Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P01185 (vasopressin)
 23,126 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Most of the large family of G-protein-coupled receptors (GPCRs) possess putative N-glycosylation sites within their N-termini. However, for the vast majority of GPCRs, it has not been determined which, if any, of the consensus glycosylation sites are actually utilized or what the functional ramifications are of modification by oligosaccharide. The occurrence and function of glycosylation of the V(1a) vasopressin receptor (V(1a)R) has been investigated in this study. Using a combination of translation systems that are either glycosylation-competent or do not support glycosylation, we established that of the four putative N-glycosylation sites at Asn(14), Asn(27), Asn(198) and Asn(333) only the first three sites are actually modified by carbohydrate. This was confirmed by disruption of consensus sites by site-directed mutagenesis, individually and in combination. The V(1a)R is not O-glycosylated. The functionality of a series of glycosylation-defective V(1a)R constructs was characterized after expression in HEK 293T cells. It was found that carbohydrate moieties are not required for the receptor to bind any of the four classes of ligand available, or for intracellular signalling. The glycosylation status of the V(1a)R did, however, regulate the level of total receptor expression and also the abundance of receptor at the cell surface. Furthermore, the nature of this regulation (increased or decreased expression) was dictated by the locus of the oligosaccharide modification. Modification of any one of the consensus sites alone, however, was sufficient for wild-type expression, indicating a redundancy within the glycosylation sites. A role for the carbohydrate in the correct folding or stabilization of the V(1a)R is indicated. Glycosylation is not required, however, for efficient trafficking of the receptor to the cell surface. This study establishes the functional importance of N-glycosylation of the V(1a)R.
 ...
PMID:Identification of the glycosylation sites utilized on the V1a vasopressin receptor and assessment of their role in receptor signalling and expression. 1141 38

We have isolated and characterized the human homolog of the rat largest urea transporter of the UT-A family (hUT-A1). The 4.2-kb hUT-A1 cDNA encodes a 920-amino acid peptide, which is 89% identical to the rat UT-A1 protein. By Northern hybridization, hUT-A1 expression is detected in the human inner medulla as a approximately 4.4-kb mRNA transcript. By Western analysis, hUT-A1 is identified as a approximately 100-kDa protein in the human inner medulla. By immunohistochemistry, hUT-A1 expression is localized to the inner medullary collecting duct (IMCD). When transfected into HEK-293 cells hUT-A1 cDNA is translated into a approximately 98-kDa protein. Expression of hUT-A1 in Xenopus oocytes results in phloretin-inhibitable uptake of (14)C-urea, which shows only modest stimulation by cAMP, suggesting that in the human IMCD vasopressin may have a limited role in the short-term regulation of hUT-A1-mediated urea transport. We determined the organization of the human Slc14a2 gene and identified 20 exons distributed over approximately 67.5 kb on chromosome 18, from which hUT-A1 and the other human urea transporter, hUT-A2, are transcribed.
 ...
PMID:Cloning and characterization of the human urea transporter UT-A1 and mapping of the human Slc14a2 gene. 1150 88

