Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The experiments presented herein were designed to identify members of the G protein-coupled receptor kinase (GRK) family that participate in the agonist-induced phosphorylation and internalization of the rat FSH receptor (rFSHR). Western blots of human kidney 293 cells (the cell line used in transfection experiments) and MSC-1 cells (a cell line derived from Sertoli cells that displays many of the differentiated functions of their normal counterparts) reveal the presence of GRK2 and GRK6 in both cell lines as well as GRK4 in MSC-1 cells. Cotransfection of 293 cells with the rFSHR and GRK2, GRK4alpha, or GRK6 resulted in an increase in the agonist-induced phosphorylation of the rFSHR. Cotransfections of the rFSHR with GRKs or arrestin-3 enhanced the agonist-induced internalization of the rFHSR, and combinations of GRKs and arrestin-3 were more effective than the individual components. To characterize the involvement of endogenous GRKs on phosphorylation and internalization, we inhibited endogenous GRK2 by overexpression of a kinase-deficient mutant of GRK2 or G alpha t, a scavenger of G betagamma. We also inhibited endogenous GRK6 by overexpression of a kinase-deficient mutant of GKR6. All three constructs were effective inhibitors of phosphorylation, but only the kinase-deficient mutant of GRK2 and G alpha t inhibited internalization. The inhibition of internalization induced by these two constructs was less pronounced than that induced by a dominant-negative mutant of the nonvisual arrrestins, however. The finding that inhibitors of GRK2 and GRK6 impair phosphorylation, but only the inhibitors of GRK2 impair internalization, suggests that different GRKs have differential effects on receptor internalization.
Mol Endocrinol 1999 Jun
PMID:Role of G protein-coupled receptor kinases on the agonist-induced phosphorylation and internalization of the follitropin receptor. 1037 86

G protein-coupled receptor kinases (GRKs) play a key role in the process of receptor homologous desensitization. In the present study, we address the question of whether a variety of receptors coupled to different G protein subtypes and naturally expressed on the same cell are selectively regulated by GRK2. The signaling stimulated by thyrotropin (TSH), alpha(1B)-adrenergic, and A(1) adenosine receptors was studied in FRTL-5 cells permanently transfected to overexpress GRK2 and GRK2-K220R, a kinase dead GRK dominant negative mutant. In FRTL-5 overexpressing GRK2, TSH-induced cyclic AMP response was attenuated, indicating that TSH receptor is desensitized by this kinase. Consistently, FRTL-5 cells overexpressing GRK2-K220R show increased TSH-induced cyclic AMP response, demonstrating that this receptor is under tonic control by GRK. Unlike TSH receptor, alpha(1B)-adrenergic receptor response was unaffected in FRTL-5 overexpressing GRK2 and GRK2-K220R. When A(1) adenosine receptors were stimulated, G(ialpha)-mediated cyclic AMP inhibition was totally unaffected by overexpression of either GRK2 or GRK2-K220R. By contrast, G(betagamma)-mediated response (activation of mitogen-activated protein kinases) was efficiently desensitized by GRK2 but was unaffected by GRK2-K220R overexpression. The present study documents that overexpression of GRK2 results in a selective regulation of different G protein-coupled receptors expressed on the same cell and that this kinase can regulate preferentially only one of the different pathways activated by the same receptor. The preferential regulation of the A(1) adenosine receptor-stimulated mitogen-activated protein kinases by GRK2 indicates that this kinase can have additional regulatory effects on G(betagamma)-stimulated pathways, possibly through direct binding and regulation of the receptor-G(betagamma) complex.
Mol Pharmacol 1999 Aug
PMID:Selective regulation of G protein-coupled receptor-mediated signaling by G protein-coupled receptor kinase 2 in FRTL-5 cells: analysis of thyrotropin, alpha(1B)-adrenergic, and A(1) adenosine receptor-mediated responses. 1041 50

