R2 cell line with a t1 / 2 = 25 min . 
Shedding of the extracellular domain of TNF-R2 was induced by 4 beta-phorbol 12-myristate 13-acetate but not by TNF-alpha or TNF-beta . 


Crystal structure of human platelet-derived growth factor BB . 
The crystal structure of the homodimeric BB isoform of human recombinant platelet-derived growth factor ( PDGF-BB ) has been determined by X-ray analysis to 3.0 A resolution . 
The polypeptide chain is folded into two highly twisted antiparallel pairs of beta-strands and contains an unusual knotted arrangement of three intramolecular disulfide bonds . 
Dimerization leads to the clustering of three surface loops at each end of the elongated dimer , which most probably form the receptor recognition sites . 


Coassociation of CD26 ( dipeptidyl peptidase IV ) with CD45 on the surface of human T lymphocytes . 
In the present report , we demonstrated that modulation of CD26 from T cell surface induced by anti CD26 ( 1F7 ) led to enhanced phosphorylation of CD3 zeta tyrosine residues and increased CD4 associated p56lck tyrosine kinase activity . 
We further showed that CD26 was comodulated on the T cell surface with CD45 , a known membrane-linked protein tyrosine phosphatase and that anti- CD26 was capable of precipitating CD45 from T cell lysates . 
These findings strongly suggest that CD26 may be closely associated with the CD45 protein tyrosine phosphatase on T cell surface and further support the notion that the interaction of CD26 with CD45 results in enhanced tyrosine kinase activity , zeta chain phosphorylation , and T cell activation . 


cDNA cloning and expression of a human FGF receptor which binds acidic and basic FGF . 
We have isolated and characterized a cDNA clone , phFGFR , encoding a human fibroblast growth factor ( FGF ) receptor. phFGFR contains an open reading frame which encodes an 820 amino acid polypeptide with three immunoglobulin-like domains in the extracellular part and an intracellular split tyrosine kinase domain . 
Transient expression in COS-1 cells and immunoprecipitation using an antiserum raised against a C-terminal peptide , gave rise to two components , representing mature ( 130 kDa ) and precursor ( 115 kDa ) forms of the phFGFR encoded polypeptide , which was denoted hFGFR-1 . 
Crosslinking of iodinated acidic FGF ( aFGF ) and basic FGF ( bFGF ) to transiently expressing COS-1 cells revealed a major band of 95 kDa , which was competed for by both aFGF and bFGF . 
From Scatchard analyses , the Kd : s for binding of aFGF and bFGF to hFGFR-1 were estimated to 25 pM and 41 pM , respectively . 
Thus , phFGFR encodes a human FGF receptor with high affinity for both aFGF and bFGF . 


Cross-family dimerization of transcription factors Fos / Jun and ATF / CREB alters DNA binding specificity . 
The Fos / Jun and ATF / CREB families of transcription factors function in coupling extracellular signals to alterations in expression of specific target genes . 
Like many eukaryotic transcription factors , these proteins bind to DNA as dimers . 
Dimerization is mediated by a structure known as the " leucine-zipper " motif . 
Although Fos / Jun and ATF / CREB were previously thought to interact preferentially with different DNA regulatory elements ( the AP-1 / TRE and ATF / CRE sites , respectively ) , we find that members of these two families form selective cross-family heterodimers . 
The resulting heterodimers display distinguishable DNA binding specificities from each other and from their parental homodimers . 
These findings indicate that the Fos / Jun and ATF / CREB families of transcription factors are not as distinct as was previously thought . 
We suggest that they can be grouped into a superfamily of transcription factors . 


Human cyclin E , a new cyclin that interacts with two members of the CDC2 gene family . 
A new human cyclin , named cyclin E , was isolated by complementation of a triple cln deletion in S. cerevisiae . 
Cyclin E showed genetic interactions with the CDC28 gene , suggesting that it functioned at START by interacting with the CDC28 protein . 
Two human genes were identified that could interact with cyclin E to perform START in yeast containing a cdc28 mutation . 
One was CDC2-HS , and the second was the human homolog of Xenopus CDK2 . 
Cyclin E produced in E. coli bound and activated the CDC2 protein in extracts from human G1 cells , and antibodies against cyclin E immunoprecipitated a histone H1 kinase from HeLa cells . 
The interactions between cyclin E and CDC2 , or CDK2 , may be important at the G1 to S transition in human cells . 


cdc2 phosphorylation is required for its interaction with cyclin . 
Activation of the cdc2 protein kinase at different stages of the cell cycle is regulated by post-translational modifications and interactions with cyclins . 
We show that in vitro translated human cdc2 binds very poorly to A and B cyclins , unless it has been preincubated with a Xenopus egg extract . 
This results in the phosphorylation of cdc2 which allows binding to cyclins . 
The replacement of Thr161 , a residue conserved and phosphorylated in other protein kinases , with valine inhibits cdc2 association with A and B cyclins . 
In addition , mutations in the amino-terminus of cdc2 and within the conserved ' PSTAIR ' region strongly inhibit binding . 
The Thr161Val mutation causes a lethal phenotype in the fission yeast Schizosaccharomyces pombe , while replacement of Thr161 with glutamic acid , potentially mimicking phosphorylation , causes uncoordination of mitosis and multiple cytokinesis . 
These results suggest that a threonine phosphorylation / dephosphorylation cycle is involved in regulating cdc2 function . 


Specific activation of cdc25 tyrosine phosphatases by B-type cyclins : evidence for multiple roles of mitotic cyclins . 
Two previously unidentified human cdc25 genes have been isolated , cdc25A and cdc25B . 
Both genes rescue a cdc25ts mutant of fission yeast . 
Microinjection of anti- cdc25A antibodies into HeLa cells causes their arrest in mitosis. cdc25A and cdc25B display endogenous tyrosine phosphatase activity that is stimulated several-fold , in the absence of cdc2 , by stoichiometric addition of either cyclin B1 or B2 but not A or D1 . 
Association between cdc25A and cyclin B1 / cdc2 was detected in the HeLa cells . 
These findings indicate that B-type cyclins are multifunctional proteins that not only act as M phase regulatory subunits of the cdc2 protein kinase , but also activate the cdc25 tyrosine phosphatase , of which cdc2 is the physiological substrate . 
A region of amino acid similarity between cyclins and tyrosine PTPases has been detected . 
This region is absent in cdc25 phosphatases . 
The motif may represent an activating domain that has to be provided to cdc25 by intermolecular interaction with cyclin B . 


Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity . 
In resting human epithelial and fibroblastic cells , c-Jun is phosphorylated on serine and threonine at five sites , three of which are phosphorylated in vitro by glycogen synthase kinase 3 ( GSK-3 ) . 
These three sites are nested within a single tryptic peptide located just upstream of the basic region of the c-Jun DNA-binding domain ( residues 227-252 ) . 
Activation of protein kinase C results in rapid , site-specific dephosphorylation of c-Jun at one or more of these three sites and is coincident with increased AP-1-binding activity . 
Phosphorylation of recombinant human c-Jun proteins in vitro by GSK-3 decreases their DNA-binding activity . 
Mutation of serine 243 to phenylalanine blocks phosphorylation of all three sites in vivo and increases the inherent trans-activation ability of c-Jun at least 10-fold . 
We propose that c-Jun is present in resting cells in a latent , phosphorylated form that can be activated by site-specific dephosphorylation in response to protein kinase C activation . 


Interaction of the regulatory subunit ( RII ) of cAMP-dependent protein kinase with RII-anchoring proteins occurs through an amphipathic helix binding motif . 
The type II cAMP-dependent protein kinase is localized to specific subcellular environments through the binding of the regulatory subunit ( RII ) dimer to RII-anchoring proteins . 
Computer-aided analysis of secondary structure , performed on four RII-anchoring protein sequences ( the microtubule-associated protein 2 , P150 , and two thyroid proteins Ht 21 and Ht 31 ) , has identified common regions of approximately 14 residues which display high probabilities of forming amphipathic helices . 
The potential amphipathic helix region of Ht 31 ( Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile ) lies between residues 494 and 507 . 
A bacterially expressed 318-amino acid fragment , Ht 31 ( 418-736 ) , containing the amphipathic helix region , was able to bind RII alpha . 
Site-directed mutagenesis designed to disrupt the secondary structure in the putative binding helix reduced binding dramatically . 
Specifically , substitution of proline for Ala-498 significantly diminished RII alpha binding , and similar mutation of Ile-502 or Ile-507 abolished interaction . 
Mutation of Ala-522 to proline , which is located outside the predicted amphipathic helix region , had no effect on RII alpha binding . 
These data suggest that anchoring proteins interact with RII alpha via an amphipathic helix binding motif . 


Phosphorylation of c-jun mediated by MAP kinases . 
The proto-oncogene c-jun is a component of the AP-1 transcription factor family involved in the mediation of nuclear events elicited by extracellular stimuli . 
The c-jun protein is negatively regulated by phosphorylation of residues near the carboxy terminus which are dephosphorylated in response to phorbol esters . 
Here we identify two serine residues in the amino terminal A1 transactivation domain which are phosphorylated in response to a variety of mitogens , phorbol esters and activated ras . 
We present evidence that mitogen-activated protein-serine ( MAP ) kinases ( pp54 and pp42 / 44 ) specifically phosphorylate these sites and that their phosphorylation positively regulates the transacting activity of c-jun . 
The MAP kinase enzymes pp54 and pp42 / 44 are regulated by tyrosine as well as serine / threonine phosphorylation . 
MAP kinase activation of c-jun may underlie the common stimulation of this transcription factor by mitogens , growth factors and oncogenes . 


A cellular protein that competes with SV40 T antigen for binding to the retinoblastoma gene product . 
Tumour-suppressor genes , such as the human retinoblastoma susceptibility gene ( Rb ) , are widely recognized as being vital in the control of cell growth and tumour formation . 
This role is indicated , in part , by the suppression of tumorigenicity of human tumour cells after retrovirus-mediated Rb replacement . 
How Rb acts to bring about this suppression is not clear but one clue is that the Rb protein forms complexes with the transforming oncoproteins of several DNA tumour viruses , and that two regions of Rb essential for such binding frequently contain mutations in tumour cells . 
These observations suggest that endogenous cellular proteins might exist that bind to the same regions of Rb and thereby mediate its function . 
We report here the identification of one such human cellular Rb-associated protein of relative molecular mass 46 , 000 ( 46K ) ( RbAP46 ) . 
Two lines of evidence support the notion that RbAP46 and simian virus 40 T antigen have homologous Rb-binding properties : first , several mutated Rb proteins that failed to bind to T also did not associate with RbAP46 ; and second , both T antigen and T peptide ( amino acids 101-118 ) were able to compete with RbAP46 for binding to Rb . 
The apparent targeting of the RbAP46-Rb interaction by oncoproteins of DNA tumour viruses strongly suggests that formation of this complex is functionally important . 


Molecular cloning and expression of a receptor for human tumor necrosis factor . 
A human tumor necrosis factor ( TNF ) binding protein from serum of cancer patients was purified to homogeneity and partially sequenced . 
Synthetic DNA probes based on amino acid sequence information were used to isolate cDNA clones encoding a receptor for TNF . 
The TNF receptor ( TNF-R ) is a 415 amino acid polypeptide with a single membrane-spanning region . 
The extracellular cysteine-rich domain of the TNF-R is homologous to the nerve growth factor receptor and the B cell activation protein Bp50 . 
Human embryonic kidney cells transfected with a TNF-R expression vector specifically bind both 125I-labeled and biotinylated TNF-alpha . 
Unlabeled TNF-alpha and TNF-beta were equally effective at displacing the binding of labeled TNF-alpha to TNF-R expressing cells . 
Northern analysis indicates a single species of mRNA for the TNF-R in a variety of cell types . 
Therefore , the soluble TNF binding protein found in human serum is probably proteolytically derived from the TNF-R . 


Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor . 
Two cDNA clones encoding a receptor for human granulocyte-macrophage colony-stimulating factor ( hGM-CSF-R ) were isolated by expression screening of a library made from human placental mRNA . 
Pools of recombinant plasmid DNA were electroporated into COS cells which were then screened for their capacity to bind radioiodinated hGM-CSF using a sensitive microscopic autoradiographic approach . 
The cloned GM-CSF-R precursor is a 400 amino acid polypeptide ( Mr 45 , 000 ) with a single transmembrane domain , a glycosylated extracellular domain and a short ( 54 amino acids ) intracytoplasmic tail . 
It does not contain a tyrosine kinase domain nor show homology with members of the immunoglobulin super gene family , but does show some significant sequence homologies with receptors for several other haemopoietic growth factors , including those for interleukin-6 , erythropoietin and interleukin-2 ( beta-chain ) and also to the prolactin receptor . 
When transfected into COS cells the cloned cDNA directed the expression of a GM-CSF-R showing a single class of affinity ( KD = 2 (-8 ) nM ) and specificity for human GM-CSF but not interleukin-3 . 
Messenger RNA coding for this receptor was detected in a variety of haemopoietic cells known to display hGM-CSF binding , and cross-linking experiments revealed a similar size for the glycosylated receptors in transfected COS and haemopoietic cells . 


Structure of tumour necrosis factor . 
Tumour necrosis factor is a trimeric molecule , each subunit of which consists of an antiparallel beta-sandwich . 
Individual subunits from the trimer by a novel edge-to-face packing of beta-sheets . 
A comparison of the subunit fold with that of other proteins reveals a remarkable similarity to the ' jelly-roll ' structural motif characteristic of viral coat proteins . 


The T-cell antigen CD5 acts as a receptor and substrate for the protein-tyrosine kinase p56lck . 
CD5 is a T-cell-specific antigen which binds to the B-cell antigen CD72 and acts as a coreceptor in the stimulation of T-cell growth . 
CD5 associates with the T-cell receptor zeta chain (TcR zeta ) /CD3 complex and is rapidly phosphosphorylated on tyrosine residues as a result of TcR zeta / CD3 ligation . 
However , despite this , the mechanism by which CD5 generates intracellular signals is unclear . 
In this study , we demonstrate that CD5 is coupled to the protein-tyrosine kinase p56lck and can act as a substrate for p56lck . 
Coexpression of CD5 with p56lck in the baculovirus expression system resulted in the phosphorylation of CD5 on tyrosine residues . 
Further , anti- CD5 and anti- p56lck coprecipitated each other in a variety of detergents , including Nonidet P-40 and Triton X-100 . 
Anti- CD5 also precipitated the kinase from various T cells irrespective of the expression of TcR zeta / CD3 or CD4 . 
No binding between p59fyn ( T ) and CD5 was detected in T cells . 
The binding of p56lck to CD5 induced a 10-to 15-fold increase in p56lck catalytic activity , as measured by in vitro kinase analysis . 
In vivo labelling with 32P ( i ) also showed a four-to fivefold increase in Y-394 occupancy in p56lck when associated with CD5 . 
The use of glutathione S-transferase-Lck fusion proteins in precipitation analysis showed that the SH2 domain of p56lck could recognize CD5 as expressed in the baculovirus expression system . 
CD5 interaction with p56lck represents a novel variant of a receptor-kinas e compl ex in which receptor can also serve as substrate. ( ABSTRACT TRUNCATED AT 250 WORDS ) AD-Division of Tumor Immunology , Dana-Farber Cancer Institute , Boston , 


Making a connection : direct binding between keratin intermediate filaments and desmosomal proteins . 
In epidermal cells , keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures . 
Desmoplakin ( DPI ) , a desmosomal component lacking a transmembrane domain , has been implicated in this interaction , although most studies have been conducted with cells that contain few or no desmosomes , and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful . 
In this report , we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes . 
We show that the carboxy terminal " tail " of DPI associates directly with the amino terminal " head " of type II epidermal keratins , including K1 , K2 , K5 , and K6 . 
We have engineered and purified recombinant K5 head and DPI tail , and we demonstrate direct interaction in vitro by solution-binding assays and by ligand blot assays . 
This marked association is not seen with simple epithelial type II keratins , vimentin , or with type I keratins , providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types . 
We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding . 
This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder . 


A newly identified member of tumor necrosis factor receptor superfamily ( TR6 ) suppresses LIGHT-mediated apoptosis . 
TR6 ( decoy receptor 3 ( DcR3 ) ) is a new member of the tumor necrosis factor receptor ( TNFR ) family . 
TR6 mRNA is expressed in lung tissues and colon adenocarcinoma , SW480 . 
In addition , the expression of TR6 mRNA was shown in the endothelial cell line and induced by phorbol 12-myristate 13-acetate / ionomycin in Jurkat T leukemia cells . 
The open reading frame of TR6 encodes 300 amino acids with a 29-residue signal sequence but no transmembrane region . 
Using histidine-tagged recombinant TR6 , we screened soluble forms of TNF-ligand proteins with immunoprecipitation . 
Here , we demonstrate that TR6 specifically binds two cellular ligands , LIGHT ( herpes virus entry mediator ( HVEM )-L ) and Fas ligand ( FasL / CD95L ) . 
These bindings were confirmed with HEK 293 EBNA cells transfected with LIGHT cDNA by flow cytometry . 
TR6 inhibited LIGHT-induced cytotoxicity in HT29 cells . 
It has been shown that LIGHT triggers apoptosis of various tumor cells including HT29 cells that express both lymphotoxin beta receptor ( LTbetaR ) and HVEM / TR2 receptors . 
Our data suggest that TR6 inhibits the interactions of LIGHT with HVEM / TR2 and LTbetaR , thereby suppressing LIGHT-mediated HT29 cell death . 
Thus , TR6 may play a regulatory role for suppressing in FasL- and LIGHT-mediated cell death . 


The retinoblastoma protein binds to RIZ , a zinc-finger protein that shares an epitope with the adenovirus E1A protein . 
The retinoblastoma protein ( Rb ) is a target of viral oncoproteins . 
To explore the hypothesis that viral proteins may be structural mimics of cellular proteins , we have searched cDNA libraries for Rb-binding proteins . 
We report here the cloning of a cDNA for the protein RIZ from rat and human cells . 
RIZ is a 250-kDa nuclear protein containing eight zinc-finger motifs . 
It contains an Rb-binding motif that shares an antigenic epitope with the C terminus of E1A . 
A domain is conserved between RIZ and the PRDI-BF1 / Blimp-1 differentiation factor . 
Other motifs of RIZ include putative GTPase and SH3 ( src homology domain 3 ) domains . 
RIZ is preferentially expressed in both adult and embryonic rat neuroendocrine tissues . 
It is also expressed in human retinoblastoma cells and at low levels in all other human cell lines examined . 
While the function of RIZ is not yet clear , its structure and pattern of expression suggest a role for RIZ in transcriptional regulation during neuronal differentiation and pathogenesis of retinoblastoma . 


FADD , a novel death domain-containing protein , interacts with the death domain of Fas and initiates apoptosis . 
Using the cytoplasmic domain of Fas in the yeast two-hybrid system , we have identified a novel interacting protein , FADD , which binds Fas and Fas-FD5 , a mutant of Fas possessing enhanced killing activity , but not the functionally inactive mutants Fas- LPR and Fas- FD8 . 
FADD contains a death domain homologous to the death domains of Fas and TNFR-1 . 
A point mutation in FADD , analogous to the lpr mutation of Fas , abolishes its ability to bind Fas , suggesting a death domain to death domain interaction . 
Overexpression of FADD in MCF7 and BJAB cells induces apoptosis , which , like Fas-induced apoptosis , is blocked by CrmA , a specific inhibitor of the interleukin-1 beta-converting enzyme . 
These findings suggest that FADD may play an important role in the proximal signal transduction of Fas . 


RIP : a novel protein containing a death domain that interacts with Fas / APO-1 ( CD95 ) in yeast and causes cell death . 
Ligation of the extracellular domain of the cell surface receptor Fas / APO-1 ( CD95 ) elicits a characteristic programmed death response in susceptible cells . 
Using a genetic selection based on protein-protein interaction in yeast , we have identified two gene products that associate with the intracellular domain of Fas : Fas itself , and a novel 74 kDa protein we have named RIP , for receptor interacting protein . 
RIP also interacts weakly with the p55 tumor necrosis factor receptor ( TNFR1 ) intracellular domain , but not with a mutant version of Fas corresponding to the murine lprcg mutation . 
RIP contains an N-terminal region with homology to protein kinases and a C-terminal region containing a cytoplasmic motif ( death domain ) present in the Fas and TNFR1 intracellular domains . 
Transient overexpression of RIP causes transfected cells to undergo the morphological changes characteristic of apoptosis . 
Taken together , these properties indicate that RIP is a novel form of apoptosis-inducing protein . 


Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue . 
Complexed with its intracellular receptor , FKBP12 , the natural product rapamycin inhibits G1 progression of the cell cycle in a variety of mammalian cell lines and in the yeast Saccharomyces cerevisae . 
Previously , a mammalian protein that directly associates with FKBP12-rapamycin has been identified and its encoding gene has been cloned from both human ( designated FRAP ) [ Brown , E.J. , Albers , M.W. , Shin , T.B. , Ichikawa , K. , Keith , C.T. , Lane , W.S. & Schreiber , S.L. ( 1994 ) Nature ( London ) 369 , 756-758 ] and rat ( designated RAFT ) [ Sabatini , D.M. , Erdjument-Bromage , H. , Lui , M. , Tempst , P. & Snyder , S.H. ( 1994 ) Cell 78 , 35-43 ] . 
The full-length FRAP is a 289-kDa protein containing a putative phosphatidylinositol kinase domain . 
Using an in vitro transcription / translation assay method coupled with proteolysis studies , we have identified an 11-kDa FKBP12-rapamycin-binding domain within FRAP . 
This minimal binding domain lies N-terminal to the kinase domain and spans residues 2025-2114 . 
In addition , we have carried out mutagenesis studies to investigate the role of Ser2035 , a potential phosphorylation site for protein kinase C within this domain . 
We now show that the FRAP Ser2035 --> Ala mutant displays similar binding affinity when compared with the wild-type protein , whereas all other mutations at this site , including mimics of phosphoserine , abolish binding , presumably due to either unfavorable steric interactions or induced conformational changes . 


Localization of functional receptor epitopes on the structure of ciliary neurotrophic factor indicates a conserved , function-related epitope topography among helical cytokines . 
By rational mutagenesis , receptor-specific functional analysis , and visualization of complex formation in solution , we identified individual amino acid side chains involved specifically in the interaction of ciliary neurotrophic factor ( CNTF ) with CNTFR alpha and not with the beta-components , gp130 and LIFR . 
In the crystal structure , the side chains of these residues , which are located in helix A , the AB loop , helix B , and helix D , are surface accessible and are clustered in space , thus constituting an epitope for CNTFR alpha . 
By the same analysis , a partial epitope for gp130 was also identified on the surface of helix A that faces away from the alpha-epitope . 
Superposition of the CNTF and growth hormone structures showed that the location of these epitopes on CNTF is analogous to the location of the first and second receptor epitopes on the surface of growth hormone . 
Further comparison with proposed binding sites for alpha- and beta-receptors on interleukin-6 and leukemia inhibitory factor indicated that this epitope topology is conserved among helical cytokines . 
In each case , epitope I is utilized by the specificity-conferring component , whereas epitopes II and III are used by accessory components . 
Thus , in addition to a common fold , helical cytokines share a conserved order of receptor epitopes that is function related . 


Evidence for a differential interaction of SHC and the insulin receptor substrate-1 ( IRS-1 ) with the insulin-like growth factor-I ( IGF-I ) receptor in the yeast two-hybrid system . 
Using the yeast two-hybrid system , a genetic assay for studying protein-protein interactions , we have examined and compared the interaction of the insulin-like growth factor-I receptor ( IGF-IR ) and the insulin receptor ( IR ) with their two known substrates p52Shc and the insulin receptor substrate-1 ( IRS-1 ) . 
We also mapped the specific domains of the IGF-IR and p52Shc participating in these interactions . 
Our findings can be summarized as follows : ( i ) the tyrosine kinase activity of the IGF-IR is essential for the interaction with p52Shc and IRS-1 , ( ii ) p52Shc and IRS-1 bind to the IGF-IR in the NPEY-juxtamembrane motif , ( iii ) contrary to p52Shc , IRS-1 binds also to the major autophosphorylation sites ( Tyr-1131 ,-1135 , and -1136 ) of the IGF-IR , and ( iv ) the amino-terminal domain of p52Shc is required for its association with the IR and the IGF-IR . 
We propose that ( i ) the IGF-IR and the IR share at least in part the same molecular mechanism underlying their interplay with their two substrates , p52Shc and IRS-1 , and ( ii ) IRS-1 interacts with the IGF-IR in a fashion that is different from that used by p52Shc . 
Finally , our data highlight the crucial role of the juxtamembrane domain in signaling by both the IR and the IGF-IR . 