By binding to agonist-activated G protein-coupled receptors (GPCRs), beta-arrestins mediate homologous receptor desensitization and endocytosis via clathrin-coated pits. Recent data suggest that beta-arrestins also contribute to GPCR signaling by acting as scaffolds for components of the ERK mitogen-activated protein kinase cascade. Because of these dual functions, we hypothesized that the stability of the receptor-beta-arrestin interaction might affect the mechanism and functional consequences of GPCR-stimulated ERK activation. In transfected COS-7 cells, we found that angiotensin AT1a and vasopressin V2 receptors, which form stable receptor-beta-arrestin complexes, activated a beta-arrestin-bound pool of ERK2 more efficiently than alpha 1b and beta2 adrenergic receptors, which form transient receptor-beta-arrestin complexes. We next studied chimeric receptors in which the pattern of beta-arrestin binding was reversed by exchanging the C-terminal tails of the beta2 and V2 receptors. The ability of the V2 beta 2 and beta 2V2 chimeras to activate beta-arrestin-bound ERK2 corresponded to the pattern of beta-arrestin binding, suggesting that the stability of the receptor-beta-arrestin complex determined the mechanism of ERK2 activation. Analysis of covalently cross-linked detergent lysates and cellular fractionation revealed that wild type V2 receptors generated a larger pool of cytosolic phospho-ERK1/2 and less nuclear phospho-ERK1/2 than the chimeric V2 beta 2 receptor, consistent with the cytosolic retention of beta-arrestin-bound ERK. In stably transfected HEK-293 cells, the V2 beta 2 receptor increased ERK1/2-mediated, Elk-1-driven transcription of a luciferase reporter to a greater extent than the wild type V2 receptor. Furthermore, the V2 beta 2, but not the V2 receptor, was capable of eliciting a mitogenic response. These data suggest that the C-terminal tail of a GPCR, by determining the stability of the receptor-beta-arrestin complex, controls the extent of beta-arrestin-bound ERK activation, and influences both the subcellular localization of activated ERK and the physiologic consequences of ERK activation.
 ...
PMID:The stability of the G protein-coupled receptor-beta-arrestin interaction determines the mechanism and functional consequence of ERK activation. 1247 60

In BRET2 (Bioluminescence Resonance Energy Transfer), a Renilla luciferase (RLuc) is used as the donor protein, while a Green Fluorescent Protein (GFP2) is used as the acceptor protein. In the presence of the cell permeable substrate DeepBlueC, RLuc emits blue light at 395 nm. If the GFP2 is brought into close proximity to RLuc via a specific biomolecular interaction, the GFP2 will absorb the blue light energy and reemit green light at 510nm. BRET2 signals are therefore easily determined by measuring the ratio of green over blue light (510/395nm) using appropriate dual channel luminometry instruments (e.g., Fusion Universal Microplate Analyzer, Packard BioScience). Since no light source is required for BRET2 assays, the technology does not suffer from high fluorescent background or photobleaching, the common problems associated with standard FRET-based assays. Using BRET2, we developed a generic G Protein-Coupled Receptor (GPCR) assay based on the observation that activation of the majority of GPCRs by agonists leads to the interaction of beta-arrestin (a protein that is involved in receptor desensitization and sequestration) with the receptor. We established a cell line stably expressing the GFP2:beta-arrestin 2 fusion protein, and showed that it can be used to monitor the activation of various transiently expressed GPCRs, in BRET2/arrestin assays. In addition, using the HEK 293/GFP2:beta-arrestin 2 cell line as a recipient, we generated a double-stable line co-expressing the vasopressin 2 receptor (V2R) fused to RLuc (V2R:RLuc) and used it for the pharmacological characterization of compounds in BRET2/arrestin assays. This approach yields genuine pharmacology and supports the BRET2/arrestin assay as a tool that can be used with recombinant cell lines to characterize ligand-GPCR interactions which can be applied to ligand identification for orphan receptors.
 ...
PMID:The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRS). 1250 39

The amiloride-sensitive epithelial sodium channel (ENaC), a multimeric plasma membrane protein composed of alpha-, beta-, and gamma-ENaC subunits, mediates Na(+) reabsorption in epithelial tissues, including the distal nephron, colon, lung, and secretory glands, and plays a critical role in pathophysiology of essential hypertension and cystic fibrosis (CF). The function of ENaC is tightly regulated by signals elicited by aldosterone, vasopressin, agents that increase intracellular cAMP levels, ions, ion channels, G-protein-coupled mechanisms, and cytoskeletal proteins. In this paper, the effects of Ca(2+) on the expression of the human ENaC subunits expressed in human embryonic kidney cells (HEK-293 cells) were examined. Incubation of cells with increased extracellular Ca(2+) and treatment of cells with A23187 and thapsigargin stimulated the expression of the monomeric ENaC subunits. Treatment of cells with Ca(2+)-chelating agents, EGTA and BAPTA-AM, reduced the levels of ENaC subunit expression. The pulse-chase experiments suggested that a rise in the intracellular Ca(2+) increases the ENaC subunit expression. Immunoblot analysis using the anti-ubiquitin antibody indicated that ENaC undergoes ubiquitination. A correlation between the processes that regulate ENaC function with the intracellular Ca(2+) was discussed.
 ...
PMID:Role of intracellular Ca2+ in the expression of the amiloride-sensitive epithelial sodium channel. 1467 Mar 68

