UNIPROT:P01185 - FACTA Search

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Query: UNIPROT:P01185 (vasopressin)
23,126 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The potential roles of protein tyrosine kinases (TKs) and of phosphotyrosine phosphatases (PTPs) in pancreatic islet function are not known. In this study, we investigated whether vanadate, a potent PTP inhibitor, affects phosphoinositide (PI) metabolism by a TK-dependent pathway in isolated mouse islets. To avoid the confounding effects of changes in Ca2+ influx, all experiments were performed in the absence of Ca2+. In the presence of 15mM glucose, vanadate, acetylcholine (ACh) or [Arg]vasopressin (AVP) strongly stimulated InsP production. Vanadate also increased PtdInsP levels in membranes. The TK inhibitor genistein (not its inactive analogues genistin and daidzein) significantly reduced vanadate effects, but was without effect in the absence of stimulation or in the presence of ACh or AVP. Islet proteins resolved by SDS/PAGE were analysed by immunobloting with anti-phosphotyrosine antibody. Under control conditions, several phosphotyrosyl-proteins (PYPs) were present. Vanadate increased phosphotyrosine residues on several PYPs, notably two proteins of 145 and 85 kDa. This effect was prevented by genistein, p145 and p85 could correspond to phospholipate Cgamma(PLCgamma) and the regulatory subunit of PtdIns-3-kinase (PtdIns-3K) respectively. Both proteins are expressed in islets, as revealed by immunoblots with specific antibodies. Tungstate, another PTP inhibitor, reproduced vanadate effects, but inhibition of PtdIns-3K by wortmannin failed to affect vanadate-increased PtdInsP levels. Incubation of the islets in the presence of 10% (v/v) fetal calf serum instead of BSA increased InsP production and this effect was prevented by genistein. These results suggest that inhibition of PTP increases InsP production in mouse islets by a TK-dependent pathway. They also provide evidence for a potential role of TK and PTP in pancreatic B-cell function.
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PMID:Possible involvement of a tyrosine kinase-dependent pathway in the regulation of phosphoinositide metabolism by vanadate in normal mouse islets. 867 Jan 31

We report here a study of photoaffinity labeling of the V1a-vasopressin receptor with high-affinity, V1-specific radioiodinated antagonist ligands: one containing an azidophenylalanine residue ([beta,beta-dimethyl-beta-mercaptopropionyl(1), p-azido-Phe2,Val4,Lys8,D-Tyr9] vasopressin), two others containing nitrophenylalanine, and one, highly similar but without a photosensitive function, as control. All analogues competed in the dark for the same binding site with vasopressin. Long-wavelength UV irradiation of rat liver membranes incubated in presence of the radio-iodinated azido photolabel produced a specifically labeled protein band at 53 kDa in SDS-PAGE. Identical experiments with the nitrophenylalanyl peptides produced only non-specific labeling and control experiments with the non-photosensitive analogue produced no labeling at all. Chemical crosslinking of 3H-VP to the same membrane preparation produced a result identical to that of the azido photolabel, confirming the receptor nature of the labeled protein. Deglycosylation of the labeled receptor with endoglycosidase F reduced the observed molecular weight of 53 kDa to 43 kDa. The molecular parameters reported herein of the presumed hepatic vasopressin receptor confirm the values deduced from the molecular cloning of the rat V1a receptor.
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PMID:Molecular weight determination of the hepatic vasopressin receptor with a high-affinity photoprobe. 891 57

Transient and stable expression in eukaryotic cells is commonly used to examine receptor function. Characterization of the V2 vasopressin receptor synthesized in transiently transfected cells revealed the presence of large quantities of immature protein and a small fraction of fully mature protein. The immature protein was characterized by its sensitivity to endoglycosidase H treatment, abnormal migration in SDS PAGE, and a tendency to form aggregates. Prevention of protein glycosylation by mutagenesis increased the fraction of mature protein produced, but did not eliminate the need for the maturation step. On the other hand, stably transfected cells produce almost exclusively mature receptor protein with a t1/2 of 6 h, while the immature form has a t1/2 of 20 min. In the absence of N-linked glycosylation the t1/2 of the mature V2 receptor in stably transfected cells was reduced to 4.5 h. In transient expression experiments the immature receptor proteins exhibited a prolonged t1/2 of about 8 h. Comparison of the half life of the immature form of the wild type and the R137H mutant V2 receptor did not reveal differences despite the lower amounts of mutant mature receptor detected by binding.
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PMID:Maturation of receptor proteins in eukaryotic expression systems. 902 6

