Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Htk is a receptor protein-tyrosine kinase that is related to the EPH subfamily of tyrosine kinases. The receptor has a wide tissue distribution including expression in several myeloid hematopoietic cell lines. Using an Htk-Fc fusion protein, a protein ligand for this receptor was expression cloned from the murine kidney mesangial cell line SV40MES 13. The Htk ligand cDNA encodes a transmembrane protein of 336 amino acids. Binding competition experiments demonstrated a Kd of 535 pM for binding of Htk-Fc to the Htk ligand. Incubation of 3T3 cells expressing Htk with COS-7 cells expressing the ligand resulted in tyrosine phosphorylation of Htk. The ligand, like its receptor, is widely expressed and may function in a variety of tissues. However, we localized hematopoietic expression of Htk to the monocytic lineage, suggesting that the ligand may play a role in differentiation and/or proliferation of these cells.
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PMID:Molecular cloning of a ligand for the EPH-related receptor protein-tyrosine kinase Htk. 753 4

"Sky" is a putative receptor tyrosine kinase predominantly expressed in the brain. Sky, like Axl/Ufo/Ark and c-Eyk, has an extracellular domain composed of two immunoglobulin-like domains and two fibronectin type III domains. Immunoblot analysis using an antibody raised against a C-terminal peptide of Sky identified a 98-kDa Sky protein in COS cells transfected with sky cDNA (COS/sky cells). A 98-kDa protein in the immunoprecipitates with anti-Sky antibody was autophosphorylated on tyrosine, by in vitro kinase reaction. When the lysates of COS/sky cells were immunoprecipitated with anti-Sky antibody and immunoblotted with an anti-phosphotyrosine antibody, a 60-kDa phosphotyrosine-containing protein, in addition to the tyrosine-phosphorylated Sky, was detected. Using the anti-Src antibody, which is reactive to Src, Fyn and Yes, we obtained evidence for an association between the Src family tyrosine kinase and the tyrosine-phosphorylated Sky receptor. These results suggest that the Src family kinase may play an important role in signal transduction of the Sky receptor.
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PMID:Autophosphorylation activity and association with Src family kinase of Sky receptor tyrosine kinase. 753 95

The binding of [3H]SR 48692, a new potent and specific nonpeptide neurotensin (NT) receptor antagonist, was characterized in membranes from mouse fibroblast LTK- cells stably transfected with the G protein-coupled rat NT receptor. The binding of [3H]SR 48692 was specific, time dependent, reversible, and saturable. Scatchard analysis of saturation experiments indicated that [3H]SR 48692 bound to a single population of sites, with a Kd of 3.4 nM and a Bmax value that was 30-40% greater than that observed in saturation experiments with [125I]NT. Two SR 48692-related enantiomers, SR 48527 and SR 49711, were 10 and 1000 times less potent, respectively, than unlabeled SR 48692 in inhibiting [3H]SR 48692. Unlabeled NT inhibited [3H]SR 48692 binding in a complex manner that was best analyzed with a three-site model, with high (Ki = 0.22 nM) and low (Ki = 57 nM) affinity NT binding sites and a site insensitive to unlabeled NT (up to 10 microM), which represented 60, 20, and 20%, respectively, of the total number of [3h]SR 48692 binding sites. Digitonin (10 micrograms/ml) markedly reduced the proportion of NT-insensitive sites without affecting [3H]SR 48692 binding. Na+ and guanosine-5'-(gamma-thio)triphosphate differentially modulated [3H]SR 48692 and [125I]NT binding and inverted the proportions of the high and low affinity NT binding sites. A mutant rat NT receptor that contained a deletion in a region (amino acids 45-60) of the amino-terminal extracellular domain near the first transmembrane helix and was expressed in COS M6 cells retained the same affinity for [3H]SR 48692 and the same stereoselectivity for SR 48527 and SR 49711 as the wild-type receptor. In contrast, it bound NT with 3000-fold lower potency. In conclusion, the data indicate that [3H]SR 48692 represents a new, potent, nonpeptide antagonist radioligand of the NT receptor that differentiates between agonist- and antagonist-receptor interactions. Furthermore, the data demonstrate that the peptide agonist and the nonpeptide antagonist bind to distinct regions of the NT receptor.
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PMID:[3H]SR 48692, the first nonpeptide neurotensin antagonist radioligand: characterization of binding properties and evidence for distinct agonist and antagonist binding domains on the rat neurotensin receptor. 774 72