FSH rapidly desensitizes the FSH-receptor (FSH-R) upon binding. Very little information is available concerning the regulatory proteins involved in this process. In the present study, we investigated whether G protein-coupled receptor kinases (GRKs) and arrestins have a role in FSH-R desensitization, using a mouse Ltk 7/12 cell line stably overexpressing the rat FSH-R as a model. We found that these cells, which express GRK2, GRK3, GRK5, and GRK6 as well as beta-arrestins 1 and 2 as detected by RT-PCR and by Western blotting, were rapidly desensitized in the presence of FSH. Overexpression of GRKs and/or beta-arrestins in Ltk 7/12 cells allowed us to demonstrate 1) that GRK2, -3, -5, -6a, and -6b inhibit the FSH-R-mediated signaling (from 71% to 96% of maximal inhibition depending on the kinase, P < 0.001); 2) that beta-arrestins 1 or 2 also decrease the FSH action when overexpressed (80% of maximal inhibition, P < 0.01) whereas dominant negative beta-arrestin 2 [319-418] potentiates it 8-fold (P < 0.001); 3) that beta-arrestins and GRKs (except GRK6a) exert additive inhibition on FSH-induced response; and 4) that FSH-R desensitization depends upon the endogenous expression of GRKs, since there is potentiation of the FSH response (2- to 3-fold, P < 0.05) with antisenses cDNAs for GRK2, -5, and -6, but not GRK3. Our results show that the desensitization of the FSH-induced response involves the GRK/arrestin system.
Mol Endocrinol 1999 Sep
PMID:Involvement of G protein-coupled receptor kinases and arrestins in desensitization to follicle-stimulating hormone action. 1047 49

The present studies were carried out to evaluate the mechanisms by which PTH/PTHrP receptor (PTHR) activation influences cell viability. In 293 cells expressing recombinant PTHRs, PTH treatment markedly reduced the number of viable cells. This effect was associated with a marked apoptotic response including DNA fragmentation and the appearance of apoptotic nuclei. Similar effects were evidenced in response to serum withdrawal or to the addition of tumor necrosis factor (TNFalpha). Addition of caspase inhibitors or overexpression of bcl-2 partially abrogated apoptosis induced by serum withdrawal. Caspase inhibitors also protected cells from PTH-induced apoptosis, but overexpression of bcl-2 did not. The effects of PTH on cell number and apoptosis were neither mimicked by activators of the cAMP pathway (forskolin, isoproterenol) nor blocked by an inhibitor (H-89). However, elevation of Ca(i)2+ by addition of thapsigargin induced rapid apoptosis, and suppression of Ca(i)2+ by overexpression of the calcium- binding protein, calbindin D28k, inhibited PTH-induced apoptosis. The protein kinase C inhibitor GF 109203X partially inhibited PTH-induced apoptosis. Regulator of G protein signaling 4 (RGS4) (an inhibitor of the activity of the alpha-subunit of Gq) suppressed apoptotic signaling by the PTHR, whereas the C-terminal fragment of GRK2 (an inhibitor of the activity of the beta(gamma)-subunits of G proteins) was without effect. Chemical mutagenesis allowed selection of a series of 293 cell lines resistant to the apoptotic actions of PTH; a subset of these were also resistant to TNFalpha. These results suggest that 1) apoptosis produced by PTHR and TNF receptor signaling involve converging pathways; and 2) Gq-mediated phospholipase C/Ca2+ signaling, rather than Gs-mediated cAMP signaling, is required for the apoptotic effects of PTHR activation.
Mol Endocrinol 2000 Feb
PMID:Apoptosis mediated by activation of the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein (PTHrP). 1067 97

We have previously shown that not only G protein-coupled receptor kinase (GRK) 2, but also a catalytically inactive Lys220Trp GRK2 decreases endothelin (ET)-1-induced inositol 1,4,5-trisphosphate (IP3) formation, and demonstrated the presence of phosphorylation-independent desensitization mechanism. To clarify the role of GRK2 other than that as a kinase, we characterized an RGS (regulator of G protein signaling)-like domain in the amino-terminus of GRK2. Both GRK2(1-181) and GRK2(54-174) suppressed Ca2+ responses induced by angiotensin II (Ang II) and ET-1, and bound directly with Galphaq but not Galphas nor Galphai3 in the presence of GDP and AlF4-. These results demonstrate that GRK2 regulates Gq-mediated signaling negatively by direct interaction between its RGS domain and the transitional state of Galphaq, as well as through phosphorylation of activated receptors by its kinase domain.
Int J Mol Med 2000 Apr
PMID:RGS domain in the amino-terminus of G protein-coupled receptor kinase 2 inhibits Gq-mediated signaling. 1071 47