Function and biochemical properties of the V2 vasopressin receptor (V2R) mutant R337ter, identified in patients suffering from X-linked recessive nephrogenic diabetes insipidus, were investigated by expression in COS.M6 or HEK293 cells. Binding assays and measurements of adenylyl cyclase activity failed to detect function for the truncated receptor, although metabolic labeling demonstrated normal levels of protein synthesis. ELISA assays performed on cells expressing the receptors tagged at the amino terminus with the HA epitope failed to detect V2R R337ter on the plasma membrane. Treatment with endoglycosidase H revealed that the receptor was present only as a precursor form because the mature R337ter V2R, resistant to endoglycosidase H treatment, was not detected. The precursor of V2R-R337ter had a longer half-life than that of the wild type V2R, suggesting that arrested maturation may slow the degradation of the precursor. Unrelated experiments had demonstrated that V2R-G345ter, containing eight additional amino acids, was expressed on the plasma membrane and functioned normally. Receptor truncations longer than 337ter revealed that four of the eight amino acids identified initially provided the minimum length required for the protein to acquire cell surface expression. This was shown by the production of mature receptor (V2R-341ter) detectable in SDS-PAGE, which mediated arginine vasopressin stimulation of adenylyl cyclase activity and bound ligand. In addition, the identity of amino acid 340 was found to play a role in this phenomenon. In conclusion, these data demonstrate that the V2R R337ter is nonfunctional because it does not reach the plasma membrane and that the minimal protein length required for translocation of the V2R to the cell surface is sufficient to confer function to the receptor protein. They also suggest the existence of a protein quality control in the endoplasmic reticulum independent of glycosylation.
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PMID:An X-linked NDI mutation reveals a requirement for cell surface V2R expression. 917 Dec 34

We have synthesized and fully characterized by fast-atom-bombardment-mass, NMR and ultraviolet spectroscopies the vasopressin antagonist 3-azidophenylpropionyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr(3I )-NH2. Easily radioiodinatable just before use, it has a high affinity for the natural rat liver V1a receptor [dissociation constant (Kd) = 54 +/- 20 pM; Carnazzi, E., Aumelas, A., Barberis, C., Guillon, G. & Seyer, R. (1994) J. Med. Chem. 37, 1841-1849] and for both the rat vasopressin V1a receptor expressed in Spodoptera frugiperda 9 cells (Sf9 cells, Kd = 688 +/- 35 pM) and in COS-7 cells (Kd = 320 +/- 20 pM). This probe labels specifically the V1a receptors in an ultraviolet-dependent manner, and binds covalently to about 12% of the receptors with high stability over several days, even in dissociation or solubilization conditions. SDS/PAGE studies and autoradiographic analyses of the photolabeled receptors reveal a single band (49.5 kDa) and two bands (63 kDa and 93.6 kDa) for receptor-probe associations obtained in Sf9 and COS-7 cells respectively. These molecular masses are consistent with non-glycosylated and highly glycosylated forms of the receptor, according to each expression system. In rat liver membranes, we have identified apparent molecular masses of about 32, 45 and more than 67 kDa. We finally demonstrated a proteolysis of the receptor that appeared to be Zn2+ and leupeptin sensitive. The high potency of this ligand is promising for the monitoring of the purification of the V1a receptor and for mapping its antagonist-binding site.
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PMID:Efficient photoaffinity labeling of the rat V1a vasopressin receptor using a linear azidopeptidic antagonist. 928 14