Wiskott-Aldrich syndrome protein physically associates with Nck through Src homology 3 domains . 
In the second of a series of experiments designed to identify p47nck-Src homology 3 ( SH3 ) -binding molecules , we report the cloning of SAKAP II ( Src A box Nck-associated protein II ) from an HL60 cDNA expression library . 
This molecule has been identified as a cDNA encoding the protein product of WASP , which is mutated in Wiskott-Aldrich syndrome patients . 
Studies in vivo and in vitro demonstrated a highly specific interaction between the SH3 domains of p47nck and Wiskott-Aldrich syndrome protein . 
Furthermore , anti- Wiskott-Aldrich syndrome protein antibodies recognized a protein of 66 kDa by Western blot ( immunoblot ) analysis . 
In vitro translation studies identified the 66-kDa protein as the protein product of WASP , and subcellular fractionation experiments showed that p66WASP is mainly present in the cytosol fraction , although significant amounts are also present in membrane and nuclear fractions . 
The main p47nck region implicated in the association with p66WASP was found to be the carboxy-terminal SH3 domain . 


Correlation of two-hybrid affinity data with in vitro measurements . 
Since their introduction , the interaction trap and other two-hybrid systems have been used to study protein-protein interactions . 
Despite their general use , little is known about the extent to which the degree of protein interaction determined by two-hybrid approaches parallels the degree of interaction determined by biochemical techniques . 
In this study , we used a set of lexAop-LEU2 and lexAop-lacZ reporters to calibrate the interaction trap . 
For the calibration , we used two sets of proteins , the Myc- Max- Mxi1 helix-loop-helix proteins , and wild-type and dimerization-defective versions of the lambda cI repressor . 
Our results indicate that the strength of interaction as predicted by the two-hybrid approach generally correlates with that determined in vitro , permitting discrimination of high-, intermediate-, and low-affinity interactions , but there was no single reporter for which the amount of gene expression linearly reflected affinity measured in vitro . 
However , some reporters showed thresholds and only responded to stronger interactions . 
Finally , some interactions were subject to directionality , and their apparent strength depended on the reporter used . 
Taken together , our results provide a cautionary framework for interpreting affinities from two-hybrid experiments . 


Interaction of p85 subunit of PI 3-kinase with insulin and IGF-1 receptors analysed by using the two-hybrid system . 
Interaction of the p85 subunit of PI 3-kinase with the insulin receptor ( IR ) and the IGF-1 receptor ( IGF-1R ) was investigated using the two-hybrid system by assessing for his3 and lacZ activation in S. cerevisiae . 
The experiments were performed with the cytoplasmic beta domain ( wild type or mutated ) of IR and IGF-1R and p85 or its subdomains ( N + C-SH2 , N-SH2 , C-SH2 , SH3 + N-SH2 ) . 
The results of his3 activation indicated that p85 , N + C-SH2 and C-SH2 interact with both IR beta and IGF-1R beta , whereas N-SH2 and SH3 + N-SH2 interact only with IR beta . 
Interaction of p85 and N+C-SH2 with IR beta ( delta C-43 ) or IGF-1R beta ( delta C-43 ) in which the C-terminal 43 amino acids ( including the YXXM motif ) were deleted , persisted . 
The internal binding site thus revealed was not altered by further mutating Y960 / F for IR or Y950 / F for IGF-1R . 
Activation of lacZ upon interaction of p85 with IR beta ( delta C-43 ) was 4-fold less as compared to IR beta . 
This activation with p85 and IGF-1R beta was 4-fold less as compared to IR beta and was somewhat increased ( 2-fold ) for IGF-1R beta ( delta C-4 3 ) . 
Thus , the C-terminal domain in IGF-1R appears to exert a negative control on binding of p85 thereby providing a possible regulatory mechanism for direct activation of the PI 3-kinase pathway . 


Interaction of the protein nucleobindin with G alpha i2 , as revealed by the yeast two-hybrid system . 
The heterotrimeric G protein , G alpha i2 , transduces signals from seven membrane spanning receptors to effectors such as adenylyl cyclase and ion channels . 
The purpose of this study was to identify these or other cellular proteins that interact with G alpha i2 by use of the yeast two-hybrid system . 
A human B cell cDNA library was screened by this system using full length G alpha i2 . 
Four positive colonies were obtained . 
Two of the four were identified as nucleobindin , a calcium binding protein and a putative antigen to which anti-nuclear antibodies are generated in mice with a disorder that resembles systemic lupus erythematosus . 
Nucleobindin has a leucine zipper , EF hands , and a signal peptide sequence and is thought to localize to the nucleus as well as being secreted . 
The specificity of intehraction between G alpha i2 and nucleobindin was confirmed by an in vitro binding assay using recombinant proteins . 
Transfection of G alpha i2 and nucleobindin in COS cells increased G alpha i2 expression relative to cells transfected with G alpha i2 and mock vector . 
Our results indicate that the yeast two-hybrid system provides a means to identify novel proteins that interact with G alpha proteins . 
Nucleobindin appears to represent one of those proteins . 


The mitogenic effects of the B beta chain of fibrinogen are mediated through cell surface calreticulin . 
We have previously shown that soluble partially degraded fibrin ( ogen ) remains in solution after fibrin clot formation and is a potent fibroblast mitogen ( Gray , A.J. , Bishop , J.E. , Reeves J.T. , Mecham , R.P. , and Laurent , G.J. ( 1995 ) Am. J. Cell Mol. Biol. 12 , 684-690 ) . 
Mitogenic sites within the fibrin ( ogen ) molecule are located on the A alpha and B beta chains of the protein ( Gray , A.J. , Bishop , J. E. , Reeves , J.T. , and Laurent , G.J. ( 1993 ) J. Cell Sci. 104 , 409-413 ) . 
However , receptor pathways through which mitogenic effects are mediated are unknown . 
The present study sought to determine the nature of fibrin ( ogen ) receptors expressed on human fibroblasts which interact with the fibrinogen B beta chain . 
Receptor complexes were isolated from 125I-surface-labeled fibroblasts and purified on a fibrinogen B beta chain affinity column . 
Subsequent high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis analysis indicated fibrinogen B beta chain bound specifically to a 60-kDa surface protein . 
Sequence analysis of the amino terminus of this protein indicated 100 % homology to human calreticulin . 
Immunoprecipitation experiments employing a polyclonal anti- calreticulin antibody provided further evidence that the 60-kDa protein isolated in this study was calreticulin . 
Further , polyclonal antibodies to human calreticulin significantly inhibited the mitogenic activity of fibrinogen B beta chain on human fibroblasts . 
The present study has shown that cell surface calreticulin binds to the B beta chain of fibrinogen mediating its mitogenic activity . 


Multiple proteins physically interact with PU.1 . 
Transcriptional synergy with NF-IL6 beta ( C / EBP delta , CRP3 ) . 
PU.1 is a transcription factor that belongs to the ets family of DNA binding proteins . 
In this study , we show by Far Western blot analyses that multiple nuclear proteins are capable of physically interacting with PU.1 . 
Using radiolabeled PU.1 protein as a probe , we screened a B cell cDNA expression library and isolated a number of clones encoding PU.1 interacting proteins . 
Three of these clones encode DNA binding proteins ( NF-IL6 beta , HMG I / Y , and SSRP ) , one clone encodes a chaperone protein , and another clone encodes a multifunctional phosphatase . 
We have characterized the physical and functional interactions between PU.1 and NF-IL6 beta , a leucine zipper transcription factor implicated in inflammatory responses . 
We found that deletion of the carboxyl-terminal 28 amino acids of PU.1 disrupted PU.1-NF-IL6 beta physical interaction . 
This deletion disrupts the PU.1 Ets domain . 
Deletion of the NF-IL6 beta leucine zipper domain also greatly diminished the interaction between these two proteins . 
In transient expression assays , we found that PU.1 and NF-IL6 beta can functionally cooperate to synergistically activate transcription . 
Electrophoretic mobility shift assays showed that PU.1 and NF-IL6 beta can simultaneously bind to adjacent DNA binding sites , but apparently do not influence the kinetics or affinity of each other 's DNA binding . 
These results suggest that transcriptional synergy is due to each protein independently influencing the basal transcription complex . 


Molecular cloning and expression of the 32-kDa subunit of human TFIID reveals interactions with VP16 and TFIIB that mediate transcriptional activation . 
Transcription factor TFIID consists of TATA binding protein ( TBP ) and at least eight TBP-associated factors ( TAFs ) . 
As TAFs are required for activated but not basal transcription , we have proposed that TAF s act as coactivators to mediate signals between activators and the basal transcription machinery . 
Here we report the cloning , expression , and biochemical characterization of the 32-kDa subunit of human ( h ) TFIID , termed hTAFII32 . 
We find that hTAFII32 is the human homologue of Drosophila TAFII40 . 
In vitro protein-protein interaction assays reveal that as observed with Drosophila TAFII40 , hTAFII32 interacts with the C-terminal 39-amino acid activation domain of the acidic transactivator viral protein 16 ( VP16 ) as well as with the general transcription factor TFIIB . 
Moreover , a partial recombinant TFIID complex containing hTAFII32 was capable of mediating in vitro transcriptional activation by the VP16 activation domain . 
These findings indicate that specific activator-coactivator interactions have been conserved between human and Drosophila and provide additional support for the function of these interactions in mediating transcriptional activation . 


A potential interaction of p75 and trkA NGF receptors revealed by affinity crosslinking and immunoprecipitation . 
Nerve growth factor binds independently to two transmembrane receptors , the p75 neurotrophin receptor and the p140trk ( trkA ) tyrosine kinase receptor , which are both co-expressed in the majority of neuronal cells that respond to NGF . 
Previous findings have suggested that appropriate co-expression of the two receptors gives rise to high affinity NGF binding sites and increased neurotrophin responsiveness ; however , evidence demonstrating a direct interaction between the two receptors in cell lines has been lacking . 
Here we have utilized affinity crosslinking agents with 125I-NGF to detect an association of trkA and p75 receptors in embryonic spinal cord and brain tissues enriched in the two receptors . 
Although multimeric complexes of trkA and p75 were not detected by affinity crosslinking , immunoprecipitation of cross-linked NGF-receptor complexes with trk-specific antibodies resulted in selective immunoprecipitation of crosslinked p75 . 
Our results indicate that the trkA and p75 receptors can potentially interact , and that such an association may be responsible for the generation of high affinity NGF binding sites . 


Crystal structure of a complex between interferon-gamma and its soluble high-affinity receptor . 
The crystal structure of interferon-gamma bound to the extracellular fragment of its high-affinity cell-surface receptor reveals the first view of a class-2 cytokine receptor-ligand complex . 
In the complex , one interferon-gamma homodimer binds two receptor molecules . 
Unlike the class-1 growth hormone receptor complex , the two interferon-gamma receptors do not interact with one another and are separated by 27 A . 
Upon receptor binding , the flexible AB loop of interferon-gamma undergoes a conformational change that includes the formation of a 3 ( 10 ) helix . 


Cloning and functional expression of a human eosinophil CC chemokine receptor . 
Eosinophils undergo chemotaxis , degranulate , and exhibit [ C2+ ] i changes in response to the human CC chemokines macrophage inflammatory protein ( MIP )-1 alpha , regulated on activation , normal T expressed and secreted ( RANTES ) , and monocyte chemoattractant protein-3 ( MCP-3 ) , but the receptors involved have not been defined . 
We have isolated a human cDNA encoding the first eosinophil-selective chemokine receptor , designated CC chemokine receptor 3 ( CC CKR3 ) . 
CC CKR3 is a seven-transmembrane domain G protein-coupled receptor most closely related to the previously reported monocyte- and neutrophil-selective receptor CC CKR1 ( also known as the MIP-1 alpha / RANTES receptor ) . 
When [ Ca2+ ] i changes were monitored in stably transfected human embryonic kidney 293 cells , ( MIP )-1 alpha MIP and RANTES were both potent agonists for CC CKR3 and CC CKR1 . 
However , MIP-1 beta was also an agonist for CC CKR3 but not CC CKR1 ; MCP-3 was an agonist for CC CKR1 but not CC CKR3 . 
CC CKR3 may be one of the host factors responsible for selective recruitment of eosinophils to sites of inflammation . 


Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex . 
The crystal structure of the human cyclinA-cyclin-dependent kinase2 ( CDK2 ) -ATP complex has been determined at 2.3 A resolution . 
CyclinA binds to one side of CDK2 ' s catalytic cleft , inducing large conformational changes in its PSTAIRE helix and T-loop . 
These changes activate the kinase by realigning active site residues and relieving the steric blockade at the entrance of the catalytic cleft . 


Receptor protein tyrosine phosphatase PTPmu associates with cadherins and catenins in vivo . 
The extracellular segment of the receptor-type type protein tyrosine phosphatase PTPmu , possesses an MAM domain , an immunoglobulin domain , and four fibronectin type-III repeats . 
It binds homophilically , i.e. , PTPmu on the surface of one cell binds to PTPmu on an apposing cell , and the binding site lies within the immunoglobulin domain . 
The intracellular segment of PTPmu has two PTP domains and a juxtamembrane segment that is homologous to the conserved intracellular domain of the cadherins . 
In cadherins , this segment interacts with proteins termed catenins to mediate association with the actin cytoskeleton . 
In this article , we demonstrate that PTPmu associates with a complex containing cadherins , alpha- and beta-catenin in mink lung ( MvLu ) cells , and in rat heart , lung , and brain tissues . 
Greater than 80 % of the cadherin in the cell is cleared from Triton X-100 lysates of MvLu cells after immunoprecipitation with antibodies to PTPmu ; however , the complex is dissociated when lysates are prepared in more stringent , SDS-containing RIPA buffer . 
In vitro binding studies demonstrated that the intracellular segment of PTPmu binds directly to the intracellular domain of E-cadherin , but not to alpha- or beta-catenin . 
Consistent with their ability to interact in vivo , PTPmu , cadherins , and catenins all localized to points of cell-cell contact in MvLu cells , as assessed by immunocytochemical staining . 
After pervanadate treatment of MvLu cells , which inhibits cellular tyrosine phosphatase activity including PTPmu , the cadherins associated with PTPm u are now found in a tyrosine-phosphorylated form , indicating that the cadherins may be an endogenous substrate for PTPmu . 
These data suggest that PTPmu may be one of the enzymes that regulates the dynamic tyrosine phosphorylation , and thus function , of the cadherin / catenin complex in vivo . 


Cloning and characterization of a human type II receptor for bone morphogenetic proteins . 
Bone morphogenetic proteins ( BMPs ) are members of the transforming growth factor beta superfamily . 
Several members of this family have been shown to transduce their signals through binding to type I and type II serine- ( threonine ) kinase receptors . 
Here we report the cDNA cloning and characterization of a human type II receptor for BMPs ( BMPR-II ) , which is distantly related to DAF-4 , a BMP type II receptor from Caenorhabditis elegans . 
In transfected COS-1 cells , osteogenic protein ( OP )-1 / BMP-7 , and less efficiently BMP-4 , bound to BMPR-II . 
BMPR-II bound ligands only weakly alone , but the binding was facilitated by the presence of previously identified type I receptors for BMPs . 
Binding of OP-1 / BMP-7 to BMPR-II was also observed in nontransfected cell lines . 
Moreover , a transcriptional activation signal was transduced by BMPR-II in the presence of type I receptors after stimulation by OP-1 / BMP-7 . 


14-3-3 proteins associate with cdc25 phosphatases . 
The cdc25 phosphatases play key roles in cell cycle progression by activating cyclin-dependent kinases . 
Two members of the 14-3-3 protein family have been isolated in a yeast two-hybrid screen designed to identify proteins that interact with the human cdc25A and cdc25B phosphatases . 
Genes encoding the human homolog of the 14-3-3 epsilon protein and the previously described 14-3-3 beta protein have been isolated in this screening . 
14-3-3 proteins constitute a family of well-conserved eukaryotic proteins that were originally isolated in mammalian brain preparations and that possess diverse biochemical activities related to signal transduction . 
We present evidence that indicates that cdc25 and 14-3-3 proteins physically interact both in vitro and in vivo . 
14-3-3 protein does not , however , affect the phosphatase activity of cdc25A . 
Raf-1 , which is known to bind 14-3-3 proteins , has recently been shown to associate with cdc25A and to stimulate its phosphatase activity . 
14-3-3 protein , however , has no effect on the cdc25A-kinase activity of Raf-1 . 
Instead , 14-3-3 may facilitate the association of cdc25 with Raf-1 in vivo , participating in the linkage between mitogenic signaling and the cell cycle machinery . 


The Cockayne syndrome group A gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH . 
The hereditary disease Cockayne syndrome ( CS ) is characterized by a complex clinical phenotype . 
CS cells are abnormally sensitive to ultraviolet radiation and are defective in the repair of transcriptionally active genes . 
The cloned CSB gene encodes a member of a protein family that includes the yeast Snf2 protein , a component of the transcriptional regulator Swi / Snf . 
We report the cloning of the CSA cDNA , which can encode a WD repeat protein . 
Mutations in the cDNA have been identified in CS-A cell lines . 
CSA protein interacts with CSB protein and with p44 protein , a subunit of the human RNA polymerase II transcription factor IIH . 
These observations suggest that the products of the CSA and CSB genes are involved in transcription . 


Cloning and expression of human TAFII250 : a TBP-associated factor implicated in cell-cycle regulation . 
Basal transcription by human RNA polymerase II requires the coordinate action of several ancillary factors ( TFIIA-J ) and can be regulated by various promoter-specific DNA binding proteins . 
An additional class of factors , called coactivators , are dispensable for basal transcription but are indispensable for regulation by transcriptional activators . 
Biochemical studies established that some coactivators are associated with the TATA-binding protein ( TBP ) to form the TFIID complex . 
We therefore set out to define the relationship between TBP and these TBP-associated factors ( TAFs ) . 
Here we describe the cloning , expression and properties of the first human TAF , hTAFII250 . 
The hTAFII250 gene is identical to a gene , CCG1 , ( ref 7 , 8 ) , implicated in cell-cycle progression . 
Recombinant hTAFII250 binds directly to TBP both in vitro and in yeast , and participates in the formation of the TFIID complex . 
This largest TAF may therefore play a central role in TFIID assembly by interacting with both TBP and other TAFs , as well as serving to link the control of transcription to the cell cycle . 


The bZIP domains of Fos and Jun mediate a physical association with the TATA box-binding protein . 
Fos and Jun oncoproteins form a complex that regulates transcription from promoters containing AP-1 binding sites . 
These two proteins , like other transcriptional activators , are likely to stimulate transcription through direct and / or indirect interactions with members of the basal transcriptional machinery . 
The ability of c-Fos and c-Jun proteins to interact directly with the TATA box-binding protein ( TBP ) , the general transcription factor required for initiating the assembly of transcription complexes , was investigated . 
Using co-immunoprecipitation and protein-protein association assays , we show that both c-Fos and c-Jun bind specifically and stably to TBP Mutational analysis demonstrates that both the basic region and leucine zipper domains of c-Fos and c-Jun are necessary and sufficient for stable association with TBP . 
A 51-residue region from the conserved C-terminal region of TBP , previously shown to be the binding site for the viral activator protein E1A , interacts with c-Fos and c-Jun proteins . 
We propose that c-Fos and c-Jun proteins function as transcriptional activators , in part by recruiting TBP to form complexes to initiate RNA synthesis . 


The retinoblastoma-susceptibility gene product binds directly to the human TATA-binding protein-associated factor TAFII250 . 
RB , the protein product of the retinoblastoma tumor-suppressor gene , regulates the activity of specific transcription factors . 
This regulation appears to be mediated either directly through interactions with specific transcription factors or through an alternative mechanism . 
Here we report that stimulation of Sp1-mediated transcription by RB is partially abrogated at the nonpermissive temperature in ts13 cells . 
These cells contain a temperature-sensitive mutation in the TATA-binding protein-associated factor TAFII250 , first identified as the cell cycle regulatory protein CCG1 . 
The stimulation of Sp1-mediated transcription by RB in ts13 cells at the nonpermissive temperature could be restored by the introduction of wild-type human TAFII250 . 
Furthermore , we demonstrate that RB binds directly to hTAFII250 in vitro and in vivo . 
These results suggest that RB can confer transcriptional regulation and possibly cell cycle control and tumor suppression through an interaction with TFIID , in particular with TAFII250 . 


Cloning and characterization of hTAFII18 , hTAFII20 and hTAFII28 : three subunits of the human transcription factor TFIID . 
We have cloned cDNAs encoding three novel TAFIIs [ TATA-binding protein ( TBP ) -associated factors ] from the human ( h ) HeLa cell TFIID complexes hTAFII28 , hTAFII20 and hTAFII18 . 
hTAFII28 is a core hTAFII present in both of the previously described hTFIID species which either lack or contain hTAFII30 ( hTFIID alpha and hTFIID beta respectively ) , and is the homologue of Drosophila ( d ) TAFII30 beta . 
hTAFII18 is a novel hTAFII which shows homology to the N-terminal region of the yeast TAFIISPT3 , but has no known Drosophila counterpart . 
In contrast to hTAFII28 , hTAFII18 is a TFIID beta-specific hTAFII . 
hTAFII20 is the homologue of p22 , an alternatively spliced form of d TAFII30 alpha ( p32 ) . 
Using a combination of protein affinity chromatography and cotransfection and immunoprecipitation assays , we have identified a series of in vitro and intracellular interactions among the novel hTAFIIs and between the novel hTAFIIs and hTAFII30 or TBP . 
We show that hTAFII28 interacts with hTAFII18 both in vitro and intracellularly ; in contrast to its Drosophila homologue , hTAFII28 also interacts directly with TBP . 
Deletion analysis indicates that TBP and hTAFII18 bind to distinct domains of hTAFII28 . 
hTAFII18 also interacts with TBP , but it interacts more strongly with hTAFII28 and hTAFII30 . 
The binding of hTAFII28 and hTAFII30 requires distinct domains of hTAFII18 . 
As observed with the homologous Drosophila proteins , hTAFII20 interacts directly with TBP ; however , additional interactions between hTAFII20 and hTAFII28 or hTAFII30 were detected . 
These results reveal differences not only in subunit composition , but also in the organization of dTFIID and hTFIID complexes . 


In vivo association of E2F and DP family proteins . 
The mammalian transcription factor E2F plays an important role in regulating the expression of genes that are required for passage through the cell cycle . 
This transcriptional activity is inhibited by association with the retinoblastoma tumor suppressor protein ( pRB ) or its relatives p107 and p103 . 
The first cDNA from the E2F family to be cloned was designated E2F-1 , and multiple E2F family members have now been identified . 
They bind to DNA as heterodimers , interacting with proteins known as DP . 
Here we demonstrate that DP is also a family of polypeptides with at least two members ( hDP-1 and hDP-2 ) . 
Both hDP-1 and hDP-2 bind to all E2F family members in vivo , and each complex is capable of activating transcription . 
However , the various E2F / DP complexes display strong differences in the ability to bind to either pRB or p107 in vivo , and the specificity of pRB or p107 binding is mediated by the E2F subunit . 


Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling . 
The helical cytokine interleukin ( IL ) 6 and its specific binding subunit IL-6R alpha form a 1 : 1 complex which , by promoting homodimerization of the signalling subunit gp130 on the surface of target cells , triggers intracellular responses . 
We expressed differently tagged forms of gp130 and used them in solution-phase binding assays to show that the soluble extracellular domains of gp130 undergo dimerization in the absence of membranes . 
In vitro receptor assembly reactions were also performed in the presence of two sets of IL-6 variants carrying amino acid substitutions in two distinct areas of the cytokine surface ( site 2 , comprising exposed residues in the A and C helices , and site 3 , in the terminal part of the CD loop ) . 
The binding affinity to IL-6R alpha of these variants is normal but their biological activity is poor or absent . 
We demonstrate here that both the site 2 and site 3 IL-6 variants complexed with IL-6R alpha bind a single gp130 molecule but are unable to dimerize it , whereas the combined site 2 / 3 variants lose the ability to interact with gp130 . 
The binding properties of these variants in vitro , and the result of using a neutralizing monoclonal antibody directed against site 3 , lead to the conclusion that gp130 dimer is formed through direct binding at two independent and differently oriented sites on IL-6 . 
Immunoprecipitation experiments further reveal that the fully assembled receptor complex is composed of two IL-6 , two IL-6R alpha and two gp130 molecules . 
We propose here a model representing the IL-6 receptor complex as hexameric , which might be common to other helical cytokines . 