Previously, we showed that the human kappa-opioid receptor (hkor) stably expressed in Chinese hamster ovary (CHO) cells underwent down-regulation after prolonged U50,488H treatment. In the present study, we determined the mechanisms underlying this process. U50, 488H caused a significant down-regulation of the hkor, although etorphine did not. Neither U50,488H nor etorphine caused down-regulation of the rat kappa-opioid receptor. Thus, similar to internalization, there are agonist and species differences in down-regulation of kappa-opioid receptors. Expression of the dominant negative mutants arrestin-2(319-418) or dynamin I-K44A significantly reduced U50,488H-induced down-regulation of the hkor. Coexpression of GRK2 or GRK2 and arrestin-2 permitted etorphine to induce down-regulation of the hkor, although expression of arrestin-2 or dynamin I alone did not. Expression of the dominant negative mutants rab5A-N133I or rab7-N125I blunted U50,488H-induced down-regulation. Pretreatment with lysosomal enzyme inhibitors [(2S, 3S)trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester or chloroquine] or proteasome inhibitors (proteasome inhibitor I, MG-132, or lactacystin) decreased the extent of U50,488H-induced down-regulation. A combination of chloroquine and proteasome inhibitor I abolished U50,488H-induced down-regulation. These results indicate that U50,488H-induced down-regulation of the hkor involves GRK-, arrestin-2-, dynamin-, rab5-, and rab7-dependent mechanisms and receptors seem to be trafficked to lysosomes and proteasomes for degradation. Thus, U50,488H-induced internalization and down-regulation of the hkor share initial common mechanisms. To the best of our knowledge, these results represent the first report on the involvement of both rab5 and rab7 in agonist-induced down-regulation of a G protein-coupled receptor. In addition, this study is among the first to show the involvement of proteasomes in agonist-induced down-regulation of a G protein-coupled receptor.
Mol Pharmacol 2000 Oct
PMID:Mechanisms of agonist-induced down-regulation of the human kappa-opioid receptor: internalization is required for down-regulation. 1099 50

Agonist-induced receptor phosphorylation is an initial step in opioid receptor desensitization, a molecular mechanism of opioid tolerance and dependence. Our previous research suggested that agonist-induced delta-opioid receptor (DOR) phosphorylation occurs at the receptor carboxyl terminal domain. The current study was carried out to identify the site of DOR phosphorylation during agonist stimulation and the kinases catalyzing this reaction. Truncation (Delta15) or substitutions (T358A, T361A, and S363G single or triple mutants) at the DOR cytoplasmic tail caused 80 to 100% loss of opioid-stimulated receptor phosphorylation, indicating that T358, T361, and S363 all contribute and are cooperatively involved in agonist-stimulated DOR phosphorylation. Coexpression of GRK2 strongly enhanced agonist-stimulated phosphorylation of the wild-type DOR (WT), but Delta15 or mutant DOR (T358A/T361A/S363G) failed to show any detectable phosphorylation under these conditions. These results demonstrate that T358, T361, and S363 are required for agonist-induced and GRK-mediated receptor phosphorylation. Agonist-induced receptor phosphorylation was severely impaired by substitution of either T358 or S363 with aspartic acid residue, but phosphorylation of the T361D mutant was comparable with that of WT. In the presence of exogenously expressed GRK2, phosphorylation levels of T358D and S363D mutants were approximately half of that of WT, whereas significant phosphorylation of the T358/S363 double-point mutant was not detected. These results indicate that both T358 and S363 residues at the DOR carboxyl terminus are capable of serving cooperatively as phosphate acceptor sites of GRK2 in vivo. Taken together, we have demonstrated that agonist-induced opioid receptor phosphorylation occurs exclusively at two phosphate acceptor sites (T358 and S363) of GRK2 at the DOR carboxyl terminus. These results represent the identification of the GRK phosphorylation site on an opioid receptor for the first time and demonstrate that GRK is the prominent kinase responsible for agonist-induced opioid receptor phosphorylation in vivo.
Mol Pharmacol 2000 Nov
PMID:Identification of G protein-coupled receptor kinase 2 phosphorylation sites responsible for agonist-stimulated delta-opioid receptor phosphorylation. 1104 53

Centrosomes maintain genomic stability by establishing the bipolar spindles during cell division and, execute accurate segregation of chromosomes during mitosis. In this study, we have demonstrated that there are three forms of STK-15 gene in breast cancer cell lines. Alternative splice positions are located in 5'-untranslated region of STK15 gene. The results of in vitro translation experiments revealed that the alternative splicing in the 5'-untranslated region of STK15 had no effect on protein translation. The differential expression patterns of these alternatively spliced STK15 in breast cell lines and primary tumors therefore suggest that STK15 gene transcription may be differentially regulated or stabilized in these cells.
Exp Mol Med 2000 Dec 31
PMID:Alternative splicing in 5'-untranslational region of STK-15 gene, encoding centrosome associated kinase, in breast cancer cell lines. 1119 Feb 69