Melanin concentrating hormone (MCH) is a cyclic peptide which regulates a broad array of functions in the mammalian brain and it may act as a paracrine factor in peripheral organs. In these studies a radiolabeled MCH derivative, the [125I]-[Phe13, Tyr19]-MCH, was synthesized and used as a tracer to perform binding experiments. A number of human or rodent cell lines displayed specific binding with [125I]-[Phe13, Tyr19]-MCH, the highest binding capacity being observed with human SVK14 keratinocytes. Saturation binding analysis with SVK14 cells indicated about 10,000 MCH binding sites per cell and a Kd of 0.7 nM for [125I]-[Phe13, Tyr19]-MCH. Surprisingly, the iodinated [Phe13, Tyr19]-MCH displayed about 10-fold higher affinity (Ki approximately 3.0 nM) for the putative MCH receptor than the noniodinated form (Ki approximately 25-30 nM). Competition binding analyses comparing various MCH-related peptides revealed a similar low binding potency for all these peptides (Ki approximately 65-160 nM). Strikingly, rat ANP and rat/human CNP but not rat BNP displaced [125I]-[Phe13, Tyr15]-MCH with Ki approximately 210-365 nM and may be due to topological similarities instead of partial sequence identities between MCH and some of the natriuretic peptides. However, other peptides such as CRF, alpha MSH, Arg-vasopressin, and MGOP-peptide I did not compete with the radioligand. Finally, the molecular mass of the MCH binding sites on SVK14 cells was estimated to be 47 kDa by crosslinking and SDS-PAGE experiments. Taken together, our data revealed the widespread expression of MCH binding sites on mammalian cells, particularly on skin carcinoma cells. However, the low affinity of these sites for the native MCH and MCH-related peptides as well as competitivity with ANP and CNP indicates that further biochemical and functional characterizations are needed to validate them as genuine physiological MCH receptors.
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PMID:Melanin-concentrating hormone binding sites in human SVK14 keratinocytes. 943 58

Aquaporin-2 (AQP2), the protein that mediates arginine vasopressin (AVP)-regulated apical water transport in the renal collecting duct, possesses a single consensus phosphorylation site for cAMP-dependent protein kinase A (PKA) at Ser256. The aim of this study was to examine whether AVP, and other agents that increase cAMP levels, could stimulate the phosphorylation of AQP2 in intact rat renal tissue. Rat renal papillae were prelabeled with 32P and incubated with vehicle or drugs, and then AQP2 was immunoprecipitated. Two polypeptides corresponding to nonglycosylated (29 kDa) and glycosylated (35-48 kDa) AQP2 were identified by SDS-PAGE. AVP caused a time- and dose-dependent increase in phosphorylation of both glycosylated and nonglycosylated AQP2. The threshold dose for a significant increase in phosphorylation was 10 pM, which corresponds to a physiological serum concentration of AVP. Maximal phosphorylation was reached within 1 min of AVP incubation. This effect on AQP2 phosphorylation was mimicked by the vasopressin (V2) agonist, 1-desamino-[8-D-arginine]vasopressin (DDAVP), or forskolin. Two-dimensional phosphopeptide mapping indicated that AVP and forskolin stimulated the phosphorylation of the same site in AQP2. Immunoblot analysis using a phosphorylation state-specific antiserum revealed an increase in phosphorylation of Ser256 after incubation of papillae with AVP. The results indicate that AVP stimulates phosphorylation of AQP2 at Ser256 via activation of PKA, supporting the idea that this is one of the first steps leading to increased water permeability in collecting duct cells.
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PMID:Arginine vasopressin stimulates phosphorylation of aquaporin-2 in rat renal tissue. 995 Sep 56

Synthesis, processing and agonist-induced modifications of the V2 vasopressin receptor were examined in stably or transiently transfected HEK293 cells. Metabolic labeling with S methionine for 30 min revealed a predominant precursor protein which subsequently gave rise to the mature receptor on the cell surface. Maturation of the receptor was unrelated to glycosylation suggesting that it was the consequence of protein refolding. In addition to monomeric forms of V2 receptor protein, oligomers of the precursor protein were also detected in SDS-PAGE. These oligomers seemed to be dimers and tetrameres, and were more apparent in transiently transfected cells that produced higher quantities of protein then stably transfected cells. No oligomers of the mature receptor were detected, and co-transfection of the wild type with a mutant V2 receptor lacking G-protein coupling activity did not alter the function of the wild type receptor. These results indicated that the formation of oligomeric was most likely a consequence of overproduction of the protein and not a required step for receptor function. Addition of vasopressin promoted phosphorylation and sequestration of the wild type receptor, and of the R137H mutant receptor which lacks coupling to G proteins. Activation of protein kinases A or C did not result in phosphorylation of un-occupied receptor. Phosphate incorporated into the protein was stable in the continuous presence of the ligand despite sequestration of the receptor protein. Deletion of the last 14 amino acids abolished receptor phosphorylation but not sequestration and desensitization, indicating that these two processes are not dependent on protein phosphorylation. Additionally, phosphorylation and sequestration of the R137H mutant receptor revealed that phosphorylation and sequestration does not require coupling to Gs. The wild type V2 vasopressin receptor was found to be palmitoylated at two cysteines at the carboxyl terminus. Either cysteine could be palmitoylated independently of each other and the presence of at least one was required to obtain receptor expression similar to the wild type. The turnover of the palmitic acid incorporated into the receptor was not altered by the addition of vasopressin demonstrating that this post-translational modification of the receptor was not altered by the ligand-promoted phosphorylation of the protein.
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PMID:Processing and ligand-induced modifications of the V2 vasopressin receptor. 1002 23