Raf1 interaction with Cdc25 phosphatase ties mitogenic signal transduction to cell cycle activation . 
The Ras and Raf1 proto-oncogenes transduce extracellular signals that promote cell growth . 
Cdc25 phosphatases activate the cell division cycle by dephosphorylation of critical threonine and tyrosine residues within the cyclin-dependent kinases . 
We show here that Cdc25 phosphatase associates with raf1 in somatic mammalian cells and in meiotic frog oocytes . 
Furthermore , Cdc25 phosphatase can be activated in vitro in a Raf1-dependent manner . 
We suggest that activation of the cell cycle by the Ras / Raf1 pathways might be mediated in part by Cdc25 . 


A direct interaction between G-protein beta gamma subunits and the Raf-1 protein kinase . 
Raf-1 is a serine / threonine protein kinase positioned downstream of Ras in the mitogen-activated protein kinase cascade . 
Using a yeast two-hybrid strategy to identify other proteins that interact with and potentially regulate Raf-1 , we isolated a clone encoding the carboxyl-terminal half of the G beta 2 subunit of heterotrimeric G-proteins . 
In vitro , purified G beta gamma subunits specifically bound to a GST fusion protein encoding amino acids 1-330 of Raf-1 ( Raf / 330 ) . 
Binding assays with truncation mutants of GST- Raf indicate that the region located between amino acids 136 and 239 is a primary determinant for interaction with G beta gamma . 
In competition experiments , the carboxyl terminus of beta-adrenergic receptor kinase ( beta ARK ) blocked the binding of G beta gamma to Raf / 330 ; however , the Raf-1-binding proteins , Ras and 14-3-3 , had no effect . 
Scatchard analysis of in vitro binding between Raf / 330 and G beta gamma revealed an affinity of interaction ( Kd = 163 + /-36 nM ) , similar to that seen between G beta gamma and beta ARK ( Kd = 87 + /-24 nM ) . 
The formation of native heterotrimeric G alpha beta gamma complexes , as measured by pertussis toxin ADP-ribosylation of G alpha , could be disrupted by increasing amounts of Raf / 330 , with an EC50 of approximately 200 nM , in close agreement with the estimated binding affinity . 
In vivo complexes of Raf-1 and G beta gamma were isolated from human embryonic kidney 293-T cells transfected with epitope-tagged G beta 2 . 
The identification and characterization of this novel interaction raises several possibilities for signaling cross-talk between growth factor receptors and those receptors coupled to heterotrimeric G-proteins . 


p53 transcriptional activation mediated by coactivators TAFII40 and TAFII60 . 
The tumor suppressor protein p53 is a transcriptional regulator that enhances the expression of proteins that control cellular proliferation . 
The multisubunit transcription factor IID ( TFIID ) is thought to be a primary target for site-specific activators of transcription . 
Here , a direct interaction between the activation domain of p53 and two subunits of the TFIID complex , TAFII40 and TAFII60 , is reported . 
A double point mutation in the activation domain of p53 impaired the ability of this domain to activate transcription and , simultaneously , its ability to interact with both TAFII40 and TAFII60 . 
Furthermore , a partial TFIID complex containing Drosophila TATA binding protein ( dTBP ) , human TAFII250 , dTAFII60 , and dTAFII40 supported activation by a Gal4-p53 fusion protein in vitro , whereas TBP or a subcomplex lacking TAFII40 and TAFII60 did not . 
Together , these results suggest that TAFII40 and TAFII60 are important targets for transmitting activation signals between p53 and the initiation complex . 


Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA . 
The Fos and Jun families of eukaryotic transcription factors heterodimerize to form complexes capable of binding 5 '-TGAGTCA-3 ' DNA elements . 
We have determined the X-ray crystal structure of a heterodimer of the bZIP regions of c-Fos and c-Jun bound to DNA . 
Both subunits form continuous alpha-helices . 
The carboxy-terminal regions form an asymmetric coiled-coil , and the amino-terminal regions make base-specific contacts with DNA in the major groove . 
Comparison of the two crystallographically distinct protein-DNA complexes show that the coiled-coil is flexibly joined to the basic regions and that the Fos-Jun heterodimer does not recognize the asymmetric 5 '-TGAGTCA-3 ' recognition element in a unique orientation . 
There is an extensive network of electrostatic interactions between subunits within the coiled-coil , consistent with proposals that these interactions determine preferential formation of the heterodimer over either of the homodimers . 


Cloning of an intrinsic human TFIID subunit that interacts with multiple transcriptional activators . 
TFIID is a multisubunit protein complex comprised of the TATA-binding protein ( TBP ) and multiple TBP-associated factors ( TAFs ) . 
The TAFs in TFIID are essential for activator-dependent transcription . 
The cloning of a complementary DNA encoding a human TFIID TAF , TAFII55 , that has no known homolog in Drosophila TFIID is now described . 
TAFII55 is shown to interact with the largest subunit ( TAFII230 ) of human TFIID through its central region and with multiple activators -- including Sp1 , YY1 , USF , CTF , adenoviral E1A , and human immunodeficiency virus-type 1 Tat proteins -- through a distinct amino-terminal domain . 
The TAFII55-interacting region of Sp1 was localized to its DNA-binding domain , which is distinct from the glutamine-rich activation domains previously shown to interact with Drosophila TAFII110 . 
Thus , this human TFIID TAF may be a co-activator that mediates a response to multiple activators through a distinct mechanism . 


Mutagenesis of the regulatory subunit ( RII beta ) of cAMP-dependent protein kinase II beta reveals hydrophobic amino acids that are essential for RII beta dimerization and / or anchoring RII beta to the cytoskeleton . 
In neurons cAMP-dependent protein kinase II beta ( PKAII beta ) is sequestered in the dendritic cytoskeleton because the regulatory subunit ( RII beta ) of the enzyme is tightly bound by A Kinase Anchor Proteins ( AKAPs ) . 
The prototypic neuronal anchor protein AKAP75 has a COOH-terminal 22-residue RII beta binding ( tethering ) site . 
A key feature of the tethering site is that several amino acids with large aliphatic side chains mediate the high-affinity binding of RII beta . 
Mutagenesis , recombinant protein expression , and physicochemical characterization were used to investigate the structural basis for the homodimerization and AKAP75 binding activities of RII beta . 
Several crucial residues are located in an NH2-terminal region that encompasses amino acids 13-36 . 
Substitution of Ala for Leu13 or Phe36 generates monomeric RII beta subunits that cannot bind AKAP75 . 
The results are not due to general misfolding since mutant RII beta monomers bind cAMP and inhibit the catalytic subunit of PKAII beta with the same affinity and efficacy as wild-type RII beta dimers . 
Moreover , substitution of Ala for Leu12 , Val20 , Leu21 , Phe31 , Leu33 , or Leu39 and replacement of Leu13 with Ile or Val did not impair the dimerization reaction . 
Evidently , large hydrophobic side chains of Leu13 and Phe36 play pivotal roles in stabilizing RII beta-RII beta interactions . 
A secondary consequence of destabilizing RII beta dimers is the loss of intracellular targeting / anchoring capacity because monomers fail to bind AKAP75 . 
Other NH2-terminal residues directly modulate the affinity of RII beta dimers for the AKAP75 tethering site . 
Replacement of Val20-Leu21 with Ala-Ala produced a dimeric RII beta protein that binds AKAP75 approximately 4 % as avidly as wild-type RII beta . 
It is possible that the aliphatic side chains of Val20 and Leu21 interact with the essential Leu and Ile residues in the AKAP75 tethering region . 


Human spleen tyrosine kinase p72Syk associates with the Src-family kinase p53 / 56Lyn and a 120-kDa phosphoprotein . 
The 72-kDa spleen tyrosine kinase ( Syk ) and Src-family kinase p53 / 56Lyn ( Lyn ) contribute to signaling via the B-cell antigen receptor complex . 
Here we show that Syk and Lyn from human B lymphocytes can interact directly . 
Syk and Lyn coimmunoprecipitated from mature and activated B-cell lines , and gel-purified Syk and Lyn reassociated in vitro , demonstrating their direct interaction . 
This Syk-Lyn interaction may be dependent on the stage of B-cell differentiation , since Syk-Lyn associations were not detected in pre-B and myeloma cell lines and Syk from an immature B-cell line did not reassociate with Lyn in vitro . 
Serine / threonine kinase activity was also associated with Syk . 
Crosslinking of cell surface IgM led to rapid activation of both tyrosine and serine / threonine protein kinase activities that resulted in phosphorylation in vitro of proteins coprecipitating with Syk-- in particular , a serine / threonine phosphorylated protein 120 kDa in size ( pp120 ) . 
Several phosphoproteins , including one of 72 kDa and one of 120 kDa , coprecipitated with phospholipase C-gamma 1 ( PLC gamma 1 ) . 
Sequential immunoprecipitation identified the 72-kDa protein associated with PLC gamma 1 as Syk . 
The 120-kDa serine / threonine phosphorylated protein that coprecipitated with PLC gamma 1 resembled the Syk-associated pp120 by several criteria . 
Thus , pp120 may serve as a link between Syk and PLC gamma 1 , coupling the B-cell antigen receptor to the phosphatidylinositol pathway . 


Cloning and functional analysis of BAG-1 : a novel Bcl-2-binding protein with anti-cell death activity . 
Using a protein interaction cloning technique , we identified cDNAs that encode a novel Bcl-2-binding protein , termed BAG-1 . 
The BAG-1 protein shares no significant homology with Bcl-2 or other Bcl-2 family proteins , which can form homo- and heterodimers . 
In gene transfer experiments using a human lymphoid cell line , Jurkat , coexpression of BAG-1 and Bcl-2 provided markedly increased protection from cell death induced by several stimuli , including staurosporine , anti-Fas antibody , and cytolytic T cells , relative to cells that contained gene transfer-mediated elevations in either BAG-1 or Bcl-2 protein alone . 
BAG-transfected 3T3 fibroblasts also exhibited prolonged cell survival in response to an apoptotic stimulus . 
The findings indicate that bag-1 represents a new type of anti-cell death gene and suggest that some routes of apoptosis induction previously ascribed to Bcl-2-independent pathways may instead reflect a need for the combination of Bcl-2 and BAG-1 . 


Syntrophin binds to an alternatively spliced exon of dystrophin . 
Dystrophin , the protein product of the Duchenne muscular dystrophy locus , is a protein of the membrane cytoskeleton that associates with a complex of integral and membrane-associated proteins . 
Of these , the 58-kD intracellular membrane-associated protein , syntrophin , was recently shown to consist of a family of three related but distinct genes . 
We expressed the cDNA of human beta 1-syntrophin and the COOH terminus of human dystrophin in reticulocyte lysates using an in vitro transcription / translation system . 
Using antibodies to dystrophin we immunoprecipitated these two interacting proteins in a variety of salt and detergent conditions . 
We demonstrate that the 53 amino acids encoded on exon 74 of dystrophin , an alternatively spliced exon , are necessary and sufficient for interaction with translated beta 1-syntrophin in our assay . 
On the basis of its alternative splicing , dystrophin may thus be present in two functionally distinct populations . 
In this recombinant expression system , the dystrophin relatives , human dystrophin related protein ( DRP or utrophin ) and the 87K postsynaptic protein from Torpedo electric organ , also bind to translated beta 1-syntrophin . 
We have found a COOH-terminal 37-kD fragment of beta 1-syntrophin sufficient to interact with translated dystrophin and its homologues , suggesting that the dystrophin binding site on beta 1-syntrophin occurs on a region that is conserved among the three syntrophin homologues . 


Interaction with RAP74 subunit of TFIIF is required for transcriptional activation by serum response factor . 
A few general transcription factors , in particular TFIID and TFIIB , have been found to bind transcriptional activators . 
Here we show that the general transcription factor TFIIF is also a target for a transcriptional activator , namely serum response factor ( SRF ) , which binds to the c-fos promoter . 
Using a yeast interaction assay , we find that SRF binds the RAP74 subunit of TFIIF and that SRF ' s transcriptional activation domain is the region involved in this binding . 
Further , RAP74 ' s central charged cluster domain is required for binding to SRF ' s activation domain . 
Deletion of this domain impairs RAP74 ' s ability to support SRF-activated transcription in vitro but has little effect on the protein 's basal transcription activity or its ability to support SP1-activated transcription . 
The correlation of SRF-RAP74 binding with transcriptional activation suggests that RAP74 is a critical target for SRF-activated transcription . 


Activity of RNA polymerase I transcription factor UBF blocked by Rb gene product . 
The protein encoded by the retinoblastoma susceptibility gene ( Rb ) functions as a tumour suppressor and negative growth regulator . 
As actively growing cells require the ongoing synthesis of ribosomal RNA , we considered that Rb might interact with the ribosomal DNA transcription apparatus . 
Here we report that ( 1 ) there is an accumulation of Rb protein in the nucleoli of differentiated U937 cells which correlates with inhibition of rDNA transcription ; ( 2 ) addition of Rb to an in vitro transcription system inhibits transcription by RNA polymerase I ; ( 3 ) this inhibition requires a functional Rb pocket ; and ( 4 ) Rb specifically inhibits the activity of the RNA polymerase I transcription factor UBF ( upstream binding factor ) in vitro . 
This last observation was confirmed by affinity chromatography and immunoprecipitation , which demonstrated an interaction between Rb and UBF . 
These results indicate that there is an additional mechanism by which Rb suppresses cell growth , namely that Rb directly represses transcription of the rRNA genes . 


Inhibin antagonizes inhibition of liver cell growth by activin by a dominant-negative mechanism . 
The beta : beta activin homodimer and alpha : beta inhibin heterodimer are mutual antagonists which share a common beta subunit . 
Recently , it has been shown that , similar to transforming growth factor-beta 1 , activin is an inhibitor of hepatocyte DNA synthesis . 
The activin receptor appears to be an obligatory complex of genetically distinct type I and II transmembrane serine / threonine kinases . 
Activin type I receptors , SKR1 and SKR2 , were first cloned from well differentiated human hepatoma cells ( HepG2 ) . 
This prompted us to investigate the binding of activin and inhibin to receptors from HepG2 cells and the effect of the two ligands on DNA synthesis . 
Here we show that beta : beta activin binds to the activin type II receptor kinase ( ActRII ) which induces activin binding to the type I receptor kinase SKR2 to form ActRII .beta : beta . 
SKR2 complexes in which an activin beta chain occupies each receptor subunit . 
Inhibin also binds to ActRII through its beta subunit , competes with the binding of activin to ActRII , but fails to form the ActRII . 
SKR2 complex . 
No specific binding site for inhibin could be demonstrated in HepG2 cells . 
Inhibin , which had no activity of its own , antagonized the inhibitory effect of activin on DNA synthesis . 
The results suggest that inhibin may be a natural antagonist of assembly of the heterodimeric activin receptor complex through a dominant-negative mechanism . 


The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA . 
The p53 tumour-suppressor protein controls the expression of a gene encoding the p21 cyclin-dependent protein kinase ( CDK ) regulator . 
Levels of p21 protein are increased in senescent cells and p21 overexpression blocks the growth of tumour cells . 
In normal human cells , but not in many tumour cells , p21 exists in a quaternary complex with a cyclin , a CDK , and the proliferating-cell nuclear antigen ( PCNA ) . 
p21 controls CDK activity , thereby affecting cell-cycle control , whereas PCNA functions in both DNA replication and repair . 
Here we use simian virus 40 DNA replication in vitro to show than p21 directly inhibits PCNA-dependent DNA replication in the absence of a cyclin / CDK . 
Furthermore , p21 blocks the ability of PCNA to activate DNA polymerase delta , the principal replicative DNA polymerase . 
This regulation results from a direct interaction between p21 and PCNA . 
Thus , during p53-mediated suppression of cell proliferation , p21 and PCNA may be important for coordinating cell-cycle progression , DNA replication and repair of damaged DNA . 


Nuclear protein CBP is a coactivator for the transcription factor CREB . 
The transcription factor CREB binds to a DNA element known as the cAMP-regulated enhancer ( CRE ) . 
CREB is activated through phosphorylation by protein kinase A ( PKA ) , but precisely how phosphorylation stimulates CREB function is unknown . 
One model is that phosphorylation may allow the recruitment of coactivators which then interact with basal transcription factors . 
We have previously identified a nuclear protein of M ( r ) 265K , CBP , that binds specifically to the PKA-phosphorylated form of CREB . 
We have used fluorescence anisotropy measurements to define the equilibrium binding parameters of the phosphoCREB : CBP interaction and report here that CBP can activate transcription through a region in its carboxy terminus . 
The activation domain of CBP interacts with the basal transcription factor TFIIB through a domain that is conserved in the yeast coactivator ADA-1 ( ref. 8 ) . 
Consistent with its role as a coactivator , CBP augments the activity of phosphorylated CREB to activate transcription of cAMP-responsive genes . 


Specific binding of the transglutaminase , platelet factor XIII , to HSP27 . 
HSP27 , the unique mammalian low molecular weight heat shock protein , is prominently phosphorylated upon activation of a wide variety of cells and has a role in thermotolerance , growth events , and regulation of actin cytoskeletal dynamics . 
In thrombin-stimulated platelets , HSP27 is rapidly and prominently phosphorylated in a manner highly correlated with platelet secretion . 
However , the function of HSP27 and the identity of proteins that interact with HSP27 remain unknown . 
To identify specific HSP27-protein interactions , a recombinant fusion protein affinity reagent was constructed and used to identify proteins associating with HSP27 from human platelet lysates and erythroleukemia cells . 
An 84-kDa protein was found to associate specifically with HSP27 and was isolated from platelet lysates , resolved on preparative gels , transferred to nitrocellulose , subjected to enzymatic digestion , and microsequenced . 
A 20-amino acid sequence derived from p84 proved identical to amino acids 484-503 of the transglutaminase , platelet Factor XIII . 
Immunoblotting studies were used to confirm the binding of FXIII from fresh platelet lysates to the HSP27 fusion protein . 
FXIII also was shown to coprecipitate with HSP27 in immunoprecipitation studies and to colocalize with HSP27 in immunofluorescence studies of intact glass-activated platelets . 
The data thus demonstrate specific binding of platelet FXIII to HSP27 and suggest that HSP27 may participate in the cellular localization and / or enzymatic regulation of platelet FXIII . 


Several hydrophobic amino acids in the p53 amino-terminal domain are required for transcriptional activation , binding to mdm-2 and the adenovirus 5 E1B 55-kD protein . 
The p53 tumor suppressor gene product is a transcriptional activator that may be associated with its ability to suppress tumor cell growth . 
The acidic amino terminus of the p53 protein has been shown to contain this trans-activation activity as well as the domains for mdm-2 and adenovirus 5 E1B 55-kD protein binding . 
An extensive genetic analysis of this amino-terminal p53 domain has been undertaken using site-specific mutagenesis . 
The results demonstrate that the acidic residues in the amino terminus of p53 may contribute to , but are not critical for , this trans-activation activity . 
Rather , the hydrophobic amino acid residues Leu-22 and Trp-23 of human p53 are both required for trans-activation activity , binding to the adenovirus E1B 55-kD protein and the human mdm-2-p53 protein in vitro . 
In addition , hydrophobic residues Leu-14 and Phe-19 are crucial for the interactions between p53 and human mdm-2 ( hdm-2 ) . 
Hydrophobic residues Trp-23 and Pro-27 are also important for binding to the adenovirus 5 ( Ad5 ) E1B 55-kD protein in vitro . 
These mutations have no impact on the ability of the p53 protein to bind to a p53-specific DNA element . 
These results suggest that 2-4 critical hydrophobic residues in the amino-terminal domain of the p53 protein interact with the transcriptional machinery of the cell resulting in transcriptional activation . 
These very same hydrophobic residues contact the hdm-2 and Ad5 E1B 55-kD oncogene products . 


Type II regulatory subunit ( RII ) of the cAMP-dependent protein kinase interaction with A-kinase anchor proteins requires isoleucines 3 and 5 . 
Compartmentalization of the type II cAMP-dependent protein kinase is maintained by association of the regulatory subunit ( RII ) with A-Kinase Anchor Proteins ( AKAPs ) . 
In previous studies ( Scott , J. D. , Stofko , R. E. , McDonald , J. R. , Comer , J. D. , Vitalis , E. A. , and Mangili J. ( 1990 ) J. Biol. Chem. 265 , 21561-21566 ) we have shown that dimerization of RII alpha was required for interaction with the cytoskeletal component microtubule-associated protein 2 . 
In this report we show that the localization and dimerization domains of RII alpha are contained within the first thirty residues of each RII protomer . 
RII des-5 ( an amino-terminal deletion mutant lacking residues 1-5 ) was unable to bind AKAPs but retained the ability to dimerize . 
RII alpha I3A , I5A ( a mutant where isoleucines 3 and 5 were replaced with alanine ) was unable to bind a variety of AKAPs . 
Mutation of both isoleucines decreased AKAP binding without affecting dimerization , cAMP binding , or the overall secondary structure of the protein . 
Measurement of RII alpha I3A , I5A interaction with the human thyroid AKAP , Ht 31 , by two independent methods suggests that mutation of isoleucines 3 and 5 decreases affinity by at least 6-fold . 
Therefore , we propose that two isoleucine side chains on each RII protomer are principle sites of contact with the conserved amphipathic helix binding domain on AKAPs . 


Characterization of an interaction between insulin receptor substrate 1 and the insulin receptor by using the two-hybrid system . 
Insulin receptor substrate 1 ( IRS-1 ) is a major substrate of the insulin receptor and has been implicated in insulin signaling . 
Although IRS-1 is thought to interact with the insulin receptor , the nature of the interaction has not been defined . 
In this study , we used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to study the interaction between human IRS-1 and the insulin receptor . 
We demonstrate that IRS-1 forms a specific complex with the cytoplasmic domain of the insulin receptor when both are expressed as hybrid proteins in yeast cells . 
We show that the interaction is strictly dependent upon receptor tyrosine kinase activity , since IRS-1 shows no interaction with a kinase-inactive receptor hybrid containing a mutated ATP-binding site . 
Furthermore , mutation of receptor tyrosine 960 to phenylalanine eliminates IRS-1 interaction in the two-hybrid assay . 
These data suggest that the interaction between IRS-1 and the receptor is direct and provide evidence that the juxtamembrane domain of the receptor is involved . 
Furthermore , we show that a 356-amino-acid region encompassed by amino acids 160 through 516 of IRS-1 is sufficient for interaction with the receptor in the two-hybrid assay . 
Lastly , in agreement with our findings for yeast cells , we show that the insulin receptor is unable to phosphorylate an IRS-1 protein containing a deletion of amino acids 45 to 516 when expressed in COS cells . 
The two-hybrid assay should provide a facile means by which to pursue a detailed understanding of this interaction . 


Activation of Raf-1 by 14-3-3 proteins . 
The protein Raf-1 , a key mediator of mitogenesis and differentiation , associates with p21ras ( refs 1-3 ) . 
However , the regulation of the serine / threonine kinase activity of Raf-1 is still not understood . 
Using the yeast two-hybrid system , we identified two structurally related proteins that interact with the aminoterminal region of Raf-1 . 
These proteins , 14-3-3 zeta ( PLA2 ) and 14-3-3 beta ( HS1 ) , are members of the 14-3-3 family of proteins . 
Expression of 14-3-3 proteins in Xenopus oocytes enhanced Raf-1 activity and promoted Raf-1-dependent oocyte maturation . 
A dominant negative mutant of Raf-1 blocked the effects of 14-3-3 protein . 


Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system . 
Interactions of the Bcl-2 protein with itself and other members of the Bcl-2 family , including Bcl-X-L , Bcl-X-S , Mcl-1 , and Bax , were explored with a yeast two-hybrid system . 
Fusion proteins were created by linking Bcl-2 family proteins to a LexA DNA-binding domain or a B42 trans-activation domain . 
Protein-protein interactions were examined by expression of these fusion proteins in Saccharomyces cerevisiae having a lacZ ( beta-galactosidase ) gene under control of a LexA-dependent operator . 
This approach gave evidence for Bcl-2 protein homodimerization . 
Bcl-2 also interacted with Bcl-X-L and Mcl-1 and with the dominant inhibitors Bax and Bcl-X-S . 
Bcl-X-L displayed the same pattern of combinatorial interactions with Bcl-2 family proteins as Bcl-2 . 
Use of deletion mutants of Bcl-2 suggested that Bcl-2 homodimerization involves interactions between two distinct regions within the Bcl-2 protein , since a LexA protein containing Bcl-2 amino acids 83-218 mediated functional interactions with a B42 fusion protein containing Bcl-2 amino acids 1-81 but did not complement a B42 fusion protein containing Bcl-2 amino acids 83-218 . 
In contrast to LexA / Bcl-2 fusion proteins , expression of a LexA / Bax protein was lethal to yeast . 
This cytotoxicity could be abrogated by B42 fusion proteins containing Bcl-2 , Bcl-X-L , or Mcl-1 but not those containing Bcl-X-S ( an alternatively spliced form of Bcl-X that lacks a well-conserved 63-amino acid region ) . 
The findings suggest a model whereby Bax and Bcl-X-S differentially regulate Bcl-2 function , and indicate that requirements for Bcl-2 / Bax heterodimerization may be different from those for Bcl-2 / Bcl-2 homodimerization . 


The rates of commitment to renaturation of rhodanese and glutamine synthetase in the presence of the groE chaperonins . 
Current models of chaperonin-assisted folding suggest that proteins undergo multiple rounds of binding and release before they are released in a form that is committed to folding to the native state . 
Using immunoprecipitation techniques , we have determined the rates at which rhodanese and glutamine synthetase ( GS ) are released from groEL in a form committed to refold to active enzyme . 
Rhodanese and glutamine synthetase were chosen as substrates because they exhibit different solution requirements for the chaperonin system and they form stable " folding arrested " complexes with groEL . 
At various times during the groE-dependent renaturations , groEL was rapidly removed from the renaturation mixture by immunoprecipitation and centrifugation ( 30 s ) . 
The conformers that are committed to the native state remained in the supernatant and were assayed after 1 h . 
At 25 degrees C , the rate profiles indicate the release and commitment to folding of GS to its native state occurs far earlier ( t1 / 2 < 1 min ) than for rhodanese ( t1 / 2 = 5 min ) . 
In light of previous results , it appears that GS monomers can attain a groE-independent assembly competent conformation after a brief interaction with the chaperonin . 
In contrast , the renaturation rate for rhodanese with the groE chaperonins mirrored the committed renaturation rates following groEL depletion . 
This suggests that rhodanese must interact with groEL throughout most of its folding reaction before it acquires a folding competent ( groE independent ) state . 
If current models of chaperonin mechanism are correct , rhodanese undergoes more rebinding and release cycles than does GS . 
Structurally , the degree of cycling and hence the rate of commitment to folding to the active form are probably dictated by the hydrophobic nature , number , and lifetimes of the folding intermediates that interact with the chaperonins . 


Interaction of IL-2R beta and gamma c chains with Jak1 and Jak3 : implications for XSCID and XCID . 
Interleukin-2 ( IL-2 ) signaling requires the dimerization of the IL-2 receptor beta ( IL-2R beta ) and common gamma ( gamma c ) chains . 
Mutations of gamma c can result in X-linked severe combined immunodeficiency ( XSCID ) . 
IL-2 , IL-4 , IL-7 ( whose receptors are known to contain gamma c ) , and IL-9 ( whose receptor is shown here to contain gamma c ) induced the tyrosine phosphorylation and activation of the Janus family tyrosine kinases Jak1 and Jak3 . 
Jak1 and Jak3 associated with IL-2R beta and gamma c , respectively ; IL-2 induced Jak3-IL-2R beta and increased Jak3-gamma c associations . 
Truncations of gamma c , and a gamma c , point mutation causing moderate X-linked combined immunodeficiency ( XCID ) , decreased gamma c- Jak3 association . 
Thus , gamma c mutations in at least some XSCID and XCID patients prevent normal Jak3 activation , suggesting that mutations of Jak3 may result in an XSCID-like phenotype . 


The X-ray structure of a growth hormone- prolactin receptor complex . 
The human pituitary hormones , growth hormone ( hGH ) and prolactin ( hPRL ) , regulate a large variety of physiological processes , among which are growth and differentiation of muscle , bone and cartilage cells , and lactation . 
These activities are initiated by hormone-receptor binding . 
The hGH and hPRL receptors ( hGHR and hPRLR , respectively ) are single-pass transmembrane receptors from class 1 of the haematopoietic receptor superfamily . 
This classification is based on sequence similarity in their extracellular domains , notably a highly conserved pentapeptide , the so-called ' WSXWS box ' , the function of which is controversial . 
All ligands in class 1 activate their respective receptors by clustering mechanisms . 
In the case of hGH , activation involves receptor homodimerization in a sequential process : the active ternary complex containing one ligand and two receptor molecules is formed by association of a receptor molecule to an intermediate 1 : 1 complex. hPRL does not bind to the hGH receptor , but hGH binds to both the hGHR and hPRLR , and mutagenesis studies have shown that the receptor-binding sites on hGH overlap . 
We present here the crystal structure of the 1 : 1 complex of hGH bound to the extracellular domain of the hPRLR . 
Comparisons with the hGH-hGHR complex reveal how hGH can bind to the two distinctly different receptor binding surfaces . 


Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes . 
Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90 , hsp70 , and three additional proteins , p54 , p50 , and p23 . 
p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein , p60 . 
Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein . 
Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice , rabbits , guinea pigs , humans , and Saccharomyces cerevisiae , indicating that p23 is a conserved protein of broad tissue distribution . 
These antibodies were used to screen a chicken brain cDNA library , resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23 . 
This partial clone was subsequently used to isolate a full-length human cDNA clone . 
The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins . 
The chicken and human cDNAs are 88 % identical at the DNA level and 96.3 % identical at the protein level . 
p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain . 
Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23 . 
These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol . 


Cloning of the complete coding region for human protein phosphatase inhibitor 2 using the two hybrid system and expression of inhibitor 2 in E. coli . 
The yeast two hybrid system has been employed to identify cDNAs encoding proteins which interact with the gamma 1 isoform of human protein phosphatase 1 . 
Here we report the isolation of cDNA encoding human protein phosphatase inhibitor . 
The deduced human sequence of 205 amino acids shows 92 % identity to inhibitor 2 from rabbit . 
Human inhibitor 2 was expressed in E. coli and purified to homogeneity . 
The expressed human protein inhibited both native and bacterially expressed PP1 , with the same Ki ( 1 nM ) as inhibitor 2 purified from skeletal muscle . 
A gene or pseudogene for inhibitor 2 may be present near the major histocompatibility complex on chromosome 6 . 


Expression cloning and characterization of a human IL-10 receptor . 
cDNA clones encoding a human IL-10R ( hIL-10R ) from a Burkitt lymphoma cell line , BJAB , express a 90 to 110 kDa polypeptide in COS7 cells that binds hIL-10 specifically . 
The predicted amino acid sequence of hIL-10R is 60 % identical and 73 % similar to mouse IL-10R ( mIL-10R ) . 
rIL-10R expressed in an IL-3-dependent mouse pro-B cell line ( Ba / F3 ) binds hIL-10 with high affinity ( 200 to 250 pM ) , and the transfected cells exhibit a proliferative response to hIL-10 . 
Mouse IL-10 does not bind to hIL-10R , and hIL-10R-expressing Ba / F3 cells do not respond to the mouse cytokine , observations consistent with the known species specificity of IL-10 . 
Expression of hIL-10R mRNA seems to be restricted mainly to human hemopoietic cells and cell lines . 
In a number of human T cell clones , expression of hIL-10R mRNA is down-regulated after activation of the cells with anti- CD3 Ab and phorbol ester . 
The hIL-10R gene is on human chromosome 11 . 
Like mIL-10R , hIL-10R is structurally related to IFNR . 
Because IL-10 inhibits macrophage activation by IFN-gamma , this relationship suggests possible shared receptor or signal transduction pathway components . 


Functional participation of the IL-2 receptor gamma chain in IL-7 receptor complexes . 
The gamma chain of the interleukin-2 ( IL-2 ) receptor is shared with the functional IL-4 receptor and is causatively related to X-linked severe combined immunodeficiency ( XSCID ) , which is ascribed to a profound T cell defect . 
Studies with monoclonal antibodies specific for the IL-2 receptor gamma chain showed that the gamma chain participates in the functional high-affinity receptor complexes for IL-7 that are involved in the differentiation of T and B cells . 
Participation of the gamma subunit in more than one receptor may enable the elucidation of the mechanisms of XSCID development and lymphocyte differentiation . 


JNK1 : a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain . 
The ultraviolet ( UV ) response of mammalian cells is characterized by a rapid and selective increase in gene expression mediated by AP-1 and NF-kappa B . 
The effect on AP-1 transcriptional activity results , in part , from enhanced phosphorylation of the c-Jun NH2-terminal activation domain . 
Here , we describe the molecular cloning and characterization of JNK1 , a distant relative of the MAP kinase group that is activated by dual phosphorylation at Thr and Tyr during the UV response . 
Significantly , Ha-Ras partially activates JNK1 and potentiates the activation caused by UV . 
JNK1 binds to the c-Jun transactivation domain and phosphorylates it on Ser-63 and Ser-73 . 
Thus , JNK1 is a component of a novel signal transduction pathway that is activated by oncoproteins and UV irradiation . 
These properties indicate that JNK1 activation may play an important role in tumor promotion . 


B61 is a ligand for the ECK receptor protein-tyrosine kinase . 
A protein ligand for the ECK receptor protein-tyrosine kinase has been isolated by using the extracellular domain ( ECK-X ) of the receptor as an affinity reagent . 
Initially , concentrated cell culture supernatants were screened for receptor binding activity using immobilized ECK-X in a surface plasmon resonance detection system . 
Subsequently , supernatants from selected cell lines were fractionated directly by receptor affinity chromatography , resulting in the single-step purification of B61 , a protein previously identified as the product of an early response gene induced by tumour necrosis factor-alpha . 
We report here that recombinant B61 induces autophosphorylation of ECK in intact cells , consistent with B61 being an authentic ligand for ECK . 
ECK is a member of a large orphan receptor protein-tyrosine kinase family headed by EPH , and we suggest that ligands for other members of this family will be related to B61 , and can be isolated in the same way . 


A lymphotoxin-beta-specific receptor . 
Tumor necrosis factor ( TNF ) and lymphotoxin-alpha ( LT-alpha ) are members of a family of secreted and cell surface cytokines that participate in the regulation of immune and inflammatory responses . 
The cell surface form of LT-alpha is assembled during biosynthesis as a heteromeric complex with lymphotoxin-beta ( LT-beta ) , a type II transmembrane protein that is another member of the TNF ligand family . 
Secreted LT-alpha is a homotrimer that binds to distinct TNF receptors of 60 and 80 kilodaltons ; however , these receptors do not recognize the major cell surface LT-alpha-LT-beta complex . 
A receptor specific for human LT-beta was identified , which suggests that cell surface LT may have functions that are distinct from those of secreted LT-alpha . 


Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor . 
A cytokine was identified that stimulated the proliferation of T lymphocytes , and a complementary DNA clone encoding this new T cell growth factor was isolated . 
The cytokine , designated interleukin-15 ( IL-15 ) , is produced by a wide variety of cells and tissues and shares many biological properties with IL-2 . 
Monoclonal antibodies to the beta chain of the IL-2 receptor inhibited the biological activity of IL-15 , and IL-15 competed for binding with IL-2 , indicating that IL-15 uses components of the IL-2 receptor . 


The POU domains of the Oct1 and Oct2 transcription factors mediate specific interaction with TBP . 
We had previously shown that the ubiquitous Oct1 and the lymphoid-specific Oct2 transcription factors stimulate transcription at the level of stable preinitiation complex formation . 
We have therefore investigated whether the octamer binding proteins might physically interact with TBP , the TATA box binding protein component of the TFIID factor . 
By using several different experimental systems we show that TBP efficiently associates with Oct1 and Oct2 . 
The interaction is direct and does not depend on the presence of DNA or additional proteins . 
N- and C-terminal deletions of the different proteins were used to localize the domains involved in the interaction . 
We show that the POU homeodomain of Oct2 and the evolutionarily conserved C-terminal core domain of TBP are both required and sufficient for the interaction . 
The Oct1 POU domain , which is highly homologous to the Oct2 POU domain , likewise mediates interaction with TBP . 
The interaction can also be observed in vivo , as TBP can be co-precipitated with Oct2 from co-transfected Cos1 cells and TBP co-immunoprecipitates with the endogenous Oct1 from HeLa cells . 
Co-transfection of human TBP and Oct2 expression vectors into B cells resulted in a synergistic activation of an octamer motif containing promoter . 


Interaction of mammalian splicing factor SF3a with U2 snRNP and relation of its 60-kD subunit to yeast PRP9 . 
In the assembly of a prespliceosome , U2 small nuclear ribonucleoprotein ( snRNP ) functions in pre-messenger RNA ( mRNA ) splicing together with splicing factors ( SFs ) 3a , SF3b , and several other proteins . 
The 17S but not the 12S form of U2 snRNP is active in splicing-complex formation . 
Here it is shown that the SF3a subunits correspond to three of the 17S U2 snRNP-specific polypeptides . 
SF3a interacts with U2 snRNP in the presence of SF3b to generate a structure similar to 17S U2 snRNP , which suggests a function for SF3a and SF3b in the incorporation of U2 snRNP into the spliceosome . 
Furthermore , the 60-kilodalton subunit of SF3a is related to the yeast splicing protein PRP9 . 


Spectrin cagliari . an Ala --> Gly substitution in helix 1 of beta spectrin repeat 17 that severely disrupts the structure and self-association of the erythrocyte spectrin heterodimer . 
The spectrin tetramer , the principal structural element of the red cell membrane skeleton , is formed by stable head-to-head self-association of two spectrin heterodimers . 
The self-association site appears to be formed by interactions between helices 1 and 2 of beta spectrin repeat 17 of one dimer with helix 3 of alpha spectrin repeat 1 of the other dimer to form two combined alpha-beta triple-helical segments . 
The head of the heterodimer appears to involve similar intradimer interactions . 
We describe the first example of an amino acid substitution in helix 1 of this combined alpha-beta triple-helical segment , which , although relatively minor , profoundly impairs tetramer formation . 
Strikingly , low angle rotary shadowing electron microscopy of isolated spectrin dimers reveals that this mutation also severely disrupts the head of the heterodimer causing it to be open . 
Following linkage studies which were most consistent with a beta spectrin gene mutation , a nucleotide change was identified in codon 2018 , resulting in an Ala --> Gly substitution in the first helical domain of beta spectrin repeat 17 . 
Because glycine is a strong helix breaker , this change is predicted to disrupt the conformation of this helical domain . 
Our results indicate that this helical domain must play direct roles in the alpha-beta interdimer interactions that form the self-association site of the tetramer and in the alpha-beta intradimer interactions at the head of the heterodimer . 


Cdi1 , a human G1 and S phase protein phosphatase that associates with Cdk2 . 
We used the interaction trap , a yeast genetic selection for interacting proteins , to isolate human cyclin-dependent kinase interactor 1 ( Cdi1 ) . 
In yeast , Cdi1 interacts with cyclin-dependent kinases , including human Cdc2 , Cdk2 , and Cdk3 , but not with Cdk4 . 
In HeLa cells , Cdi1 is expressed at the G1 to S transition , and the protein forms stable complexes with Cdk2 . 
Cdi1 bears weak sequence similarity to known tyrosine and dual specificity phosphatases . 
In vitro , Cdi1 removes phosphate from tyrosine residues in model substrates , but a mutant protein that bears a lesion in the putative active site cysteine does not . 
Overexpression of wild-type Cdi1 delays progression through the cell cycle in yeast and HeLa cells ; delay is dependent on Cdi1 phosphatase activity . 
These experiments identify Cdi1 as a novel type of protein phosphatase that forms complexes with cyclin-dependent kinases . 


The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases . 
The cyclin-dependent kinase Cdk2 associates with cyclins A , D , and E and has been implicated in the control of the G1 to S phase transition in mammals . 
To identify potential Cdk2 regulators , we have employed an improved two-hybrid system to isolate human genes encoding Cdk-interacting proteins ( Cips ) . 
CIP1 encodes a novel 21 kd protein that is found in cyclin A , cyclin D1 , cyclin E , and Cdk2 immunoprecipitates . 
p21CIP1 is a potent , tight-binding inhibitor of Cdks and can inhibit the phosphorylation of Rb by cyclin A-Cdk2 , cyclin E-Cdk2 , cyclin D1-Cdk4 , and cyclin D2-Cdk4 complexes . 
Cotransfection experiments indicate that CIP1 and SV40 T antigen function in a mutually antagonistic manner to control cell cycle progression . 


Evidence for physical interaction between the zinc-finger transcription factors YY1 and Sp1 . 
Two promoter elements are important for basal-level transcription , the TATA motif typically located 30 nucleotides upstream of the transcription initiation site and the initiator ( Inr ) element encompassing the start site . 
The mechanism of how Inr elements work is poorly understood , partly because very few proteins that bind to Inr elements have been identified and isolated . 
The recently cloned YY1 is such an Inr-binding protein . 
YY1 is able to direct transcription upon binding to its recognition sequence in vitro . 
The ability of YY1 to initiate transcription is augmented by the presence of a TATA motif or binding sites for transcription factor Sp1 . 
To study the mechanism underlying the apparent functional cooperation between YY1 and Sp1 , we explored the possibility of protein-protein interactions between these two transcription factors . 
We found that YY1 and Sp1 can form a physical complex . 
In addition , we identified domains within YY1 and Sp1 that mediate their interactions with each other . 
The physical interaction between YY1 and Sp1 may thus form the basis for the functional interplay observed previously . 


Involvement of IL-6 signal transducer gp130 in IL-11-mediated signal transduction . 
IL-11 is a novel cytokine with a variety of biofunctions which overlap with those of IL-6 . 
We have previously identified IL-11 specific binding protein which is distinct from that of IL-6 in a number of cell lines . 
The similarities in biofunctions and differences in ligand binding proteins between IL-11 and IL-6 prompted us to investigate whether IL-11 shares common signal transduction mechanisms with IL-6 . 
We have examined early signals triggered by IL-11 or IL-6 in a multifactor-dependent human erythroleukemic cell line , TF-1 . 
The results showed that IL-11 and IL-6 can both stimulate cell proliferation , induce similar pattern of protein tyrosine phosphorylation , and activate the same proto-oncogene ( junB ) expression in TF-1 cells . 
These findings imply that IL-11 and IL-6 share similar early signaling events with the possibility of using the same signal transducer , gp130 . 
We next tested whether IL-11 induced signaling can be inhibited by anti- gp130 antibodies which blocked IL-6-mediated functions . 
It was observed that anti- gp130 antibodies abolished cell proliferation , protein tyrosine phosphorylation , and junB gene expression elicited by IL-11 or IL-6 in TF-1 cells . 
The same antibodies , however , had no effect on granulocyte-macrophage colony stimulating factor or erythropoietin-induced TF-1 cell proliferation . 
Finally , anti- IL-6R antibody inhibited the ability of IL-6 , but not IL-11 , to transduce early signals in TF-1 cells . 
These results demonstrate that IL-11 and IL-6 utilize different ligand binding proteins , but share common signal transducer , gp130 , in TF-1 cells . 


The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit . 
The retinoblastoma protein ( p110RB ) interacts with many cellular proteins in complexes potentially important for its growth-suppressing function . 
We have developed and used an improved version of the yeast two-hybrid system to isolate human cDNAs encoding proteins able to bind p110RB . 
One clone encodes a novel type 1 protein phosphatase catalytic subunit ( PP-1 alpha 2 ) , which differs from the originally defined PP-1 alpha by an amino-terminal 11-amino-acid insert . 
In vitro-binding assays demonstrated that PP-1 alpha isoforms preferentially bind the hypophosphorylated form of p110RB . 
Moreover , similar p110RB sequences are required for binding PP-1 alpha 2 and SV40 large T antigen . 
Cell cycle synchrony experiments revealed that this association occurs from mitosis to early G1 . 
The implications of these findings on the regulation of both proteins are discussed . 


Crystal structure of the soluble human 55 kd TNF receptor-human TNF beta complex : implications for TNF receptor activation . 
The X-ray crystal structure of the complex of the extracellular domain of the human 55 kd tumor necrosis factor ( TNF ) receptor with human TNF beta has been determined at 2.85 A resolution . 
The complex has three receptor molecules bound symmetrically to one TNF beta trimer . 
The receptor fragment , a very elongated end to end assembly of four similar folding domains , binds in the groove between two adjacent TNF beta subunits . 
The structure of the complex defines the orientation of the ligand with respect to the cell membrane and provides a model for TNF receptor activation . 
The novel fold of the TNF receptor structure is likely to be representative of the nerve growth factor ( NGF ) / TNF receptor family as a whole . 


Characterization of a novel high affinity human IL-7 receptor . 
Expression on T cells and association with IL-7 driven proliferation . 
Although both unstimulated and activated human T cells express high affinity IL-7R , only activated T cells can proliferate to IL-7 . 
This responsiveness may occur as a direct result of changes in the structure of the IL-7R during T-cell activation . 
We have previously demonstrated such changes by affinity cross-linking studies , and have shown that unstimulated human T cells express a single IL-7R of 90 kDa , whereas activated T cells express an additional 76-kDa IL-7 binding protein . 
In this study the origin and function of the p90 and p76 molecules have been investigated . 
To determine the role of each of these receptors in IL-7 driven proliferation , IL-7R expression and proliferative capacity were monitored during mitogenic stimulation . 
These analyses showed that the ability of PBMC to proliferate to IL-7 correlated with expression of the p76 IL-7R , and not with expression of the p90 IL-7R . 
IL-7-driven proliferation is mediated via high affinity IL-7R , and accordingly , Scatchard analysis revealed that , like the p90 IL-7R , the p76 IL-7R bound IL-7 with dual ( high ; Kd 38 pM and low ; Kd 360 pM ) affinity . 
Deglycosylation studies showed that the p90 and p76 IL-7R are not simply differently glycosylated isoforms of a single receptor . 
In agreement , mAb to the previously cloned IL-7R were found to stain unstimulated T cells that express only the p90 IL-7R but not T-cell clones that express predominantly the p76 IL-7R . 
These antibodies also immunoprecipitated the cloned IL-7R as a 90-kDa species from both 125-I-surface-labeled resting and activated T cells , but were unable to precipitate the 76-kDa IL-7R . 
In addition , PCR analysis of p76-expressing cells could not detect splicing of the extracellular domain of the cloned IL-7R , thereby excluding the possibility that the p76 IL-7R is a previously undescribed splice variant of the cloned IL-7R . 
These data demonstrate that the p90 IL-7R is the T-cell homologue of the cloned IL-7R , and imply that the p90 and p76 IL-7R have different extracellular domains . 
Taken together these data suggest that the 76-kDa receptor is a novel high affinity IL-7R that may be necessary for IL-7 driven proliferation in human T cells . 


Characterization of the retinoblastoma binding proteins RBP1 and RBP2 . 
The retinoblastoma gene product , pRB , regulates cell proliferation by binding to and inhibiting the activity of key growth promoting proteins . 
Several cellular proteins have been shown to bind directly to pRB and the genes encoding a number of them have been isolated . 
The protein product of one of these genes is the transcription factor E2F . 
We have now isolated cDNA clones that contain the full-length coding sequence of two other proteins , RBP1 and RBP2 , cloned originally by their interaction with pRB . 
The products of the RBP1 and RBP2 genes are ubiquitously expressed , large ( 200 kDa for RBP1 and 195 kDa for RBP2 ) nuclear phosphoproteins with structural motifs that suggest a role in transcriptional regulation . 
In addition we have been able to identify complexes of pRB and RBP1 in vivo that are dissociated in the presence of purified human papillomavirus E7 protein . 