During short term agonist exposure, the alpha 2A-adrenergic receptor (alpha 2AAR) undergoes rapid functional desensitization caused by phosphorylation of the receptor by the beta-adrenergic receptor kinase (beta ARK). This signal quenching is similar in nature to that found with a number of G-protein coupled receptors in which agonist-promoted desensitization is due to beta ARK phosphorylation; like these other receptors, the precise molecular determinants of the receptor required for beta ARK phosphorylation are not known. To delineate such a motif in the human alpha 2AAR (alpha 2C10), we constructed six mutated receptors consisting of deletions or substitutions of Ser-296-299 in the EESSSS sequence of the third intracellular loop of the receptor. These were expressed in Chinese hamster ovary and COS-7 cells, and agonist-promoted desensitization and receptor phosphorylation were assessed. Deletion of the EESSSS sequence and substitution of alanine for all four serines resulted in a total loss of phosphorylation and desensitization. Mutant receptors that retained two of the original serines (AASS and SSAA) underwent agonist-promoted phosphorylation of 55 +/- 7% and 57 +/- 8% of the phosphorylation found for wild type alpha 2C10. Additional substitution mutants (SSSA and SAAA) underwent 77 +/- 1% and 27 +/- 4% of wild type phosphorylation, respectively. Thus, substitution of alanine for each additional serine decreased overall phosphorylation as compared with wild type alpha 2C10 by approximately 25%, which is consistent with all 4 serines being phosphorylated. Mutated receptors that only partially phosphorylated (as compared with wild type) failed to undergo agonist-promoted desensitization. Thus, beta ARK-mediated phosphorylation of alpha 2C10 occurs at Ser-296-299 in the third intracellular loop, and this represents the critical step in rapid agonist-promoted desensitization. A number of other G-protein coupled receptors that undergo desensitization have a sequence motif similar to that which we have found for beta ARK-mediated phosphorylation of alpha 2C10, suggesting that these receptors may also be substrates for beta ARK.
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PMID:Four consecutive serines in the third intracellular loop are the sites for beta-adrenergic receptor kinase-mediated phosphorylation and desensitization of the alpha 2A-adrenergic receptor. 787 39

We demonstrate that purified fibroblast growth factor (FGF) 3 from Xenopus laevis (XFGF3) activates the mitogen-activated protein kinase pathway and induces DNA synthesis in quiescent cells. To characterize the high affinity cell surface receptors that mediate these responses, the ligand binding domains of different FGF receptors (FGFR) were expressed on COS-1 cells, and their affinity for XFGF3 was determined. Unlabeled XFGF3 efficiently competed with 125I-FGF1 for binding to the IIIb and IIIc isoforms of FGFR2, giving 50% displacement (ID50) at 0.3-0.8 nM. Higher XFGF3 concentrations were needed to displace 125I-FGF1 from FGFR3 and FGFR1 (ID50 approximately 4 and 21 nM, respectively), indicating that XFGF3 has a lower affinity for these receptors. No association of XFGF3 with FGFR4 was found using this assay. FGFR2 isoforms isolated from both mouse and Xenopus showed similar high affinity binding of XFGF3 as determined by direct binding assays (Kd values in the range of 0.2-0.6 nM). These results indicate that the binding specificity of XFGF3 is different from that of other FGFs, and identifies FGFR2 as its high affinity receptor.
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PMID:Fibroblast growth factor (FGF) 3 from Xenopus laevis (XFGF3) binds with high affinity to FGF receptor 2. 789 24

Although a role for the beta gamma-subunits of heterotrimeric G proteins (G beta gamma) in signal transduction by several cellular systems has been established, the structural features of cellular proteins interacting with G beta gamma have yet to be fully elucidated. The G beta gamma-binding region of beta-adrenergic receptor kinase (beta ARK), a cytosolic enzyme recruited to the membrane receptor substrate by G beta gamma, has been localized to the carboxyl terminus of the enzyme. Here, we demonstrate that the amino terminus of phosducin, a 33-kDa G beta gamma-binding retinal phosphoprotein, contains sequences homologous with the G beta gamma-binding domain of beta ARK. Accordingly, a glutathione S-transferase-fusion protein containing only the amino-terminal 105 amino acids of phosducin displayed G beta gamma binding ability. This domain of phosducin contains a protein kinase A (PKA) phosphorylation site, and upon phosphorylation, the binding of full-length phosducin to G beta gamma is reduced. In addition, transient expression of phosducin in COS-7 cells significantly inhibits G beta gamma-mediated phosphoinositide hydrolysis. This inhibitory effect is completely reversed by pretreatment of cells with dibutyryl cAMP, an activator of PKA. Thus, the binding of G beta gamma to phosducin can be regulated by PKA-phosphorylation in an intact cell model system.
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PMID:Determination of the G beta gamma-binding domain of phosducin. A regulatable modulator of G beta gamma signaling. 796 75