NMDA receptor activation produces a characteristic pattern of neuronal firing in magnocellular neuroendocrine cells (MNCs) of the supraoptic nucleus of the hypothalamus (SON) which has been associated with greater hormone release in vivo and in vitro. In addition, i.c.v. administered NMDA receptor blockers suppress the dehydration-induced rise in plasma vasopressin and drinking. To investigate the role of NMDA receptor subunits in the neuroendocrine functions of the magnocellular neuroendocrine cells of the hypothalamus, we examined the effects of osmotic stimulation on the protein expression of the NMDA receptor subunits, NR1 and NR2B, important in binding glycine and glutamate, respectively. Homogenates of SON, paraventricular nucleus of the hypothalamus (PVN), cortex and lateral hypothalamus from control rats and rats given 2% saline water to drink for 4-10 days were subjected to SDS-PAGE and Western blot analysis. This saline water drinking regimen produced a significant rise in plasma osmolality levels. NR1 and NR2B immunoreactivity was detected in SON, PVN, lateral hypothalamus and cortex but not in liver homogenates using subunit-specific polyclonal antibodies and quantified using computer-assisted densitometry. Mean NR2B immunoreactivity was significantly lower in SON (29%) and PVN homogenates (23%) from saline-treated rats than in those from control rats. In addition, the effect of dehydration on NR2B was regionally specific since no significant changes in NR2B expression were observed in homogenates of cortex and lateral hypothalamus. Rehydration allowed recovery of plasma osmolality as well as NR2B protein levels in the SON. These results suggest that changes in NMDA receptor subunit expression contribute to the plasticity manifested by in magnocellular neuroendocrine cells in response to osmotic activation of the hypothalamo-neurohypophysial system. In addition, our results indicate that NMDA receptors on SON and PVN MNCs may contribute to neuroendocrinological functions associated with body fluid homeostasis.
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PMID:Osmotic activation of the hypothalamo-neurohypophysial system reversibly downregulates the NMDA receptor subunit, NR2B, in the supraoptic nucleus of the hypothalamus. 1040 67

A Porphyromonas endodontalis ATCC 35406 protease was purified from Triton X-114 cell extracts by preparative SDS-PAGE followed by electroelution. The purified enzyme exhibits a molecular size of 88 kDa and was dissociated into two polypeptides of 43 and 41 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The protease (pH optimum 7.5-8.0) degraded the extracellular matrix proteins fibrinogen and fibronectin. Collagen IV was also degraded at 37 degrees C but not at 28 degrees C. The protease also cleaved the bioactive peptide angiotensin at amino acid residue phenylalanine-8 and tyrosine-4 but failed to hydrolyze bradykinin, vasopressin and synthetic chromogenic substrates with phenylalanine or tyrosine at the P1 position. In addition, two peptidases were detected in P. endodontalis cells: a proline aminopeptidase that remained associated with the cell pellet after detergent extraction and peptidase/s that partitioned into the Triton X-114 phase after phase separation and degraded the bioactive peptides bradykinin and vasopressin. These P. endodontalis peptidases and proteases may play an important role in both the nutrition and pathogenicity of these assacharolytic microorganisms. The inactivation of bioactive peptides and degradation of extracellular matrix proteins by bacterial enzymes may contribute to the damage of host tissues accompanied with endodontic infections.
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PMID:The purification and characterization of an 88-kDa Porphyromonas endodontalis 35406 protease. 1173 54

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