Two potentially oncogenic cyclins , cyclin A and cyclin D1 , share common properties of subunit configuration , tyrosine phosphorylation and physical association with the Rb protein PG-1377-84 AB-Originally identified as a ' mitotic cyclin ' , cyclin A exhibits properties of growth factor sensitivity , susceptibility to viral subversion and association with a tumor-suppressor protein , properties which are indicative of an S-phase-promoting factor ( SPF ) as well as a candidate proto-oncogene . 
Other recent studies have identified human cyclin D1 ( PRAD1 ) as a putative G1 cyclin and candidate proto-oncogene . 
However , the specific enzymatic activities and , hence , the precise biochemical mechanisms through which cyclins function to govern cell cycle progression remain unresolved . 
In the present study we have investigated the coordinate interactions between these two potentially oncogenic cyclins , cyclin-dependent protein kinase subunits ( cdks ) and the Rb tumor-suppressor protein . 
The distribution of cyclin D isoforms was modulated by serum factors in primary fetal rat lung epithelial cells . 
Moreover , cyclin D1 was found to be phosphorylated on tyrosine residues in vivo and , like cyclin A , was readily phosphorylated by pp60c-src in vitro . 
In synchronized human osteosarcoma cells , cyclin D1 is induced in early G1 and becomes associated with p9Ckshs1 , a Cdk-binding subunit . 
Immunoprecipitation experiments with human osteosarcoma cells and Ewing 's sarcoma cells demonstrated that cyclin D1 is associated with both p34cdc2 and p33cdk2 , and that cyclin D1 immune complexes exhibit appreciable histone H1 kinase activity . 
Immobilized , recombinant cyclins A and D1 were found to associate with cellular proteins in complexes that contain the p105Rb protein . 
This study identifies several common aspects of cyclin biochemistry , including tyrosine phosphorylation and the potential to interact directly or indirectly with the Rb protein , that may ultimately relate membrane-mediated signaling events to the regulation of gene expression . 


The SH2 / SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc : implications for insulin control of ras signalling . 
GRB2 , a small protein comprising one SH2 domain and two SH3 domains , represents the human homologue of the Caenorhabditis elegans protein , sem-5 . 
Both GRB2 and sem-5 have been implicated in a highly conserved mechanism that regulates p21ras signalling by receptor tyrosine kinases . 
In this report we show that in response to insulin , GRB2 forms a stable complex with two tyrosine-phosphorylated proteins . 
One protein is the major insulin receptor substrate IRS-1 and the second is the SH2 domain-containing oncogenic protein , Shc . 
The interactions between GRB2 and these two proteins require ligand activation of the insulin receptor and are mediated by the binding of the SH2 domain of GRB2 to phosphotyrosines on both IRS-1 and Shc . 
Although GRB2 associates with IRS-1 and Shc , it is not tyrosine-phosphorylated after insulin stimulation , implying that GRB2 is not a substrate for the insulin receptor . 
Furthermore , we have identified a short sequence motif ( YV / IN ) present in IRS-1 , EGFR and Shc , which specifically binds the SH2 domain of GRB2 with high affinity . 
Interestingly , both GRB2 and phosphatidylinositol-3 ( PI-3 ) kinase can simultaneously bind distinct tyrosine phosphorylated regions on the same IRS-1 molecule , suggesting a mechanism whereby IRS-1 could provide the core for a large signalling complex . 
We propose a model whereby insulin stimulation leads to formation of multiple protein -- protein interactions between GRB2 and the two targets IRS-1 and Shc . 
These interactions may play a crucial role in activation of p21ras and the control of downstream effector molecules . 


Regulation of the Ets-related transcription factor Elf-1 by binding to the retinoblastoma protein . 
The retinoblastoma gene product ( Rb ) is a nuclear phosphoprotein that regulates cell cycle progression . 
Elf-1 is a lymphoid-specific Ets transcription factor that regulates inducible gene expression during T cell activation . 
In this report , it is demonstrated that Elf-1 contains a sequence motif that is highly related to the Rb binding sites of several viral oncoproteins and binds to the pocket region of Rb both in vitro and in vivo . 
Elf-1 binds exclusively to the underphosphorylated form of Rb and fails to bind to Rb mutants derived from patients with retinoblastoma . 
Co-immunoprecipitation experiments demonstrated an association between Elf-1 and Rb in resting normal human T cells . 
After T cell activation , the phosphorylation of Rb results in the release of Elf-1 , which is correlated temporally with the activation of Elf-1-mediated transcription . 
Overexpression of a phosphorylation-defective form of Rb inhibited Elf-1-dependent transcription during T cell activation . 
These results demonstrate that Rb interacts specifically with a lineage-restricted Ets transcription factor . 
This regulated interaction may be important for the coordination of lineage-specific effector functions such as lymphokine production with cell cycle progression in activated T cells . 


Cloning of two proximal sequence element-binding transcription factor subunits ( gamma and delta ) that are required for transcription of small nuclear RNA genes by RNA polymerases II and III and interact with the TATA-binding protein . 
The proximal sequence element ( PSE )-binding transcription factor ( PTF ) specifically recognizes the PSEs of both RNA polymerase II- and RNA polymerase III-transcribed small nuclear RNA ( snRNA ) genes . 
We previously have shown that PTF purified from human HeLa cells is a multisubunit complex of four polypeptides designated PTF alpha ,-beta ,-gamma , and -delta . 
We now report the isolation and expression of cDNAs encoding PTF gamma and PTF delta , as well as functional studies with cognate antibodies that recognize the native PTF complex in HeLa extracts . 
Immunoprecipitation studies confirm that the four PTF subunits originally found to copurify during conventional chromatography indeed form a tightly associated complex ; they further show that the PTF so defined , including the gamma and delta subunits specifically , is essential for transcription of both class II and class III snRNA genes . 
Immunoprecipitation assays also show a weak substoichiometric association of the TATA-binding protein ( TBP ) with PTF , consistent with the previous report of a PTF-related complex ( SNAPc ) containing substoichiometric levels of TBP and a component ( SNAPc43 ) identical in sequence to the PTF gamma reported here . 
Glutathione S-transferase pulldown assays further indicate relatively strong direct interactions of both recombinant PTF gamma and PTF delta with TBP , consistent either with the natural association of TBP with PTF in a semistable TBP- TBP-associated factor complex or with possible functional interactions between PSE-bound PTF and TATA-bound TBP during promoter activation . 
In addition , we show that in extracts depleted of TBP and TBP-associated factors , transcription from the U1 promoter is restored by recombinant TBP but not by TFIID or TFIIIB , indicating that transcription of class II snRNA genes requires a TBP complex different from the one used for mRNA-encoding genes . 


TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways . 
Tumor necrosis factor ( TNF ) can induce apoptosis and activate NF-kappa B through signaling cascades emanating from TNF receptor 1 ( TNFR1 ) . 
TRADD is a TNFR1-associated signal transducer that is involved in activating both pathways . 
Here we show that TRADD directly interacts with TRAF2 and FADD , signal transducers that activate NF-kappa B and induce apoptosis , respectively . 
A TRAF2 mutant lacking its N-terminal RING finger domain is a dominant-negative inhibitor of TNF-mediated NF-kappa B activation , but does not affect TNF-induced apoptosis . 
Conversely , a FADD mutant lacking its N-terminal 79 amino acids is a dominant-negative inhibitor of TNF-induced apoptosis , but does not inhibit NF-kappa B activation . 
Thus , these two TNFR1- TRADD signaling cascades appear to bifurcate at TRADD . 


Characterization of adenosine deaminase binding to human CD26 on T cells and its biologic role in immune response . 
CD26 , a T cell activation Ag , also known as dipeptidyl peptidase IV is directly associated with adenosine deaminase ( ADA ) on the surface of T cells and T cell lines . 
In the present study , we examined both the binding of ADA and CD26 and the functional consequences of this interaction . 
We found that ADA was associated with CD26 on T cell lines lacking either ADA or dipeptidyl peptidase IV enzymatic activity , indicating that the association between dipeptidyl peptidase IV and ADA did not require enzymatic activity . 
Moreover , using immunoelectron microscopy , we demonstrated that CD26 and ADA co-localized on the cell surface , but not inside cells , suggesting that CD26 did not transport ADA to the surface . 
In keeping with this observation , we showed that human CD26-transfected murine pre-B cell lines lacking human ADA acquired ADA from an extracellular source . 
More importantly , adenosine in the absence of cell surface ADA inhibited T cell proliferation and IL-2 production induced by various stimuli . 
On the other hand , cells expressing ADA and CD26 on the surface were much more resistant to the inhibitory effect of adenosine . 
These data suggest that ADA on the cell surface is involved in an important immunoregulatory mechanism by which released ADA binds to cell surface CD26 , and this complex is capable of reducing the local concentration of adenosine . 


The three human syntrophin genes are expressed in diverse tissues , have distinct chromosomal locations , and each bind to dystrophin and its relatives . 
The syntrophins are a biochemically heterogeneous group of 58-kDa intracellular membrane-associated dystrophin-binding proteins . 
We have cloned and characterized human acidic ( alpha 1-) syntrophin and a second isoform of human basic ( beta 2-) syntrophin . 
Comparison of the deduced amino acid structure of the three human isoforms of syntrophin ( together with the previously reported human beta 1-syntrophin ) demonstrates their overall similarity . 
The deduced amino acid sequences of human alpha 1- and beta 2-syntrophin are nearly identical to their homologues in mouse , suggesting a strong functional conservation among the individual isoforms , Much like beta 1-syntrophin , human beta 2-syntrophin has multiple transcript classes and is expressed widely , although in a distinct pattern of relative abundance . 
In contrast , human alpha 1-syntrophin is most abundant in heart and skeletal muscle , and less so in other tissues . 
Somatic cell hybrids and fluorescent in situ hybridization were both used to determine their chromosomal locations : beta 2-syntrophin to chromosome 16q22-23 and alpha 1-syntrophin to chromosome 20q11.2 . 
Finally , we used in vitro translated proteins in an immunoprecipitation assay to show that , like beta 1-syntrophin , both beta 2- and alpha 1-syntrophin interact with peptides encoding the syntrophin-binding region of dystrophin , utrophin / dystrophin related protein , and the Torpedo 87K protein . 


A histone octamer-like structure within TFIID . 
The general transcription factor TFIID nucleates initiation complex formation through direct core promoter binding , commits promoters within chromatin to transcription , and mediates the action of transcriptional activators , a phenomenon that may correlate with enhanced TFIID recruitment or conformational changes in TFIID-promoter complexes . 
Molecular studies of the multiprotein TFIID complex have identified a primary TATA binding subunit ( TBP ) , TBP-associated factors ( TAFs ) that interact with and mediate the function of activators and intersubunit interactions but have yielded relatively little insight into the structural organization of the complex or the actual mechanism of transcriptional activation . 
Here we present biochemical evidence for the structural relevance of histone homologies in the human TFIID subunits hTAF80 , hTAF31 and hTAF20 / 15 . 
Together with analyses of native TFIID complexes and accompanying crystallographic studies , the results suggest that there is a histone octamer-like TAF complex within TFIID . 


IRAK : a kinase associated with the interleukin-1 receptor . 
The pleiotropic biological activities of interleukin-1 ( IL-1 ) are mediated by its type I receptor ( IL-1RI ) . 
When the ligand binds , IL-1RI initiates a signaling cascade that results in the activation of the transcription regulator nuclear factor kappa B ( NF-kappa B ) . 
A protein kinase designated IRAK ( IL-1 receptor-associated kinase ) was purified , and its complementary DNA was molecularly cloned . 
When human embryonic kidney cells ( cell line 293 ) over-expressing IL-1RI or HeLa cells were exposed to IL-1 , IRAK rapidly associated with the IL-1RI complex and was phosphorylated . 
The primary amino acid sequence of IRAK shares similarity with that of pelle a protein kinase that is essential for the activation of a NF-kappa B homolog in Drosophila . 


Coordination of three signaling enzymes by AKAP79 , a mammalian scaffold protein . 
Multivalent binding proteins , such as the yeast scaffold protein Sterile-5 , coordinate the location of kinases by serving as platforms for the assembly of signaling units . 
Similarly , in mammalian cells the cyclic adenosine 3 ' , 5 '-monophosphate-dependent protein kinase ( PKA ) and phosphatase 2B [ calcineurin ( CaN ) ] are complexed by an A kinase anchoring protein , AKAP79 . 
Deletion analysis and binding studies demonstrate that a third enzyme , protein kinase C ( PKC ) , binds AKAP79 at a site distinct from those bound by PKA or CaN . 
The subcellular distributions of PKC and AKAP79 were similar in neurons . 
Thus , AKAP79 appears to function as a scaffold protein for three multifunctional enzymes . 


TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex . 
The death domain of tumor necrosis factor ( TNF ) receptor-1 ( TNFR1 ) triggers distinct signaling pathways leading to apoptosis and NF-kappa B activation through its interaction with the death domain protein TRADD . 
Here , we show that TRADD interacts strongly with RIP , another death domain protein that was shown previously to associate with Fas antigen . 
We also show that RIP is a serine-threonine kinase that is recruited by TRADD to TNFR1 in a TNF-dependent process . 
Overexpression of the intact RIP protein induces both NF-kappa B activation and apoptosis . 
However , expression of the death domain of RIP Induces apoptosis , but potently inhibits NF-kappa B activation by TNF . 
These results suggest that distinct domains of RIP participate in the TNF signaling cascades leading to apoptosis and NF-kappa B activation . 


Characterization of the dystrophin-syntrophin interaction using the two-hybrid system in yeast . 
The carboxy-terminal region of dystrophin has previously been shown to interact directly with alpha1 syntrophin , a cytoplasmic component of the dystrophin-glycoprotein complex , by in vitro biochemical studies such as overlay assay or immunoprecipitation . 
Using the two-hybrid system , we have isolated from a human heart cDNA library the entire coding sequence of human alpha1 syntrophin , therefore confirming for the first time this interaction via an in vivo approach . 
In addition , we have reduced the interaction domain to the distal half of alpha1 syntrophin . 


Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors . 
Generation of receptor-selective VEGF variants by site-directed mutagenesis . 
Vascular endothelial growth factor ( VEGF ) expression in various cell types is induced by hypoxia and other stimuli . 
VEGF mediates endothelial cell proliferation , angiogenesis , vascular growth , and vascular permeability via the endothelial cell receptors , kinase insert domain-containing receptor ( KDR ) / fetal liver kinase 1 ( Flk-1 ) and FLT-1 . 
Alanine-scanning mutagenesis was used to identify a positively charged surface in VEGF that mediates binding to KDR / Flk-1 . 
Arg82 , Lys84 and His86 , located in a hairpin loop , were found to be critical for binding KDR / Flk-1 , while negatively charged residues , Asp63 , Glu64 , and Glu67 , were associated with FLT-1 binding . 
A VEGF model based on PDGFb indicated these positively and negatively charged regions are distal in the monomer but are spatially close in the dimer . 
Mutations within the KDR site had minimal effect on FLT-1 binding , and mutants deficient in FLT-1 binding did not affect KDR binding . 
Endothelial cell mitogenesis was abolished in mutants lacking KDR affinity ; however , FLT-1 deficient mutants induced normal proliferation . 
These results suggest dual sets of determinants in the VEGF dimer that cross-link cell surface receptors , triggering endothelial cell growth and angiogenesis . 
Furthermore , this mutational analysis implicates KDR , but not FLT-1 , in VEGF induction of endothelial cell proliferation . 


Use of the two hybrid system to detect the association of the protein-tyrosine-phosphatase , SHPTP2 , with another SH2-containing protein , Grb7 . 
SHPTP2 is a ubiquitously-expressed SH2-containing tyrosine phosphatase that is tyrosine phosphorylated in response to activation of various receptor and nonreceptor tyrosine kinases . 
SHPTP2 associates with the platelet-derived growth factor ( PDGF ) receptor after ligand stimulation , and binding of SHPTP2 to this receptor promotes tyrosine phosphorylation of SHPTP2 . 
The yeast two-hybrid system was modified to identify partners of tyrosine-phosphorylated proteins . 
Using SHPTP2 as bait and supplying an exogenous tyrosine kinase gene to the yeast cells , we have found that SHPTP2 interacts with another signaling protein , Grb7 . 
We have localized the region of interaction to tyrosine 580 in the carboxyl end of SHPTP2 and to the SH2 domain in the carboxy-terminus of Grb7 . 
We demonstrate that Grb7 binds to SHPTP2 in vitro under conditions where the latter is tyrosine-phosphorylated . 
These experiments show that this modified two hybrid technique may be useful for the identification of proteins involved in tyrosine kinase signal transduction cascades . 


Modulation of the transcriptional activity of thyroid hormone receptors by the tumor suppressor p53 . 
Thyroid hormone nuclear receptors ( TRs ) are ligand-dependent transcriptional factors that regulate growth , differentiation , and development . 
The molecular mechanisms by which TRs mediate these effects are unclear . 
One prevailing hypothesis suggests that TRs may cooperate with other transcriptional factors to mediate their biological effects . 
In this study , we tested this hypothesis by examining whether the activity of TRs is modulated by the tumor suppressor p53 . 
p53 is a nuclear protein that regulates gene expression via sequence-specific DNA binding and / or direct protein-protein interaction . 
We found that the human TR subtype beta 1 ( h-TR beta 1 ) physically interacted with p53 via its DNA binding domain . 
As a result of this physical interaction , binding of h-TR beta 1 to its hormone response elements either as homodimer or as a heterodimer with the retinoic X receptor was inhibited by p53 in a concentration-dependent manner . 
In transfected cells , wild-type p53 repressed the hormone-dependent transcriptional activation of h-TR beta 1 . 
In contrast , mutant p53 either had no effect or activated the transcriptional activity of h-TR beta 1 depending on the type of hormone response elements . 
These results indicate the gene regulating activity of TRs was modulated by p53 , suggesting that the cross talk between these two transcriptional factors may play an important role in the biology of normal and cancer cells . 


Enhanced degradation of EGF receptors by a sorting nexin , SNX1 . 
The vectorial movement of proteins requires specific recognition by components of the vesicular trafficking machinery . 
A protein , sorting nexin-1 ( SNX1 ) , was identified in a human cell line that bound to a region of the epidermal growth factor receptor ( EGFR ) containing the lysosomal targeting code . 
SNX1 contains a region of homology to a yeast vacuolar sorting protein , and overexpression of SNX1 decreased the amount of EGFR on the cell surface as a result of enhanced rates of constitutive and ligand-induced degradation . 
Thus , SNX1 is likely to play a role in sorting EGFR to lysosomes . 


The splicing factor U2AF35 mediates critical protein-protein interactions in constitutive and enhancer-dependent splicing . 
The splicing factor U2AF ( U2 snRNP auxiliary factor ) is a heterodimer with subunits of 65 and 35 kD ( U2AF65 and U2AF35 ) . 
U2AF65 binds specifically to 3 ' splice sites , but previous studies failed to demonstrate a function for U2AF35 . 
Here , we report that U2AF35 is required for constitutive splicing and also functions as a mediator of enhancer-dependent splicing . 
Nuclear extracts deficient in U2AF35 were inactive ; however , both constitutive and enhancer-dependent splicing could be restored by the addition of purified recombinant U2AF35 . 
In vitro protein-RNA interaction studies with pre-mRNAs containing either a constitutive or regulated splicing enhancer revealed that U2AF35 directly mediates interactions between U2AF65 and proteins bound to the enhancers . 
Thus , U2AF35 functions as a bridge between U2AF65 and the enhancer complex to recruit U2AF65 to the adjacent intron . 


Interactions involving the human RNA polymerase II transcription / nucleotide excision repair complex TFIIH , the nucleotide excision repair protein XPG , and Cockayne syndrome group B (CSB ) protein . 
The human basal transcription factor TFIIH plays a central role in two distinct processes . 
TFIIH is an obligatory component of the RNA polymerase II ( RNAP II ) transcription initiation complex . 
Additionally , it is believed to be the core structure around which some if not all the components of the nucleotide excision repair ( NER ) machinery assemble to constitute a nucleotide excision repairosome . 
At least two of the subunits of TFIIH ( XPB and XPD proteins ) are implicated in the disease xeroderma pigmentosum ( XP ) . 
We have exploited the availability of the cloned XPB , XPD , p62 , p44 , and p34 genes ( all of which encode polypeptide subunits of TFIIH ) to examine interactions between in vitro-translated polypeptides by co-immunoprecipitation . 
Additionally we have examined interactions between TFIIH components , the human NER protein XPG , and the CSB protein which is implicated in Cockayne syndrome ( CS ) . 
Our analyses demonstrate that the XPB , XPD , p44 , and p62 proteins interact with each other . 
XPG protein interacts with multiple subunits of TFIIH and with CSB protein . 


Characterization of the receptor binding sites of human leukemia inhibitory factor and creation of antagonists . 
Residues in human leukemia inhibitory factor ( hLIF ) crucial for binding to both the human LIF receptor ( R ) and gp130 were identified by analysis of alanine scanning mutants of hLIF in assays for both receptor binding and bioactivity . 
The region of hLIF most important for binding to the hLIF-R is composed of residues from the amino terminus of the D-helix , carboxyl terminus of the B-helix , and C-D loop . 
This site forms a distinct surface at the end of the four-helix bundle in the tertiary structure of the closely related murine LIF . 
The two residues of hLIF that contribute the majority of free energy for hLIF-R binding , Phe-156 and Lys-159 are surrounded by other residues which have only a moderate impact . 
This arrangement of a few key residues surrounded by less important ones is analogous to the functional binding epitope of human growth hormone for its receptor . 
A second region of hLIF that includes residues from the carboxyl terminus of the D-helix and A-B loop also had a weak influence on hLIF-R binding . 
Residues in hLIF from both the A- and C-helices are involved in binding the gp130 co-receptor . 
Abolition of the gp130 binding site in hLIF created antagonists of LIF action . 


The glut 1 glucose transporter interacts with calnexin and calreticulin . 
Calnexin is an integral membrane protein that acts as a chaperone during glycoprotein folding in the endoplasmic reticulum . 
Cross-linking studies were carried out with the aim of investigating the interactions of calnexin with glycoproteins in vitro . 
A truncated version of the integral membrane glycoprotein Glut 1 ( GT155 ) was synthesized in a rabbit reticulocyte translation system in the presence of canine pancreatic microsomes . 
Following immunoprecipitation with an anticalnexin antiserum , a cross-linker-independent association was observed between GT155 and calnexin . 
In addition , the anti-calnexin antiserum immunoprecipitated a UV-dependent cross-linking product consisting of GT155 and a protein of approximately 60 kDa designated CAP-60 ( calnexin-associated protein of 60 kDa ) . 
Both the GT155-calnexin and the GT155-CAP-60 interactions were dependent on the presence of a correctly modified oligosaccharide group on GT155 , a characteristic of many calnexin interactions . 
A GT155 mutant that was not glycosylated ( AGGT155 ) did not associate with calnexin or CAP-60 . 
Calreticulin , the soluble homologue of calnexin , was also shown to interact with GT155 only when the protein bore a correctly modified oligosaccharide group . 
Thus , our data show that both calnexin and calreticulin interact with Glut 1 in a glycosylation-dependent manner . 


Identification of a critical ligand binding determinant of the human erythropoietin receptor . 
Evidence for common ligand binding motifs in the cytokine receptor family . 
The erythropoietin receptor ( EPOR ) is a member of a family of cytokine and growth factor receptors that share conserved features in their extracellular and cytoplasmic domains . 
We have used site-specific mutagenesis within the extracellular domain of the EPOR to search for amino acid residues involved in erythropoietin ( EPO ) binding . 
Mutant proteins were expressed in bacteria as soluble EPO binding proteins ( EBP ) and characterized for EPO binding activity in a number of different assays . 
Substitution of phenylalanine at position 93 ( Phe93 ) with alanine ( F93A mutation ) resulted in a drastic reduction in EPO binding in the EBP . 
More conservative tyrosine or tryptophan substitutions at Phe93 resulted in much less dramatic effects on EPO binding . 
Biophysical studies indicated that the F93A mutation does not result in gross structural alterations in the EBP . 
Furthermore , the F93A mutation in full-length EPOR expressed in COS cells abolished detectable EPO binding . 
This was not a result of processing or transport defects , since mutant receptor was present on the surface of the cells . 
Mutations in the region immediately around Phe93 and in residues homologous to other reported ligand binding determinants of the cytokine receptor family had small to moderate effects on EPO binding . 
These data indicate that Phe93 is a critical EPO binding determinant of the EPOR . 
Furthermore , since Phe93 aligns with critical ligand binding determinants in other receptors of the cytokine receptor family , these data suggest that receptors of this family may use common structural motifs to bind their cognate ligands . 