The beta-adrenoceptor (beta-AR) mediated signal transduction pathway in cardiomyocytes is known to involve beta1- and beta2-ARs, stimulatory (Gs) and inhibitory (Gi) guanine nucleotide binding proteins, adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA). The activation of beta1- and beta2-ARs has been shown to increase heart function by increasing Ca2+ -movements across the sarcolemmal membrane and sarcoplasmic reticulum through the stimulation of Gs-proteins, activation of AC and PKA enzymes and phosphorylation of the target sites. The activation of PKA has also been reported to increase phosphorylation of some myofibrillar proteins (for promoting cardiac relaxation) and nuclear proteins (for cardiac hypertrophy). The activation of beta2-AR has also been shown to affect Gi-proteins, stimulate mitogen activated protein kinase and increase protein synthesis by enhancing gene expression. Beta1- and beta2-ARs as well as AC are considered to be regulated by PKA- and protein kinase C (PKC)-mediated phosphorylations directly; both PKA and PKC also regulate beta-AR indirectly through the involvement of beta-AR kinase (betaARK), beta-arrestins and Gbeta gamma-protein subunits. Genetic manipulation of different components and regulators of beta-AR signal transduction pathway by employing transgenic and knockout mouse models has provided insight into their functional and regulatory characteristics in cardiomyocytes. The genetic studies have also helped in understanding the pathophysiological role of PARK in heart dysfunction and therapeutic role of betaARK inhibitors in the treatment of heart failure. Varying degrees of defects in the beta-AR signal transduction system have been identified in different types of heart failure to explain the attenuated response of the failing heart to sympathetic stimulation or catecholamine infusion. A decrease in beta1-AR density, an increase in the level of G1-proteins and overexpression of betaARK are usually associated with heart failure; however, these attenuations have been shown to be dependent upon the type and stage of heart failure as well as region of the heart. Both local and circulating renin-angiotensin systems, sympathetic nervous system and endothelial cell function appears to regulate the status of beta-AR signal transduction pathway in the failing heart. Thus different components and regulators of the beta-AR signal transduction pathway appears to represent important targets for the development of therapeutic interventions for the treatment of heart failure.
Mol Cell Biochem 2000 Nov
PMID:Modification of beta-adrenoceptor signal transduction pathway by genetic manipulation and heart failure. 1119 84

Traditional mechanisms thought to underlie opioid tolerance include receptor phosphorylation/down-regulation, G-protein uncoupling, and adenylyl cyclase superactivation. A parallel line of investigation also indicates that opioid tolerance development results from a switch from predominantly opioid receptor G(i alpha) inhibitory to G(beta gamma) stimulatory signaling. As described previously, this results, in part, from the increased relative abundance of G(beta gamma)-stimulated adenylyl cyclase isoforms as well as from a profound increase in their phosphorylation [Chakrabarti, S., Rivera, M., Yan, S.-Z., Tang, W.-J. & Gintzler, A. R. (1998) Mol. Pharmacol. 54, 655-662; Chakrabarti, S., Wang, L., Tang, W.-J. & Gintzler, A. R. (1998) Mol. Pharmacol. 54, 949--953]. The present study demonstrates that chronic morphine administration results in the concomitant phosphorylation of three key signaling proteins, G protein receptor kinase (GRK) 2/3, beta-arrestin, and G(beta), in the guinea pig longitudinal muscle myenteric plexus tissue. Augmented phosphorylation of all three proteins is evident in immunoprecipitate obtained by using either anti-GRK2/3 or G(beta) antibodies, but the phosphorylation increment is greater in immunoprecipitate obtained with G(beta) antibodies. Analyses of coimmunoprecipitated proteins indicate that phosphorylation of GRK2/3, beta-arrestin, and G(beta) has varying consequences on their ability to associate. As a result, increased availability of and signaling via G(beta gamma) could occur without compromising the membrane content (and presumably activity) of GRK2/3. Induction of the concomitant phosphorylation of multiple proteins in a multimolecular complex with attendant modulation of their association represents a novel mechanism for increasing G(beta gamma) signaling and opioid tolerance formation.
 ...
PMID:Chronic morphine induces the concomitant phosphorylation and altered association of multiple signaling proteins: a novel mechanism for modulating cell signaling. 1127 43


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>