We have expressed in COS-1 cells mutants of neprilysin (neutral endopeptidase-24.11; NEP) in which the hydrophilic sequence S-Q-N-S was either substituted for V42-T-M-I or inserted after T38 in the signal peptide/membrane anchor (SA) domain. These mutations were introduced in full-length NEP (mutants NEP(H1) and NEP(H2), respectively) and a form of NEP lacking its cytosolic tail (mutants NEP delta cyto(H1) and NEP delta cyto(H2), respectively). Immunoblotting showed that NEP(H1) was membrane-bound while NEP delta cyto(H1), NEP(H2), and NEP delta cyto(H2) were secreted. Furthermore, carbonate treatment of isolated intracellular membranes suggested that cleavage of the SA domain was performed in the endoplasmic reticulum, presumably by signal peptidase. Sequencing of the secreted proteins indicated that cleavage of the SA domain mostly occurred at the carboxy side of Ala46 but also at the carboxy side of Ala41 in NEP(H2) and NEP delta cyto(H2). We conclude that the position of the S-Q-N-S sequence influences the accessibility of the cleavage site and, in the case of NEP(H1) and NEP(H2), the efficiency of cleavage of the SA domain.
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PMID:Insertion of hydrophilic amino acid residues in the signal peptide/membrane anchor domain of neprilysin (neutral endopeptidase-24.11) results in its cleavage: role of the position of insertion. 798 81

Six tyrosine residues (Y28, Y143, Y292, Y293, Y308, Y432(1)) which are conserved in all mammalian glucose transporters were substituted for phenylalanine by site-directed mutagenesis, and mutant glucose transporters were transiently expressed in COS-7 cells. Glucose transport activity as assessed by reconstitution of the solubilized transporters into lecithin liposomes was reduced by 70% in the mutant Y143F and appeared to be abolished in Y293F, but was not affected by substitution of Y28, Y292, Y308 and Y432. In contrast, covalent binding of the photolabel 125IAPS-forskolin was normal in all mutants. Stable expression of the mutants Y143F, Y293F, and Y292F in LTK cells yielded identical results. These data indicate that only two of the 6 conserved helical tyrosine residues, located in helices 4 and 7, are essential for full activity, but not for IAPS-forskolin binding of the GLUT4.
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PMID:Substitution of conserved tyrosine residues in helix 4 (Y143) and 7 (Y293) affects the activity, but not IAPS-forskolin binding, of the glucose transporter GLUT4. 803 25

Neutral endopeptidase (NEP, EC 3.4.24.11) is a major ectoenzyme of the brush-border membrane. The ectodomain of NEP contains five putative N-glycosylation sites. In order to determine the role of the addition of sugar moieties on the activity and intracellular transport of NEP, we have used site-directed mutagenesis to remove all or some of the five potential sites of sugar addition in membrane-bound and secreted forms of the enzyme. Expression of NEP glycosylation mutants in COS-1 cells showed that all five sites are used for sugar addition. Immunoblotting of NEP in COS-1 cell extracts or culture media indicated that total expression of normal membrane-bound NEP was not affected by mutations at glycosylation sites, whereas this expression level appeared to be strictly dependent on the number of glycosylation sites retained on the soluble form. The transport to the cell surface was also reduced by decreased glycosylation, but again the phenomenon appeared more drastic in the case of the soluble form than for the membrane-bound enzyme. Enzyme activity was decreased by deglycosylation. However, the presence of either of two crucial sites (sites 1 and 5; numbered from the N-terminus of the protein) was sufficient to recover close-to-normal enzymic activities. Transport to the cell surface and enzyme activity of NEP are thus both dependent on sugar residues, probably through different conformational constraints. These constraints seem to be local for enzyme activity but more global for transport to the cell surface.
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PMID:Role of glycosylation in transport and enzymic activity of neutral endopeptidase-24.11. 809 97

Cloned sequences encoding a truncated form of the HER2 receptor were obtained from cDNA libraries derived from two HER2-overexpressing human breast cancer cell lines, BT-474 and SK-BR-3. The 5' 2.1 kb of the encoded transcript is identical to that of full-length 4.6-kb HER2 transcript and would be expected to produce a secreted form of HER2 receptor containing only the extracellular ligand binding domain (ECD). The 3' end of the truncated transcript diverges 61 nucleotides before the receptor's transmembrane region, reads through a consensus splice donor site containing an in-frame stop codon, and contains a poly(A) addition site, suggesting that the truncated transcript arises by alternative RNA processing. S1 nuclease protection assays show a 40-fold variation in the abundance of the truncated 2.3-kb transcript relative to full-length 4.6-kb transcript in a panel of eight HER2-expressing tumor cell lines of gastric, ovarian, and breast cancer origin. Expression of this truncated transcript in COS-1 cells produces both secreted and intracellular forms of HER2 ECD; however, immunofluorescent labeling of HER2 ECD protein in MKN7 tumor cells that natively overexpress the 2.3-kb transcript suggests that transcriptionally generated HER2 ECD is concentrated within the perinuclear cytoplasm. Metabolic labeling and endoglycosidase studies suggest that this HER2 ECD (100 kDa) undergoes differential trafficking between the endoplasmic reticulum and Golgi compartments compared with full-length (185-kDa) HER2 receptor. Transfection studies indicate that excess production of HER2 ECD in human tumor cells overexpressing full-length HER2 receptor can result in resistance to the growth-inhibiting effects of anti-HER2 monoclonal antibodies such as muMAb4D5. These findings demonstrate alternative processing of the HER2 transcript and implicate a potentially important growth regulatory role for intracellularly sequestered HER2 ECD in HER2-amplified human tumors.
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PMID:A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells. 809 58


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