Cloning and characterization of a specific interleukin ( IL )-13 binding protein structurally related to the IL-5 receptor alpha chain . 
Interleukin-13 ( IL-13 ) is a cytokine secreted by activated T lymphocytes that shares many , but not all , biological activities with IL-4 . 
These overlapping activities are probably due to the existence of common receptor components . 
Two proteins have been described as constituents of the IL-4 receptor , a approximately 140-kDa glycoprotein ( IL-4R ) and the gamma chain ( gammac ) of the IL-2 receptor , but neither of these proteins binds IL-13 . 
We have cloned a cDNA encoding an IL-13 binding protein ( IL-13R ) from the Caki-1 human renal carcinoma cell line . 
The cloned cDNA encodes a 380-amino acid protein with two consensus patterns characteristic of the hematopoietic cytokine receptor family and a short cytoplasmic tail . 
The IL-13R shows homology with the IL-5 receptor , and to a lesser extent , with the prolactin receptor . 
COS-7 cells transfected with the IL-13R cDNA bind IL-13 with high affinity but do not bind IL-4 . 
COS-7 cells co-transfected with the cloned IL-13R cDNA and IL-4R cDNA resulted in the reconstitution of a small number of receptors that recognized both IL-4 and IL-13 . 
Reverse transcription-polymerase chain reaction analysis detected the receptor transcript only in cell lines known to bind IL-13 . 


Elimination of false negative results in the two-hybrid system in the phagocyte NADPH oxidase . 
The yeast two-hybrid system is finding increased use in the study of interactions between proteins . 
In this method , two polypeptides are expressed in yeast as fusion proteins to a transcriptional activator DNA-binding domain ( bd ) and activating domain ( ad ) , respectively . 
Interaction between the two polypeptides reconstitutes function of a transactivator which controls expression of reporters . 
The phagocyte NADPH oxidase is a complex of membrane cytochrome b558 ( comprised of subunits p22-phox and gp91-phox ) and three cytosol proteins ( p47-phox , p67-phox , and p21rac ) that translocate to membrane and bind to cytochrome b558 . 
This is the first report to demonstrate that two of cytosolic components of cytochrome b558 , p47-phox binding to p67-phox each other . 
We encountered several methodological problems in the two-hybrid system which are the focus of this report . 


Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex . 
The crystal structure of the human p27Kip1 kinase inhibitory domain bound to the phosphorylated cyclin A- cyclin-dependent kinase 2 ( Cdk2 ) complex has been determined at 2.3 angstrom . 
p27Kip1 binds the complex as an extended structure interacting with both cyclin A and Cdk2 . 
On cyclin A , it binds in a groove formed by conserved cyclin box residues . 
On Cdk2 , it binds and rearranges the amino-terminal lobe and also inserts into the catalytic cleft , mimicking ATP . 


Vascular endothelial growth factor-related protein : a ligand and specific activator of the tyrosine kinase receptor Flt4 . 
The tyrosine kinases Flt4 , Flt1 , and Flk1 ( or KDR ) constitute a family of endothelial cell-specific receptors with seven immunoglobulin-like domains and a split kinase domain . 
Flt1 and Flk1 have been shown to play key roles in vascular development ; these two receptors bind and are activated by vascular endothelial growth factor ( VEGF ) . 
No ligand has been identified for Flt4 , whose expression becomes restricted during development to the lymphatic endothelium . 
We have identified cDNA clones from a human glioma cell line that encode a secreted protein with 32 % amino acid identity to VEGF . 
This protein , designated VEGF-related protein ( VRP ) , specifically binds to the extracellular domain of Flt4 , stimulates the tyrosine phosphorylation of Flt4 expressed in mammalian cells , and promotes the mitogenesis of human lung endothelial cells . 
VRP fails to bind appreciably to the extracellular domain of Flt1 or Flk1 . 
The protein contains a C-terminal , cysteine-rich region of about 180 amino acids that is not found in VEGF . 
A 2.4-kb VRP mRNA is found in several human tissues including adult heart , placenta , ovary , and small intestine and in fetal lung and kidney . 


Two-hybrid system as a model to study the interaction of beta-amyloid peptide monomers . 
The kinetics of amyloid fibril formation by beta-amyloid peptide ( Abeta ) are typical of a nucleation-dependent polymerization mechanism . 
This type of mechanism suggests that the study of the interaction of Abeta with itself can provide some valuable insights into Alzheimer disease amyloidosis . 
Interaction of Abeta with itself was explored with the yeast two-hybrid system . 
Fusion proteins were created by linking the Abeta fragment to a LexA DNA-binding domain ( bait ) and also to a B42 transactivation domain ( prey ) . 
Protein-protein interactions were measured by expression of these fusion proteins in Saccharomyces cerevisiae harboring lacZ ( beta-galactosidase ) and LEU2 ( leucine utilization ) genes under the control of LexA-dependent operators . 
This approach suggests that the Abeta molecule is capable of interacting with itself in vivo in the yeast cell nucleus . 
LexA protein fused to the Drosophila protein bicoid ( LexA- bicoid ) failed to interact with the B42 fragment fused to Abeta , indicating that the observed Abeta-Abeta interaction was specific . 
Specificity was further shown by the finding that no significant interaction was observed in yeast expressing LexA- Abeta bait when the B42 transactivation domain was fused to an Abeta fragment with Phe-Phe at residues 19 and 20 replaced by Thr-Thr ( AbetaTT ) , a finding that is consistent with in vitro observations made by others . 
Moreover , when a peptide fragment bearing this substitution was mixed with native Abeta- ( 1-40 ) , it inhibited formation of fibrils in vitro as examined by electron microscopy . 
The findings presented in this paper suggest that the two-hybrid system can be used to study the interaction of Abeta monomers and to define the peptide sequences that may be important in nucleation-dependent aggregation . 


Influence of interleukin-6 ( IL-6 ) dimerization on formation of the high affinity hexameric IL-6 .receptor complex . 
The high affinity interleukin-6 ( IL-6 ) signaling complex consists of IL-6 and two membrane-associated receptor components : a low affinity but specific IL-6 receptor and the affinity converter / signal transducing protein gp130 . 
Monomeric ( IL-6M ) and dimeric ( IL-6D ) forms of Escherichia coli-derived human IL-6 and the extracellular ( " soluble " ) portions of the IL-6 receptor ( sIL-6R ) and gp130 have been purified in order to investigate the effect of IL-6 dimerization on binding to the receptor complex . 
Although IL-6D has a higher binding affinity for immobilized sIL-6R , as determined by biosensor analysis employing surface plasmon resonance detection , IL-6M is more potent than IL-6D in a STAT3 phosphorylation assay . 
The difference in potency is significantly less pronounced when measured in the murine 7TD1 hybridoma growth factor assay and the human hepatoma HepG2 bioassay due to time-dependent dissociation at 37 ? C of IL-6 dimers into active monomers . 
The increased binding affinity of IL-6D appears to be due to its ability to cross-link two sIL-6R molecules on the biosensor surface . 
Studies of the IL-6 ternary complex formation demonstrated that the reduced biological potency of IL-6D resulted from a decreased ability of the IL-6D ( sIL-6R ) 2 complex to couple with the soluble portion of gp130 . 
These data imply that IL-6-induced dimerization of sIL-6R is not the driving force in promoting formation of the hexameric ( IL-6 IL-6R gp130 ) 2 complex . 
A model is presented whereby the trimeric complex of IL-6R , gp130 , and IL-6M forms before the functional hexamer . 
Due to its increased affinity for the IL-6 R but its decreased ability to couple with gp130 , we suggest that a stable IL-6 dimer may be an efficient IL-6 antagonist . 


Physical interaction of mammalian CDC37 with CDK4 PG-22030-4 AB- CDC37 was originally identified as a Start gene in budding yeast and has been shown to be required for association of CDC28 with cyclins . 
The exact functional mechanism by which CDC37 promotes this association , however , remains unknown . 
CDK4 is a cyclin D-dependent kinase that controls progression through G1 of the mammalian cell cycle . 
We have detected a specific association of CDK4 with the molecular chaperon HSP90 and a 44-kDa protein that we identify as mammalian CDC37 . 
A physical interaction between CDC37 and CDK4 suggests that CDC37 may regulate the mammalian cell cycle through a direct effect on CDK4 . 
Association of CDK4 with both CDC37 and HSP90 may also imply a mechanistic link between the functions of CDC37 and HSP90 . 


Interactions of p60 , a mediator of progesterone receptor assembly , with heat shock proteins hsp90 and hsp70 . 
Previous studies on the assembly of progesterone receptor ( PR ) complexes in vitro have suggested that PR assembly is a dynamic , ordered process involving at least eight nonreceptor proteins . 
One of these proteins , p60 , appears transiently during assembly and is not a component of functionally mature PR complexes . 
In the present study we observe that a monoclonal antibody specific for p60 can , on the one hand , inhibit formation of mature PR complexes containing heat shock protein 90 ( hsp90 ) , p23 , and immunophilins and , on the other , enhance recovery of early PR complexes containing hsp70 and Hip ( p48 ) . 
This observation supports a model in which p60 functions at an intermediate stage of PR assembly to facilitate formation of subsequent PR complexes lacking p60 . 
Since p60 is typically found in a complex with hsp90 and hsp70 , we have further characterized its interactions with these proteins . 
P60 can bind either hsp70 or hsp90 independently and in an ATP-independent manner . 
Since hsp90 and hsp70 do not readily associate on their own , it appears that p60 is the central organizing component of an hsp90-p60-hsp70 complex . 
Mutational analysis of p60 indicates that the N terminus is required for hsp70 binding , and a central region containing tetratricopeptide repeat motifs is necessary for binding hsp90 and hsp70 . 
The hsp90-p60-hsp70 multichaperone complex is highly dynamic and does not appear to be affected by the hsp90-binding drug geldanamycin . 
The interactions of hsp70 and hsp90 in intermediate PR complexes are shown to be distinct from their separate interactions in early PR complexes ( hsp70 ) or in mature PR complexes ( hsp90 ) . 
From these results , it appears that p60 is a key mediator in the chaperoned assembly and functional maturation of PR complexes . 


Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system . 
Many biological processes rely upon protein-protein interactions . 
Hence , detailed analysis of these interactions is critical for their understanding . 
Due to the complexities involved , genetic approaches are often needed . 
In yeast and phage , genetic characterizations of protein complexes are possible . 
However , in multicellular organisms , such characterizations are limited by the lack of powerful selection systems . 
Herein we describe genetic selections that allow single amino acid changes that disrupt protein-protein interactions to be selected from large libraries of randomly generated mutant alleles . 
The strategy , based on a yeast reverse two-hybrid system , involves a first-step negative selection for mutations that affect interaction , followed by a second-step positive selection for a subset of these mutations that maintain expression of full-length protein ( two-step selection ) . 
We have selected such mutations in the transcription factor E2F1 that affect its ability to heterodimerize with DP1 . 
The mutations obtained identified a putative helix in the marked box , a region conserved among E2F family members , as an important determinant for interaction . 
This two-step selection procedure can be used to characterize any interaction domain that can be tested in the two-hybrid system . 


Formation of p27-CDK complexes during the human mitotic cell cycle . 
In eukaryotic cells , the coordinated activation of different cyclin-dependent kinases regulates entry into S-phase . 
In vitro and in nonproliferating cells , p27 associates with and inhibits cyclin / cycin-dependent kinase ( CDK ) holoenzymes containing either CDK4 , CDK6 , or CDK2 . 
Although many different types of proliferating cells contain p27 protein , neither the interactions of p27 with cyclin / CDK complexes nor the consequences of this interaction during the mitotic cycle have been fully explored . 
We report that , in MANCA cells , the amount of p27 is constant during the cell cycle . 
In addition , p27 associates with three different CDKs : CDK2 , CDK4 , and CDK6 . 
Furthermore , the amount of p27 is significantly lower than the amount of cyclin D3 in these cells . 
The amount of CDK4 and CDK6 associated with p27 does not change in a cell cycle-dependent fashion ; in contrast , the amount of CDK2 associated with p27 is lowest in early G1 cells and increases to a maximum in mid-G1 phase , reaching a steady-state level in late G1-phase cells . 
After mid-G1 phase , the amount of each p27 / CDK complex remains constant through the remainder of the cell cycle . 
p27-immunoprecipitates contain an Rb-kinase activity . 
The substrate specificity , the expression pattern of this kinase , and the ability to deplete 50 % of this kinase activity with a CDK6-specific antibody suggest that the CDK6 protein mediates , in part , the p27-associated Rb-kinase activity . 
In contrast , p27 complexes containing CDK2 are incapable of phosphorylating histone H1 . 
These data are consistent with a model wherein cyclin D / CDK complexes sequester the CDK2-dependent kinase inhibitory activity of p27 . 


Gastrin stimulates tyrosine phosphorylation of insulin receptor substrate 1 and its association with Grb2 and the phosphatidylinositol 3-kinase . 
The growth-promoting effects of gastrin on normal and neoplastic gastrointestinal tissues have been shown to be mediated by the gastrin / CCKB receptor , which belongs to the family of G protein-coupled receptors . 
However , the downstream signaling pathways activated by gastrin are not well characterized . 
In the present study , we demonstrate that gastrin stimulates tyrosine phosphorylation of insulin receptor substrate 1 ( IRS-1 ) , the major cytoplasmic substrate of the insulin receptor . 
The gastrin-induced phosphorylation of IRS-1 was rapid and transient , occurring within 30 s of treatment and diminishing thereafter . 
IRS-1 binds several proteins containing Src homology 2 domains through its multiple tyrosine phosphorylation sites . 
Following gastrin stimulation , we observed a time- and dose-dependent association of IRS-1 with the p85 regulatory subunit of phosphatidylinositol 3-kinase ( PI 3-kinase ) . 
In addition , activation of PI 3-kinase was detected in anti- IRS-1 immunoprecipitates from gastrin-treated cells , suggesting that tyrosine phosphorylation of IRS-1 , which leads to the rapid recruitment of p85 , might be one mechanism used by gastrin to activate PI 3-kinase . 
We have previously reported that tyrosine phosphorylation of Shc and its association with the Grb2-Sos complex may contribute to the activation of the mitogen-activated protein kinase pathway by gastrin . 
We report here that Grb2 also interacts with tyrosine-phosphorylated IRS-1 in response to gastrin . 
Taken together , our results suggest that IRS-1 may serve as a converging target in the signaling pathways stimulated by receptors that belong to different families , such as the gastrin / CCKB G protein-coupled receptor and the insulin receptor . 


Use of a two-hybrid system to investigate molecular interactions of GAP-43 . 
We used the ' interaction trap ' ( two-hybrid system ) to identify polypeptides that interact with the neuronal phosphoprotein , GAP-43 , in an intracellular environment . 
GAP-43 ( neuromodulin , B-50 , F1 ) , a protein kinase C ( PKC ) substrate important for the growth and plasticity of neuronal connections , has been implicated in vitro in several signal transduction pathways . 
In the yeast-based cloning system , the only strong interaction that was detected between GAP-43 and the calcium effector protein , calmodulin ( CaM ) . 
PKC phosphorylates GAP-43 on serine 41 . 
When we changed this serine to an aspartate residue to mimic constitutive phosphorylation , the interaction with CaM was blocked . 
Surprisingly , the N-terminal third of GAP-43 alone bound CaM more strongly than did intact GAP-43 , suggesting that the protein 's C-terminus may play a role in modulating the interaction with CaM . 
These results , along with other recent findings , suggest a novel role for the interaction between GAP-43 and CaM . 


Structure of the p53 tumor suppressor bound to the ankyrin and SH3 domains of 53BP2 . 
Mutations in the p53 tumor suppressor are among the most frequently observed genetic alterations in human cancer and map to the 200-amino acid core domain of the protein . 
The core domain contains the sequence-specific DNA binding activity and the in vitro 53BP2 protein binding activity of p53 . 
The crystal structure of the p53 core domain bound to the 53BP2 protein , which contains an SH3 ( Src homology 3 ) domain and four ankyrin repeats , revealed that ( i ) the SH3 domain binds the L3 loop of p53 in a manner distinct from that of previously characterized SH3-polyproline peptide complexes , and ( ii ) an ankyrin repeat , which forms an L-shaped structure consisting of a beta hairpin and two alpha helices , binds the L2 loop of p53 . 
The structure of the complex shows that the 53BP2 binding site on the p53 core domain consists of evolutionarily conserved regions that are frequently mutated in cancer and that it overlaps the site of DNA binding . 
The six most frequently observed p53 mutations disrupt 53BP2 binding in vitro . 
The structure provides evidence that the 53BP2-p53 complex forms in vivo and may have a critical role in the p53 pathway of tumor suppression . 


Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain . 
The MDM2 oncoprotein is a cellular inhibitor of the p53 tumor suppressor in that it can bind the transactivation domain of p53 and downregulate its ability to activate transcription . 
In certain cancers , MDM2 amplification is a common event and contributes to the inactivation of p53 . 
The crystal structure of the 109-residue amino-terminal domain of MDM2 bound to a 15-residue transactivation domain peptide of p53 revealed that MDM2 has a deep hydrophobic cleft on which the p53 peptide binds as an amphipathic alpha helix . 
The interface relies on the steric complementarity between the MDM2 cleft and the hydrophobic face of the p53 alpha helix and , in particular , on a triad of p53 amino acids-Phe19 , Trp23 , and Leu26-which insert deep into the MDM2 cleft . 
These same p53 residues are also involved in transactivation , supporting the hypothesis that MDM2 inactivates p53 by concealing its transactivation domain . 
The structure also suggests that the amphipathic alpha helix may be a common structural motif in the binding of a diverse family of transactivation factors to the TATA-binding protein-associated factors . 


The cytokine-activated tyrosine kinase JAK2 activates Raf-1 in a p21ras-dependent manner . 
JAK2 , a member of the Janus kinase superfamily was found to interact functionally with Raf-1 , a central component of the ras / mitogen-activated protein kinase signal transduction pathway . 
Interferon-gamma and several other cytokines that are known to activate JAK2 kinase were also found to stimulate Raf-1 kinase activity toward MEK-1 in mammalian cells . 
In the baculovirus coexpression system , Raf-1 was activated by JAK2 in the presence of p21ras . 
Under these conditions , a ternary complex of p21ras , JAK2 , and Raf-1 was observed . 
In contrast , in the absence of p21ras , coexpression of JAK2 and Raf-1 resulted in an overall decrease in the Raf-1 kinase activity . 
In addition , JAK2 phosphorylated Raf-1 at sites different from those phosphorylated by pp60v-src . 
In mammalian cells treated with either erythropoietin or interferon-gamma , a small fraction of Raf-1 coimmunoprecipitated with JAK2 in lysates of cells in which JAK2 was activated as judged by its state of tyrosine phosphorylation . 
Taken together , these data suggest that JAK2 and p21ras cooperate to activate Raf-1 . 


CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor  CCR-5 . 
For efficient entry into target cells , primary macrophage-tropic and laboratory-adapted human immunodeficiency viruses type 1 ( HIV-1 ) require particular chemokine receptors , CCR-5 and CXCR-4 , respectively , as well as the primary receptor CD4 ( refs 1-6 ) . 
Here we show that a complex of gp120 , the exterior envelope glycoprotein , of macrophage-tropic primary HIV-1 and soluble CD4 interacts specifically with CCR-5 and inhibits the binding of the natural CCR-5 ligands , macrophage inflammatory protein ( MIP )-1alpha and MIP-1beta ( refs 7 , 8 ) . 
The apparent affinity of the interaction between gp120 and CCR-5 was dramatically lower in the absence of soluble CD4 . 
Additionally , in the absence of gp120 , an interaction between a two-domain CD4 fragment and CCR-5 was observed . 
A gp120 fragment retaining the CD4-binding site and overlapping epitopes was able to interact with CCR-5 only if the V3 loop , which can specify HIV-1 tropism and chemokine receptor choice , was also present on the molecule . 
Neutralizing antibodies directed against either CD4-induced or V3 epitopes on gp120 blocked the interaction of gp12O-CD4 complexes with CCR-5 . 
These results suggest that HIV-1 attachment to CD4 creates a high-affinity binding site for CCR-5 , leading to membrane fusion and virus entry . 


Stat3 recruitment by two distinct ligand-induced , tyrosine-phosphorylated docking sites in the interleukin-10 receptor intracellular domain . 
Recent work has shown that IL-10 induces activation of the JAK-STAT signaling pathway . 
To define the mechanism underlying signal transducer and activator of transcription ( STAT ) protein recruitment to the interleukin 10 ( IL-10 ) receptor , the STAT proteins activated by IL-10 in different cell populations were first defined using electrophoretic mobility shift assays . 
In all cells tested , IL-10 activated Stat1 and Stat3 and induced the formation of three distinct DNA binding complexes that contained different combinations of these two transcription factors . 
IL-10 also activated Stat5 in Ba / F3 cells that stably expressed the murine IL-10 receptor . 
Using a structure-function mutagenesis approach , two tyrosine residues ( Tyr427 and Tyr477 ) in the intracellular domain of the murine IL-10 receptor were found to be redundantly required for receptor function and for activation of Stat3 but not for Stat1 or Stat5 . 
Twelve amino acid peptides encompassing either of these two tyrosine residues in phosphorylated form coprecipitated Stat3 but not Stat1 and blocked IL-10-induced Stat3 phosphorylation in a cell-free system . 
In contrast , tyrosine-phosphorylated peptides containing Tyr374 or Tyr396 did not interact with Stat3 or block Stat3 activation . 
These data demonstrate that Stat3 but not Stat1 or Stat5 is directly recruited to the ligand-activated IL-10 receptor by binding to specific but redundant receptor intracellular domain sequences containing phosphotyrosine . 
This study thus supports the concept that utilization of distinct STAT proteins by different cytokine receptors is dependent on the expression of particular ligand-activatable , tyrosine-containing STAT docking sites in receptor intracellular domains . 


cDNA cloning and characterization of the human interleukin 13 receptor alpha chain . 
We have cloned cDNAs corresponding to the human interleukin 13 receptor alpha chain ( IL-13Ralpha ) . 
The protein has 76 % homology to murine IL-13Ralpha , with 95 % amino acid identity in the cytoplasmic domain . 
Only weak IL-13 binding activity was found in cells transfected with only IL-13Ralpha ; however , the combination of both IL-13Ralpha and IL-4Ralpha resulted in substantial binding activity , with a Kd of approximately 400 pM , indicating that both chains are essential components of the IL-13 receptor . 
Whereas IL-13Ralpha serves as an alternative accessory protein to the common cytokine receptor gamma chain ( gammac ) for IL-4 signaling , it could not replace the function of gammac in allowing enhanced IL-2 binding activity . 
Nevertheless , the overall size and length of the cytoplasmic domain of IL-13Ralpha and gammac are similar , and like gammac , IL-13Ralpha is located on chromosome X . 


Interaction of transthyretin with amyloid beta-protein : binding and inhibition of amyloid formation . 
Aggregated amyloid beta-protein ( A beta ) is a key component of the amyloid depositions found in the brains of patients with Alzheimer 's disease . 
In contrast , in cerebrospinal fluid ( CSF ) , A beta is found in a soluble form . 
The analysis of complexes of A beta with CSF proteins in a KBr gradient revealed an association of A beta only with free proteins and not with lipoprotein particles . 
Transthyretin ( TTR ) , a second major CSF protein , formed SDS-stable complexes with A beta and significantly decreased the rate of A beta fibril formation . 
In physiological buffers and CSF , TTR exclusively decreased the level of A beta pentamers . 
Endogenous TTR-A beta complexes were detected in human CSF by immunoprecipitation . 
Using site-directed mutagenesis and computer-assisted modelling , we identified amino acid residues on the surface of the TTR monomer that interact with A beta . 
Specific TTR immunoreactivity was detected in multiple cortical neurons and astrocytes in the human brain . 
We propose that A beta binding proteins play a key role in the modulation of A beta aggregation and normally prevent amyloid formation in biological fluids and in the brain . 


SREBP transcriptional activity is mediated through an interaction with the CREB-binding protein . 
The sterol regulatory element binding proteins ( SREBP-1 and -2 ) activate transcription of genes whose products are involved in the cellular uptake and synthesis of cholesterol . 
Although considerable effort has been exerted to define the events regulating the levels of active SREBP , little is known about the transcriptional cofactors mediating SREBP function . 
In an unbiased search for potential coactivators of SREBP , we isolated a protein of 265 kD from HeLa cells that directly bound SREBP-1 and SREBP-2 . 
Peptide sequencing and Western blot analysis established that the 265-kD protein was CBP ( CREB-binding protein ) a recently identified transcriptional coactivator . 
The putative activation domain of SREBP was shown to bind specifically to amino-terminal domains of recombinant CBP and p300 ( a CBP-related protein ) . 
Moreover , transfection studies demonstrated that CBP enhances the ability of SREBP to activate transcription of reporter genes in HeLa cells . 
Together , these data suggest that CBP mediates SREBP transcriptional activity , thus revealing a new step in the biochemical pathway regulating cholesterol metabolism . 


Associations of UBE2I with RAD52 , UBL1 , p53 , and RAD51 proteins in a yeast two-hybrid system . 
The yeast RAD52-dependent pathway is involved in DNA recombination and double-strand break repair . 
Yeast ubiquitin-conjugating enzyme UBC9 participates in S- and M-phase cyclin degradation and mitotic control . 
Using the human RAD52 protein as the " bait " in a yeast two-hybrid system , we have identified a human homolog of yeast UBC9 , designated UBE2I , that interacts with RAD52 , RAD51 , p53 , and a ubiquitin-like protein UBL1 . 
These interactions are UBE2I-specific , since another DNA repair-related ubiquitin-conjugating enzyme , RAD6 ( UBC2 ) , does not interact with these proteins . 
The interaction of UBE2I with RAD52 is mediated by RAD52 ' s self-association region . 
These results suggest that the RAD52-dependent processes , cell cycle control , p53-mediated pathway ( s ) , and ubiquitination interact through human UBE2I . 


Discrete protein interactions with the Grb2 / c-Cbl complex in SCF- and TPO-mediated myeloid cell proliferation . 
Hemopoietic cell proliferation is mediated by non-tyrosine and tyrosine kinases that signal via uncommon and common sets of downstream effector molecules including the Grb2 / c-Cbl . 
In the present study we evaluated tyrosine phosphorylation of c-Cbl and the interaction of the Grb2 / c-Cbl complex with signaling proteins upon activation of non-tyrosine ( c-Mpl ) and tyrosine kinase ( c-Kit ) receptors leading to myeloid cell proliferation . 
By using the growth factor dependent M-07e cell line , we found that both c-Mpl and c-Kit ligands , namely : SCF and TPO , induce c-Cbl tyrosine phosphorylation . 
In these cells the adaptor protein Grb2 constitutively binds a substantial fraction of c-Cbl through the N-terminal SH3 domain . 
In vitro experiments showed that the stable Grb2 / c-Cbl complex interacts , through the Grb2 SH2 domain , with the SCF-activated c-Kit . 
By contrast stimulation with TPO leads to the formation of a Grb2 complex containing JAK2 . 
In vitro and in vivo experiments support the hypothesis that Grb2 mediates the association of c-Kit with c-Cbl . 
Moreover we found that , upon SCF stimulation , the Grb2 / c-Cbl complex recruits Shc , probably via Grb2 . 
By contrast the Ras exchanger factor ( Sos1 ) was not detected in anti- c-Cbl immunoprecipitates suggesting that Grb2 / Sos1 and Grb2 / c-Cbl are present in different complexes . 
Taken together our results demonstrate that c-Cbl plays an important role in coupling both tyrosine and non-tyrosine kinase receptors to downstream effector molecules and that different signaling molecules interact with Grb2 / c-Cbl complex when non-tyrosine or tyrosine kinase receptors are activated . 


Efficient interaction of HIV-1 with purified dendritic cells via multiple chemokine coreceptors . 
HIV-1 actively replicates in dendritic cell ( DC ) -T cell cocultures , but it has been difficult to demonstrate substantial infection of purified mature DCs . 
We now find that HIV-1 begins reverse transcription much more efficiently in DCs than T cells , even though T cells have higher levels of CD4 and gp120 binding . 
DCs isolated from skin or from blood precursors behave similarly . 
Several M-tropic strains and the T-tropic strain IIIB enter DCs efficiently , as assessed by the progressive formation of the early products of reverse transcription after a 90-min virus pulse at 37 degrees C . 
However , few late gag-containing sequences are detected , so that active viral replication does not occur . 
The formation of these early transcripts seems to follow entry of HIV-1 , rather than binding of virions that contain viral DNA . 
Early transcripts are scarce if DCs are exposed to virus on ice for 4 h , or for 90 min at 37 degrees C , conditions which allow virus binding . 
Also the early transcripts once formed are insensitive to trypsin . 
The entry of a M-tropic isolates is blocked by the chemokine RANTES , and the entry of IIIB by SDF-1 . 
RANTES interacts with CCR5 and SDF-1 with CXCR4 receptors . 
Entry of M-tropic but not T-tropic virus is ablated in DCs from individuals who lack a functional CCR5 receptor . 
DCs express more CCR5 and CXCR4 mRNA than T cells . 
Therefore , while HIV-1 does not replicate efficiently in mature DCs , viral entry can be active and can be blocked by chemokines that act on known receptors for M- and T-tropic virus . 


Activation of interferon-gamma inducing factor mediated by interleukin-1beta converting enzyme . 
The interleukin-1beta (IL-1beta ) converting enzyme ( ICE ) processes the inactive IL-1beta precursor to the proinflammatory cytokine . 
ICE was also shown to cleave the precursor of interferon-gamma inducing factor ( IGIF ) at the authentic processing site with high efficiency , thereby activating IGIF and facilitating its export . 
Lipopolysaccharide-activated ICE-deficient ( ICE-/-) Kupffer cells synthesized the IGIF precursor but failed to process it into the active form . 
Interferon-gamma and IGIF were diminished in the sera of ICE-/-mice exposed to Propionibacterium acnes and lipopolysaccharide . 
The lack of multiple proinflammatory cytokines in ICE-/-mice may account for their protection from septic shock . 


Physical and functional interactions between the transcriptional inhibitors Id3 and ITF-2b . 
Evidence toward a novel mechanism regulating muscle-specific gene expression . 
We have used an interaction cloning strategy to identify an inhibitory isoform of the ITF-2 transcription factor , ITF-2b , that interacts with the transcriptional inhibitor Id3 / HLH462 . 
The interaction was confirmed in vitro , and inside intact myogenic C2C12 cells . 
As expected , overexpression of either Id3 / HLH462 or ITF-2b effectively inhibited the activation of the muscle-specific creatine kinase promoter by the myogenic transcription factor MyoD . 
However , when overexpressed simultaneously , ITF-2b and Id3 / HLH462 counteracted each other 's inhibitory effect to produce a reduced overall inhibition . 
Moreover , while ITF-2b inhibited the creatine kinase promoter , it acted as a weak transactivator on an artificial promoter consisting of three tandem copies of the consensus myogenic factor DNA binding site . 
Further investigation indicated that the ITF-2b / MyoD heterodimer bound to its specific DNA binding site in vitro , and the DNA binding was effectively blocked by Id3 / HLH462 . 
Additional analysis revealed the presence of transcripts for both the activating ( ITF-2a ) and inhibitory ( ITF-2b ) isoforms in differentiating C2C12 cultures , suggesting that both isoforms might participate in regulating the differentiation process . 
Taken together , this study reveals a more complex pattern of regulatory interactions involving the helix-loop-helix proteins than was previously anticipated . 


Bcl-x ( L ) forms an ion channel in synthetic lipid membranes . 
Bcl-2-related proteins are critical regulators of cell survival that are localized to the outer mitochondrial , outer nuclear and endoplasmic reticulum membranes . 
Despite their physiological importance , the biochemical function of Bcl-2-related proteins has remained elusive . 
The three-dimensional structure of Bcl-xL , an inhibitor of apoptosis , was recently shown to be similar to the structures of the pore-forming domains of bacterial toxins . 
A key feature of these pore-forming domains is the ability to form ion channels in biological membranes . 
Here we demonstrate that Bcl-xL shares this functional feature . 
Like the bacterial toxins , Bcl-xL can insert into either synthetic lipid vesicles or planar lipid bilayers and form an ion-conducting channel . 
This channel is pH-sensitive and becomes cation-selective at physiological pH . 
The ion-conducting channel ( s ) formed by Bcl-xL display multiple conductance states that have identical ion selectivity . 
Together , these data suggest that Bcl-xL may maintain cell survival by regulating the permeability of the intracellular membranes to which it is distributed . 


Competitive binding of alpha-actinin and calmodulin to the NMDA receptor . 
The mechanisms by which neurotransmitter receptors are immobilized at postsynaptic sites in neurons are largely unknown . 
The activity of NMDA ( N-methyl-D-aspartate ) receptors is mechanosensitive and dependent on the integrity of actin , suggesting a functionally important interaction between NMDA receptors and the postsynaptic cytoskeleton . 
alpha-Actinin-2 , a member of the spectrin / dystrophin family of actin-binding proteins , is identified here as a brain postsynaptic density protein that colocalizes in dendritic spines with NMDA receptors and the putative NMDA receptor-clustering molecule PSD-95 . 
alpha-Actinin-2 binds by its central rod domain to the cytoplasmic tail of both NR1 and NR2B subunits of the NMDA receptor , and can be immunoprecipitated with NMDA receptors and PSD-95 from rat brain . 
Intriguingly , NR1- alpha-actinin binding is directly antagonized by Ca2+ / calmodulin . 
Thus alpha-actinin may play a role in both the localization of NMDA receptors and their modulation by Ca2+ . 


HOX11 interacts with protein phosphatases PP2A and PP1 and disrupts a G2 / M cell-cycle checkpoint . 
Hox11 is an orphan homeobox gene that controls the genesis of the spleen . 
HOX11 is also oncogenic , having been isolated from a chromosomal breakpoint in human T-cell leukaemia . 
Transgenic mice that redirected HOX11 to the thymus demonstrated cell-cycle aberration and progression to malignancy . 
We observed that the protein HOX11 interacted with protein serine-threonine phosphatase 2A catalytic subunit ( PP2AC ) , as well as protein phosphatase 1 ( PP1C ) in mammalian cells . 
Inhibition of PP2A can regulate the cell cycle and control the activation of maturation-promoting factor in Xenopus oocytes . 
Microinjection of HOX11 into Xenopus oocytes arrested at the G2 phase of the cell cycle promoted progression to the M phase . 
G2 arrest can be induced by gamma-irradiation , but is eliminated by expression of HOX11 within a T-cell line . 
Thus HOX11 is a cellular oncogene that targets PP2A and PP1 , both of which are targets for oncogenic viruses and chemical tumour promoters . 
This interaction suggests a mechanism by which a homeobox can alter the cell cycle . 


Analysis of the human interleukin-6 / human interleukin-6 receptor binding interface at the amino acid level : proposed mechanism of interaction . 
The interaction between interleukin-6 ( IL-6 ) and IL-6 receptor ( IL-6R ) is the initial and most specific step in the IL-6 signaling pathway . 
Understanding its mechanism at the amino acid level is the basis for developing small IL-6-inhibiting molecules . 
We studied the human IL-6 ( hIL-6 ) / hIL-6R binding interface by a combination of molecular modelling and site-directed mutagenesis . 
Our model suggests that the center of the interface between the two molecules consists of hydrophobic contacts predicted to account for most of the binding-free energy . 
These contacts can be regarded as a hydrophobic core shielded by hydrophilic residues that are also needed for recognition . 
Following this hypothesis , we altered in hIL-6 and hIL-6R residues predicted to reside in the contact region and to interact with each other . 
We studied the capacity of these mutants to form an IL-6 / IL-6R complex and their ability to transduce the signal . 
This combined approach has led to the identification of certain residue-clusters in the binding interface and to a rational explanation of their specific interactions , suggesting therein a likely mechanism of complex formation . 
The results confirm the predictive model and strongly support our hypothesis . 
Comparison with other cytokines and their alpha-subunit receptors suggests that the structural location of certain binding sites are conserved . 


Mutagenesis studies of the human erythropoietin receptor . 
Establishment of structure-function relationships . 
Mutagenesis of the erythropoietin receptor ( EPOR ) permits analysis of the contribution that individual amino acid residues make to erythropoietin ( EPO ) binding . 
We employed both random and site-specific mutagenesis to determine the function of amino acid residues in the extracellular domain ( referred to as EPO binding protein , EBP ) of the EPOR . 
Residues were chosen for site-specific alanine substitution based on the results of the random mutagenesis or on their homology to residues that are conserved or have been reported to be involved in ligand binding in other receptors of the cytokine receptor family . 
Site-specific mutants were expressed in Escherichia coli as soluble EBP and analyzed for EPO binding in several different assay formats . 
In addition , selected mutant proteins were expressed as full-length EPOR on the surface of COS cells and analyzed for 125I- EPO binding in receptor binding assays . 
Using these methods , we have identified residues that appear to be involved in EPO binding as well as other residues , most of which are conserved in receptors of the cytokine receptor family , that appear to be necessary for the proper folding and / or stability of the EPOR . 
We present correlations between these mutagenesis data and the recently solved crystal structure of the EBP with a peptide ligand . 


Signal transduction pathway of human fibroblast growth factor receptor 3 . 
Identification of a novel 66-kDa phosphoprotein . 
Stimulation of fibroblast growth factor receptor 3 ( FGFR3 ) results in a variety of functional effects , including regulation of epithelial cell growth and differentiation . 
In order to characterize the signaling pathway through which FGFR3 regulates cell growth , L6 cells lacking any endogenous FGFR were stably transfected with the two different human isoforms , FGFR3 IIIb and FGFR3 IIIc , that result from alternative splicing of exon III of the FGFR3 gene encoding the ligand binding domain . 
Expression of FGFR3 IIIc in stably transfected L6 cells conferred growth responses to several members of the FGF family including FGF-1 ,-2 ,-4 , and -6 , while FGFR3 IIIb-expressing cells responded only to FGF-1 . 
Activation of FGFR3 upon ligand binding resulted in activation of mitogen-activated protein kinase pathway . 
FGFR3 utilizes two different pools of adapter protein GRB2 to link to Ras . 
Activated FGFR3 predominantly interacts with GRB2 . Sos in complex with a previously identified 90-kDa protein and designated protein 80K-H . 
In addition , 80K-H . GRB2 . Sos complex was found to contain a novel 66-kDa protein . 
Tyrosine phophorylation of the 66-kDa protein was dependent on ligand activation of FGFR3 , suggesting that the 66-kDa protein may play an important role in FGFR3-specific signaling . 
In addition to this unique pathway , FGFR3 also links to GRB2 . 
Sos complex via the adapter protein Shc . 
Furthermore , activated FGFR3 was not able to induce dissociation of GRB2 . 
Sos complex following Sos phosphorylation . 
In summary , FGFR3 signaling pathway utilizes two GRB2-containing complexes ; Shc . 
GRB2 . Sos and 80K-H . pp66 . GRB2 . Sos  ; these two complexes may alternatively link FGFG3 to mitogen-activated protein kinase . 
Finally , activated FGFR3 was also found to result in phosphorylation of phospholipase C-gamma but reduced phosphorylation of c-Src . 


Identification of interaction partners for the basic-helix-loop-helix protein E47 . 
Helix-loop-helix proteins constitute a family of transcription factors with the potential to form homo- and hetero-dimers mediated by the helix-loop-helix domain . 
Oncogenic mutations in such genes can disrupt the equilibrium of protein-protein interactions in the affected cell . 
In order to assess the biological consequences of such mutations , the full complement of interacting proteins must be known . 
To identify proteins interacting with the basic-helix-loop-helix domain of the ubiquitously expressed E47 protein , a ' sandwich '-screening procedure was developed which distinguishes between homo- and hetero-oligomers , and specifically excludes the detection of complexes which cannot bind DNA . 
Nine distinct cDNAs were identified which encode proteins with apparent basic-helix-loop-helix domains , including a novel clone termed eip1 which is distantly related in the basic-helix-loop-helix domain to the Drosophila enhancer-of-split m7 protein . 
Using epitope-tagging , interaction of E47 basic-helix-loop-helix protein with the eip1 protein encoded by this novel cDNA was confirmed by immunoprecipitation experiments in COS7 cells . 
Interaction was also observed in the yeast two-hybrid system . 
Three cDNAs encoding proteins without basic-helix-loop-helix domains were also found to interact in the sandwich-expression screen . 
Interactions with human PARP and mouse replication factor 1a were confirmed using glutathione transferase-tagged cDNAs . 
A cDNA encoding part of the nucleolin protein sequence interacted with the E47 basic-helix-loop-helix only when fused to a beta-galactosidase tag . 


Hepatitis B virus X protein is a transcriptional modulator that communicates with transcription factor IIB and the RNA polymerase II subunit 5 . 
Hepatitis B virus X protei n ( HBx ) transactivates viral and cellular genes through a wide variety of cis-elements . 
However , the mechanism is still obscure . 
Our finding that HBx directly interacts with RNA polymerase II subunit 5 ( RPB5 ) , a common subunit of RNA polymerases , implies that HBx directly modulates the function of RNA polymerase ( Cheong , J. H. , Yi , M. , Lin , Y. , and Murakami , S. ( 1995 ) EMBO J. 14 , 142-150 ) . 
In this context , we examined the possibility that HBx and RPB5 interact with other general transcription factors . 
HBx and RPB5 specifically bound to transcription factor IIB ( TFIIB ) in vitro , both of which were detected by either far-Western blotting or the glutathione S-transferase-resin pull-down assay . 
Delineation of the binding regions of these three proteins revealed that HBx , RPB5 , and TFIIB each has two binding regions for the other two proteins . 
Co-immunoprecipitation using HepG2 cell lysates that express HBx demonstrated trimeric interaction in vivo . 
Some HBx substitution mutants , which had severely impaired transacting activity , exhibited reduced binding affinity with either TFIIB or RPB5 in a mutually exclusive manner , suggesting that HBx transactivation requires the interactions of both RPB5 and TFIIB . 
These results indicated that HBx is a novel virus modulator that facilitates transcriptional initiation by stabilizing the association between RNA polymerase and TFIIB through communication with RPB5 and TFIIB . 


Crystal structure of the type-I interleukin-1 receptor complexed with interleukin-1beta . 
Interleukin-1 ( IL-1 ) is an important mediator of inflammatory disease . 
The IL-1 family currently consists of two agonists , IL-1alpha and IL-1beta , and one antagonist , IL-1ra . 
Each of these molecules binds to the type I IL-1 receptor ( IL1R ) . 
The binding of IL-1alpha or IL-1beta to IL1R is an early step in IL-1 signal transduction and blocking this interaction may therefore be a useful target for the development of new drugs . 
Here we report the three-dimensional structure of IL-1beta bound to the extracellular domain of IL1R ( s-IL1R ) at 2.5 A resolution . 
IL-1beta binds to s-IL1R with a 1 : 1 stoichiometry . 
The crystal structure shows that s-IL1R consists of three immunoglobulin-like domains which wrap around IL-1beta in a manner distinct from the structures of previously described cytokine-receptor complexes . 
The two receptor-binding regions on IL-1beta identified by site-directed mutagenesis both contact the receptor : one binds to the first two domains of the receptor , while the other binds exclusively to the third domain . 


A new cytokine-receptor binding mode revealed by the crystal structure of the IL-1 receptor with an antagonist . 
Inflammation , regardless of whether it is provoked by infection or by tissue damage , starts with the activation of macrophages which initiate a cascade of inflammatory responses by producing the cytokines interleukin-1 ( IL-1 ) and tumour necrosis factor-alpha ( ref. 1 ) . 
Three naturally occurring ligands for the IL-1 receptor ( IL1R ) exist : the agonists IL-1alpha and IL-1beta and the IL-1-receptor antagonist IL1RA ( ref. 2 ) . 
IL-1 is the only cytokine for which a naturally occurring antagonist is known . 
Here we describe the crystal structure at 2.7 A resolution of the soluble extracellular part of type-I IL1R complexed with IL1RA . 
The receptor consists of three immunoglobulin-like domains . 
Domains 1 and 2 are tightly linked , but domain three is completely separate and connected by a flexible linker . 
Residues of all three domains contact the antagonist and include the five critical IL1RA residues which were identified by site-directed mutagenesis . 
A region that is important for biological function in IL-1beta , the ' receptor trigger site ' is not in direct contact with the receptor in the IL1RA complex . 
Modelling studies suggest that this IL-1beta trigger site might induce a movement of domain 3 . 


The catalytic domain of activated collagenase I ( MMP-1 ) is absolutely required for interaction with its specific inhibitor , tissue inhibitor of metalloproteinases-1 ( TIMP-1 ) . 
Here , we describe the production of recombinant human tissue inhibitor of metalloproteinases-1 ( rTIMP-1 ) and wild-type and mutant human collagenase type I ( rMMP-1 ) proteins in SF9 cells by the baculovirus expression system . 
Wild-type MMP-1 , as well as the MMP-1 mutant lacking the C-terminal hemopexin-like domain [ des- ( 248-450 ) - MMP-1 ] , exhibit enzymatic activity upon cleavage of the prodomain by treatment with trypsin or 4-aminophenylmercuric acetate . 
Enzyme activity of both proteins can be inhibited by addition of rTIMP . 
Deletion of the complete active-site [ des- ( 161-228 ) - MMP-1 ] within the catalytic domain , or mutation of a single His residue of the Zn2+ binding domain ( His199 ) , generates stable forms of MMP-1 proteins which are unable to digest collagen type I or beta-casein . 
In addition to co-immunoprecipitation analysis , we have established a rapid and sensitive ELISA assay using immobilized rTIMP to determine the structural requirements of MMP-1 to form complexes with its inhibitor . 
Only the activated and not the latent forms of wild-type and C-terminal mutant des- ( 248-450 ) -MMP-1 proteins are able to form complexes with TIMP . 
Neither mutation of His199 , nor deletion mutants des- ( 161-228 ) - MMP-1 and des- ( 161-228 / 248-450 ) - MMP-1 , interact with TIMP . 
This demonstrates that the C-terminal hemopexin domain of MMP-1 , in contrast to the corresponding regions of gelatinase A and gelatinase B , does not interact with TIMP-1 . 
In summary , we have shown that the integrity of the catalytic domain of MMP-1 and its ability to bind Zn2+ is absolutely required for complex formation with TIMP-1 , which further underlines the importance of this region for proper regulation of enzymatic activity of MMP-1 . 


Thymocyte activation induces the association of phosphatidylinositol 3-kinase and pp120 with CD5 . 
CD5 is a glycoprotein expressed on thymocytes , T cells , and a subset of B cells . 
Antibody-mediated cross-linking studies or studies on CD5 knockout mice implicate CD5 as a co-stimulatory or negative regulatory molecule . 
CD5 is rapidly phosphorylated on tyrosine ( Y ) residues following Tcell activation . 
Y429 and Y441 occur in an imperfect immunoreceptor tyrosine-based activation motif ( ITAM ) -like sequence . 
We investigated whether phosphatidylinositol ( PI ) 3-kinase , which binds to tyrosine-phosphorylated ITAM , interacts with CD5 following T cell activation . 
PI 3-kinase activity and the regulatory p85 subunit of PI 3-kinase associated with CD5 in pervanadate-stimulated , but not in unstimulated thymocytes . 
Cellular p85 as well as the recombinant Src homology 2 ( SH2 ) domains of p85 bound a tyrosine-phosphorylated peptide encompassing Y463 with approximately threefold greater affinity than a doubly tyrosine-phosphorylated Y429-Y441 peptide . 
Binding of the C-SH2 domain to the Y463 phosphopeptide , together with preferential binding of the N-SH2 domain to the Y429-Y441 phosphopeptide , suggests a bivalent interaction . 
A 120-kDa phosphoprotein ( pp120 ) associated with CD5 and specifically with the Y429-Y441 phosphopeptide in stimulated thymocytes . 
We conclude that stimulation of thymocytes with pervanadate induces the recruitment of PI 3-kinase and pp120 to CD5 . 


Functional significance of CD9 association with beta 1 integrins in human epidermal keratinocytes . 
CD9 is a member of the tetraspan ( TM4 ) family of proteins and is abundantly expressed in the epidermis . 
As CD9 forms complexes with beta 1 integrins and the integrins are known to regulate keratinocyte behaviour , we investigated CD9 expression and function in human epidermal keratinocytes . 
CD9 was present in all the living layers of the epidermis , whereas the beta 1 integrins were largely confined to the basal layer ; the same relative distribution was found in stratified cultures of keratinocytes . 
There was extensive co-localisation of CD9 and beta 1 integrins on microvilli and at cell-cell borders of basal keratinocytes ; however , in contrast to the integrins , CD9 was not found in focal adhesions . 
CD9 was detected in beta 1 integrin immunoprecipitates and also in immunoprecipitates of CD44 and syndecan , but not of cadherins . 
CD9 was associated with alpha 3 beta 1 but not alpha 5 beta 1 ; small amounts of CD9 also co-immunoprecipitated with antibodies to alpha 2 beta 1 and alpha 6 beta 4 . 
Antibodies to CD9 did not affect the proportion of keratinocytes that adhered to laminin 1 , type IV collagen and fibronectin , but did inhibit motility of keratinocytes on tissue culture plastic . 
Like antibodies to the beta 1 integrin subunit , anti- CD9 inhibited suspension-induced terminal differentiation . 
These results suggest that CD9 may play a role in regulating keartinocyte motility and differentiation . 


MyoD prevents cyclinA / cdk2 containing E2F complexes formation in terminally differentiated myocytes . 
Withdrawal from the cell cycle of differentiating myocytes is regulated by the myogenic basic helix-loop-helix ( bHLH ) protein MyoD and the pocket proteins pRb , p107 and pRb2 / p130 . 
Downstream effectors of ' pocket ' proteins are the components of the E2F family of transcription factors , which regulate the G1 / S-phase transition . 
We analysed by EMSA the composition of E2F complexes in cycling , quiescent undifferentiated and differentiated C2C12 skeletal muscle cells . 
An E2F complex containing mainly E2F4 and pRb2 / p130 ( E2F-G0 / G1 complex ) appears when DNA synthesis arrests , replacing the cyclinA / cdk2 containing E2F complex of proliferating myoblasts ( E2F-G1 / S complex ) . 
Serum stimulation reinduces DNA synthesis and the re-appearance of E2F-G1 / S complexes in quiescent myoblasts but not in differentiated C2C12 myotubes . 
In differentiating C2C12 cells , E2F complexes switch and DNA synthesis in response to serum are prevented when MyoD DNA binding activity and the cdks inhibitor MyoD downstream effector p21 are induced . 
Thus , during myogenic differentiation , formation of E2F4 and pRb2 / p130 containing complexes is an early event , but not enough on its own to prevent the reactivation of DNA synthesis . 
Using a subclone of C3H10T1 / 2 mouse fibroblasts stably expressing Estrogen Receptor- MyoD ( ER- MyoD ) chimerae , we found that estrogen directed MyoD activation prevents the reassociation of cyclinA / cdk2 to the E2F4 containing complex following serum stimulation and this correlates with suppression of E2F activity and the inability of cells to re-enter the cell cycle . 
Our data indicate that , in differentiating myocytes , one mechanism through which MyoD induces permanent cell cycle arrest involves p21 upregulation and suppression of the proliferation-associated cdks-containing E2F complexes formation . 


Early events in TNF signaling : a story of associations and dissociations . 
At the cellular level , the multifunctional cytokine tumor necrosis factor ( TNF ) modulates growth and activates genes through various intermediates , including protein kinases , protein phosphatases , reactive oxygen intermediates , phospholipases , proteases , sphingomyelinases , and transcription factors . 
Unlike many cytokine receptors , however , the cytoplasmic domain ( CD ) of the TNF receptors lacks an intrinsic protein kinase activity and yet on interaction with ligand it phosphorylates various proteins . 
Although the kinetics of most of these activities differ , their interactions are coordinated through the selective interplay between the CD of the receptors and the associated proteins . 
A unique pathway has been identified by the ability of the TNF receptors to associate with a novel family of proteins . 
Two distinct families of proteins have emerged , the TNF receptor-associated factors ( TRAFs ) and the death domain homologues . 
The cloning of members of these gene families and the identification of the protein-interaction motifs found within their gene products has initiated the molecular identity of factors ( TRADD , FADD / MORT , RIP , FLICE / MACH , and TRAFs ) associated with both of the p60 and p80 forms of the TNF receptor and with other members of the TNF receptor superfamily . 
In this review , we summarize these and other TNF receptor-associated proteins and their potential roles in regulating the activation of nuclear factor-kappaB and apoptosis , two major responses activated by engagement of TNF receptors by the ligand . 


Protein binding and signaling properties of RIN1 suggest a unique effector function . 
Human RIN1 was first characterized as a RAS binding protein based on the properties of its carboxyl-terminal domain . 
We now show that full-length RIN1 interacts with activated RAS in mammalian cells and defines a minimum region of 434 aa required for efficient RAS binding . 
RIN1 interacts with the " effector domain " of RAS and employs some RAS determinants that are common to , and others that are distinct from , those required for the binding of RAF1 , a known RAS effector . 
The same domain of RIN1 that binds RAS also interacts with 14-3-3 proteins , extending the similarity between RIN1 and other RAS effectors . 
When expressed in mammalian cells , the RAS binding domain of RIN1 can act as a dominant negative signal transduction blocker . 
The amino-terminal domain of RIN1 contains a proline-rich sequence similar to consensus Src homology 3 ( SH3 ) binding regions . 
This RIN1 sequence shows preferential binding to the ABL-SH3 domain in vitro . 
Moreover , the amino-terminal domain of RIN1 directly associates with , and is tyrosine phosphorylated by , c-ABL . 
In addition , RIN1 encodes a functional SH2 domain that has the potential to activate downstream signals . 
These data suggest that RIN1 is able to mediate multiple signals . 
A differential pattern of expression and alternate splicing indicate several levels of RIN1 regulation . 


Cross-family interaction between the bHLHZip USF and bZip Fra1 proteins results in down-regulation of AP1 activity . 
Heterodimerization among the basic-leucine zipper ( bZIP ) proteins or among the basic-helix-loop-helix-leucine zipper ( bHLHZip ) proteins confers a multitude of combinational activities to these transcription factors . 
To further examine the function of the bHLHZip protein , USF , we screened for cellular proteins which could directly interact with USF using the yeast two-hybrid system . 
A bZip protein , Fra1 , was found to efficiently interact with USF . 
USF specifically interacts with Fra1 but not with other closely related family members , c-Fos , Fra2 , FosB , or with c-Jun . 
Both the bHLHZip and the N-terminal regions of Fra1 are required for efficient interaction with USF . 
In vivo association between USF and Fra1 has been demonstrated by co-immunoprecipitation . 
Expression of exogenous USF led to a decrease in AP1-dependent transcription in F9 cells . 
Co-expression of exogenous Fra1 restored the AP1 activity in a dose-dependent manner . 
These data show that USF and Fra1 physically and functionally interact demonstrating that cross-talk occurs between factors of distantly related transcription families . 


Ligands for ErbB-family receptors encoded by a neuregulin-like gene . 
Neuregulins ( also called ARIA , GGF , heregulin or NDF ) are a group of polypeptide factors that arise from alternative RNA splicing of a single gene . 
Through their interaction with the ErbB family of receptors ( ErbB2 , ErbB3 and ErbB4 ) , neuregulins help to regulate cell growth and differentiation in many tissues . 
Here we report the cloning of a second neuregulin-like gene , neuregulin-2 . 
The encoded product of the neuregulin-2 gene has a motif structure similar to that of neuregulins and an alternative splicing site in the epidermal growth factor ( EGF ) -like domain gives rise to two isoforms ( alpha and beta ) . 
Northern blot and in situ hybridization analysis of adult rat tissues indicate that expression of neuregulin-2 is highest in the cerebellum , and the expression pattern is different from that of neuregulins . 
Recombinant neuregulin-2beta induces the tyrosine-phosphorylation of ErbB2 , ErbB3 and ErbB4 in cell lines expressing all of these ErbB-family receptors . 
However , in cell lines with defined combinations of ErbBs , neuregulin-2beta only activates those with ErbB3 and / or ErbB4 , suggesting that signalling by neuregulin-2 is mediated by ErbB3 and / or ErbB4 receptors . 


Interaction between ATM protein and c-Abl in response to DNA damage . 
The gene mutated in the autosomal recessive disorder ataxia telangiectasia ( AT ) , designated ATM ( for ' AT mutated ' ) , is a member of a family of phosphatidylinositol-3-kinase-like enzymes that are involved in cell-cycle control , meiotic recombination , telomere length monitoring and DNA-damage response . 
Previous results have demonstrated that AT cells are hypersensitive to ionizing radiation and are defective at the G1 / S checkpoint after radiation damage . 
Because cells lacking the protein tyrosine kinase c-Abl are also defective in radiation-induced G1 arrest , we investigated the possibility that ATM might interact with c-Abl in response to radiation damage . 
Here we show that ATM binds c-Abl constitutively in control cells but not in AT cells . 
Our results demonstrate that the SH3 domain of c-Abl interacts with a DPAPNPPHFP motif ( residues 1 , 373-1 , 382 ) of ATM . 
The results also reveal that radiation-induction of c-Abl tyrosine kinase activity is diminished in AT cells . 
These findings indicate that ATM is involved in the activation of c-Abl by DNA damage and this interaction may in part mediate radiation-induced G1 arrest . 


Multiple mechanisms of transcriptional repression by YY1 . 
The four C-terminal GLI-Kruppel type zinc fingers of YY1 have been identified as a transcriptional repression domain . 
Previous reports have proposed DNA-bending and activator-quenching mechanisms for this zinc finger-mediated repression . 
In addition , previous work indicated that p300 and CBP might be involved in YY1-mediated repression . 
We have analyzed these possible models for the zinc finger-mediated repression . 
The role of each zinc finger in the repression and DNA-binding functions was determined by using a structure-and-function approach . 
We show that zinc finger 2 of YY1 plays a central role in both DNA binding and transcriptional repression . 
However , a survey of a panel of YY1 mutants indicates that these two functions can be separated , which argues against the DNA-bending model for repression . 
We show that the physical interaction between YY1 and p300 , a coactivator for CREB , is not sufficient for repression of CREB-mediated transcription . 
Our studies indicate that YY1 functions as an activator-specific repressor . 
Repression of CTF-1-directed transcription may be accomplished through direct physical interaction between YY1 and this activator . 
In contrast , physical interaction is not necessary for YY1 to repress Sp1- and CREB-mediated transcription . 
Rather , the repression likely reflects an ability of YY1 to interfere with communication between these activators and their targets within the general transcription machinery . 
Taken together , our results suggest that YY1 employs multiple mechanisms to achieve activator-specific repression . 


Proto-oncoprotein Vav interacts with c-Cbl in activated thymocytes and peripheral T cells . 
The molecular adapter c-Cbl is rapidly tyrosine phosphorylated following stimulation through the TCR and associates with Src homology domain-2 ( SH2 ) / SH3 domain-containing adapters such as Grb2 , Crk , and Crk-L , which interact with guanine nucleotide exchange factors specific for the Ras family . 
This suggests that c-Cbl may link TCR activation to molecules that regulate GTP binding proteins . 
The SH2 / SH3-containing protein Vav also contains a guanine nucleotide exchange factor domain , and Vav has a crucial role in thymocyte development and activation of peripheral T cells following stimulation through the TCR . 
Here we show that Vav and c-Cbl form inducible molecular complexes in TCR-activated murine thymocytes and peripheral T cells as well as pervanadate-treated T cells . 
Vav / c-Cbl interactions are also detectable in freshly isolated T cells from gene-targeted mice that lack the T cell-specific inhibitory receptor CTLA-4 , in which c-Cbl is hyperphosphorylated on tyrosine residues . 
The interaction between Vav and c-Cbl is directly mediated via the SH2 domain of Vav and is dependent on tyrosine phosphorylation of c-Cbl . 
In addition , we show that the conserved motif Y699 MTP present in c-Cbl is the binding site for the Vav SH2 domain in vitro . 
These data imply that c-Cbl is a molecular adapter that regulates the function of Vav in thymocytes and peripheral T cells . 


Induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5 . 
Structural changes in the extracellular matrix are necessary for cell migration during tissue remodeling and tumor invasion . 
Specific cleavage of laminin-5 ( Ln-5 ) by matrix metalloprotease-2 ( MMP2 ) was shown to induce migration of breast epithelial cells . 
MMP2 cleaved the Ln-5 gamma2 subunit at residue 587 , exposing a putative cryptic promigratory site on Ln-5 that triggers cell motility . 
This altered form of Ln-5 is found in tumors and in tissues undergoing remodeling , but not in quiescent tissues . 
Cleavage of Ln-5 by MMP2 and the resulting activation of the Ln-5 cryptic site may provide new targets for modulation of tumor cell invasion and tissue remodeling . 


The Ras-RasGAP complex : structural basis for GTPase activation and its loss in oncogenic Ras mutants . 
The three-dimensional structure of the complex between human H-Ras bound to guanosine diphosphate and the guanosine triphosphatase ( GTPase ) -activating domain of the human GTPase-activating protein p120GAP ( GAP-334 ) in the presence of aluminum fluoride was solved at a resolution of 2.5 angstroms . 
The structure shows the partly hydrophilic and partly hydrophobic nature of the communication between the two molecules , which explains the sensitivity of the interaction toward both salts and lipids . 
An arginine side chain ( arginine-789 ) of GAP-334 is supplied into the active site of Ras to neutralize developing charges in the transition state . 
The switch II region of Ras is stabilized by GAP-334 , thus allowing glutamine-61 of Ras , mutation of which activates the oncogenic potential , to participate in catalysis . 
The structural arrangement in the active site is consistent with a mostly associative mechanism of phosphoryl transfer and provides an explanation for the activation of Ras by glycine-12 and glutamine-61 mutations . 
Glycine-12 in the transition state mimic is within van der Waals distance of both arginine-789 of GAP-334 and glutamine-61 of Ras , and even its mutation to alanine would disturb the arrangements of residues in the transition state . 


Characterization of Fas ( Apo-1 , CD95 ) -Fas ligand interaction . 
The death-inducing receptor Fas is activated when cross-linked by the type II membrane protein Fas ligand ( FasL ) . 
When human soluble FasL ( sFasL , containing the extracellular portion ) was expressed in human embryo kidney 293 cells , the three N-linked glycans of each FasL monomer were found to be essential for efficient secretion . 
Based on the structure of the closely related lymphotoxin alpha-tumor necrosis factor receptor I complex , a molecular model of the FasL homotrimer bound to three Fas molecules was generated using knowledge-based protein modeling methods . 
Point mutations of amino acid residues predicted to affect the receptor-ligand interaction were introduced at three sites . 
The F275L mutant , mimicking the loss of function murine gld mutation , exhibited a high propensity for aggregation and was unable to bind to Fas . 
Mutants P206R , P206D , and P206F displayed reduced cytotoxicity toward Fas-positive cells with a concomitant decrease in the binding affinity for the recombinant Fas-immunoglobulin Fc fusion proteins . 
Although the cytotoxic activity of mutant Y218D was unaltered , mutant Y218R was inactive , correlating with the prediction that Tyr-218 of FasL interacts with a cluster of three basic amino acid side chains of Fas . 
Interestingly , mutant Y218F could induce apoptosis in murine , but not human cells . 


Mutational analysis of thrombopoietin for identification of receptor and neutralizing antibody sites . 
Thrombopoietin ( TPO ) is a hematopoietin important for megakaryocyte proliferation and production of blood platelets . 
We sought to characterize how TPO binds and activates its receptor , myeloproliferative leukemia virus receptor . 
The erythropoietin-like domain of TPO ( TPO1-153 ) has been fused to the gIII coat protein of M13 bacteriophage . 
Forty residues were chosen for mutation to alanine using the criteria that they were charged residues or predicted to be solvent-exposed , based on a homology model . 
Phage enzyme-linked immunosorbent assay was used to determine affinities for binding to both the TPO receptor and five anti- TPO1-153 monoclonal antibodies . 
Mutations at mostly positively charged residues ( Asp8 , Lys14 , Lys52 , Lys59 , Lys136 , Lys138 , Arg140 ) caused the greatest reduction in receptor-binding affinity . 
Most of these residues mapped to helices-1 and -4 and a loop region between helix-1 and helix-2 . 
Two of the monoclonal antibodies that blocked TPO binding and bioactivity had determinants in helix-4 . 
In contrast , the other three monoclonal antibodies , which were effective at blocking TPO activity but did not block initial binding of TPO to its receptor , had epitopes predominantly on helix or 3 . 
These results suggest that TPO has two distinct receptor-binding sites that function to dimerize TPO receptors in a sequential fashion . 


14-3-3 zeta negatively regulates raf-1 activity by interactions with the Raf-1 cysteine-rich domain . 
Although Raf-1 is a critical effector of Ras signaling and transformation , the mechanism by which Ras promotes Raf-1 activation is complex and remains poorly understood . 
We recently reported that Ras interaction with the Raf-1 cysteine-rich domain ( Raf-CRD , residues 139-184 ) may be required for Raf-1 activation . 
The Raf-CRD is located in the NH2-terminal negative regulatory domain of Raf-1 and is highly homologous to cysteine-rich domains found in protein kinase C family members . 
Recent studies indicate that the structural integrity of the Raf-CRD is also critical for Raf-1 interaction with 14-3-3 proteins . 
However , whether 14-3-3 proteins interact directly with the Raf-CRD and how this interaction may mediate Raf-1 function has not been determined . 
In the present study , we demonstrate that 14-3-3 zeta binds directly to the isolated Raf-CRD . 
Moreover , mutation of Raf-1 residues 143-145 impairs binding of 14-3-3 , but not Ras , to the Raf-CRD . 
Introduction of mutations that impair 14-3-3 binding resulted in full-length Raf-1 mutants with enhanced transforming activity . 
Thus , 14-3-3 interaction with the Raf-CRD may serve in negative regulation of Raf-1 function by facilitating dissociation of 14-3-3 from the NH2 terminus of Raf-1 to promote subsequent events necessary for full activation of Raf-1 . 


Interactions between brain-derived neurotrophic factor and the TRKB receptor . 
Identification of two ligand binding domains in soluble TRKB by affinity separation and chemical cross-linking . 
The extracellular domain of the human neurotrophin TRKB receptor expressed in Chinese hamster ovary cells is a highly glycosylated protein , possessing binding ability for brain-derived neurotrophic factor ( BDNF ) . 
Two distinct ligand binding domains of TRKB were isolated from proteolytic digests of the receptor by affinity separation on immobilized BDNF . 
One of these domains consists of amino acid residues 103-181 and contains both the third leucine-rich motif and the second cysteine cluster domain . 
The second domain is close to the second immunoglobulin-like domain ( amino acid residues 342-394 ) . 
Each of these two domains can bind BDNF independently . 
Disulfide linkages present in the first domain are necessary for BDNF binding , probably because of preservation of the native conformation . 
To study the second domain in greater detail , a truncated form of TRKB containing the second immunoglobulin-like domain ( residues 248-398 ) was expressed in Escherichia coli . 
This domain was cross-linked to BDNF through a 1-ethyl-3- ( 3-dimethylaminopropyl ) carbodiimide coupling reaction . 
Several synthetic peptides corresponding to amino acid residues 343-379 were able to bind immobilized BDNF . 
Amino acid substitution and cross-linking analysis indicated that amino acids Phe347 , Asp354 , and Tyr361 are intimately involved in BDNF binding . 
These results , obtained from a variety of experimental techniques , highlight the importance of two distinct regions of the extracellular domain of the TRKB receptor in binding BDNF . 


A region of the beta subunit of the interferon alpha receptor different from box 1 interacts with Jak1 and is sufficient to activate the Jak-Stat pathway and induce an antiviral state . 
Coexpression of the alpha and betaL subunits of the human interferon alpha ( IFNalpha ) receptor is required for the induction of an antiviral state by human IFNalpha . 
To explore the role of the different domains of the betaL subunit in IFNalpha signaling , we coexpressed wild-type alpha subunit and truncated forms of the betaL chain in L-929 cells . 
Our results demonstrated that the first 82 amino acids ( AAs ) ( AAs 265-346 ) of the cytoplasmic domain of the betaL chain are sufficient to activate the Jak-Stat pathway and trigger an antiviral state after IFNalpha2 binding to the receptor . 
This region of the betaL chain , required for Jak1 binding and activation , contains the Box 1 motif that is important for the interaction of some cytokine receptors with Jak kinases . 
However , using glutathione S-transferase fusion proteins containing amino- and carboxyl-terminal deletions of the betaL cytoplasmic domain , we demonstrate that the main Jak1-binding region ( corresponding to AAs 300-346 on the beta subunit ) is distinct from the Box 1 domain ( AAs 287-295 ) . 


A c-Cbl yeast two hybrid screen reveals interactions with 14-3-3 isoforms and cytoskeletal components . 
The protein product of c-cbl proto-oncogene is known to interact with several proteins , including Grb2 , Crk and PI3 kinase , and is thought to regulate signalling by many cell surface receptors . 
The precise function of c-Cbl in these pathways is not clear , although a genetic analysis in Caenorhabditis elegans suggests that c-Cbl is a negative regulator of the epidermal growth factor receptor . 
Here we describe a yeast two hybrid screen performed with c-Cbl in an attempt to further elucidate its role in signal transduction . 
The screen identified interactions involving c-Cbl and two 14-3-3 isoforms , cytokeratin 18 , human unconventional myosin IC , and a recently identified SH3 domain containing protein , SH3 P17 . 
We have used the yeast two hybrid assay to localise regions of c-Cbl required for its interaction with each of the proteins . 
Interaction with 14-3-3 is demonstrated in mammalian cell extracts . 


Interaction between cell cycle regulator , E2F-1 , and NF-kappaB mediates repression of HIV-1 gene transcription . 
The NF-kappaB / Rel family of transcription factors is one of the main targets of cytokines and other agents that induce HIV-1 gene expression . 
Some of these extracellular stimuli arrest cells in the G1 phase of the mitotic division cycle and modulate the activity of the tumor suppressor protein Rb and its partner E2F-1 . 
Earlier studies indicated that E2F-1 , a transcription factor that stimulates expression of S-phase-specific genes , is able to repress transcription directed by the human immunodeficiency virus ( HIV-1 ) type-1 promoter in a variety of cells , including those of glial and lymphocytic origin . 
Here , we demonstrate that E2F-1 may regulate the activity of the HIV-1 long terminal repeat through its ability to bind sequences in the NF-kappaB enhancer region and to interact with the NF-kappaB subunit , p50 . 
Gel retardation and methylation interference assays show that E2F-1 is able to bind specifically to a site embedded within the two NF-kappaB elements . 
Gel retardation / immunoblot analysis using purified E2F-1 and p50 homodimers reveals the presence of complexes containing both proteins . 
Affinity chromatography and co-immunoprecipitation assays provide evidence for direct interaction of E2F-1 and p50 in the absence of their DNA target sequences . 
In vitro transcription assay demonstrates that E2F-1 represses NF-kappaB mediated transcription in a cell-free system . 
Functional studies in Jurkat T lymphocytic cells point to the importance of both the E2F and NF-kappaB binding sites in E2F-1 mediated repression of HIV-1 promoter , in vivo . 
The results of this study suggest that NF-kappaB activity may be regulated by its interaction with the cell cycle regulatory protein , E2F-1 . 


TAP- and tapasin-dependent HLA-E surface expression correlates with the binding of an MHC class I leader peptide . 
BACKGROUND : The human major histocompatibility complex ( MHC ) class lb molecule HLA-E is transcribed in most tissues but little is known about its localisation within the cell . 
We have recently shown that HLA-E binds signal-sequence-derived peptides from human MHC class I molecules in vitro . 
RESULTS : Using a newly characterised antibody recognising HLA-E , we show that HLA-E is expressed at the cell surface . 
We demonstrate that HLA-E surface expression is correlated with the presence of MHC class I molecules which provide suitable leader sequence peptides capable of binding to HLA-E . 
Further studies on the interaction of HLA-E with molecules in the endoplasmic reticulum revealed that HLA-E associates with the transporter associated with antigen processing ( TAP ) and calreticulin , and that HLA-E expression is TAP-dependent and tapasin-dependent . 
In addition , HLA-E dissociates from TAP upon binding of MHC class I leader sequence peptides . 
CONCLUSION : These experiments establish that surface expression of HLA-E is regulated by the binding of a restricted pool of peptides from the leader sequence of MHC class I molecules . 
The correlation between HLA-E and MHC class I surface expression might be relevant to the function of HLA-E . 
Our results also show that , although these HLA-E binding peptides are derived from signal sequences , they may be released back into the cytosol and subsequently translocated by the TAP complex and loaded onto HLA-E molecules . 