The internalization of G protein-coupled receptors is regulated by several important proteins that act in concert to finely control this complex cellular process. Here, we have applied the RNA interference approach to demonstrate that ADP-ribosylation factor 6 (ARF6) is essential for the endocytosis of a broad variety of receptors. Reduction of endogenous expression of ARF6 in HEK 293 cells resulted in a correlated inhibition of the beta(2) -adrenergic receptor internalization previously characterized as being sequestered via the clathrin-coated vesicle pathway. Furthermore, other receptors internalizing via this endocytic route, namely the angiotensin type 1 receptor and the vasopressin type 2 receptor, were also impaired in their ability to be sequestered when levels of endogenous ARF6 in cells were reduced. Interestingly, endocytosis of the endothelin type B receptor, characterized as being internalized via the caveolae pathway, was also markedly inhibited in ARF6-depleted cells. In contrast, internalization of the vasoactive intestinal peptide receptor was unaffected by reduced levels of ARF6. Finally, internalization of the acetylcholine-muscarinic type 2 receptor via the non-clathrin-coated vesicle pathway was also inhibited in ARF6-depleted cells. Taken together, our results demonstrate that ARF6 proteins play an essential role in the internalization process of most G protein-coupled receptors regardless of the endocytic route being utilized. However, this phenomenon is not general. In some cases, another ARF isoform or other proteins may be essential to regulate the endocytic process.
 ...
PMID:G protein-coupled receptor endocytosis in ADP-ribosylation factor 6-depleted cells. 1559 Jun 45

The highly conserved "Asp-Arg-Tyr" triplet in the distal region of the third transmembrane region of most G-protein-coupled receptors is implicated in their activation process and mediation of G-protein signaling. The aim of this study was to determine whether specific features at this locus are important for the vasopressin V(1a) receptor (V(1a)R) by performing site-directed mutagenesis. In transfected HEK 293T cells, mutation of Asp (D148A) resulted in a misfolded receptor that was nonfunctional, localized intracellularly, and not constitutively active. Nonconservative (D148R) substitution was not expressed, whereas asparagine (D148N) partially restored cell surface expression, although no specific ligand-binding or inositol phosphate signaling was detected. In contrast, conservative (D148E) substitution was expressed moderately higher, bound ligands, and signaled similarly to a hemagglutinin epitope-tagged wild-type receptor. However, D148E showed a greater tendency to be internalized once it was delivered to the membrane. Individual replacements of the conserved arginine and tyrosine (R149A, Y150A) led to decreased signal transduction without affecting surface expression, agonist affinity, or internalization or increasing basal signaling activity. Incorporation of aspartate (R149D) or reversal of charges (D148R/R149D) were nonfunctional, localized intracellularly, and indicated the absence of an ionic interaction between Asp-148 and Arg-149. It is noteworthy that an important role of arginine was identified for regulating agonist-mediated internalization when a histidine (R149H) was present. This mutant was expressed on the cell surface but was rapidly internalized after agonist treatment. This study highlights the importance of specific charged residues within this motif that provide important determinants for cell surface delivery, internalization and for normal V(1a)R function.
 ...
PMID:Charged residues of the conserved DRY triplet of the vasopressin V1a receptor provide molecular determinants for cell surface delivery and internalization. 1604 68

Pharmacological chaperones represent a new class of ligand with the potential to facilitate the delivery of misfolded, but still active, G-protein-coupled receptors to the cell surface. Using transfected HEK 293T cells, treatment with a nonpeptide antagonist, SR49059, dramatically increased ( approximately 60-fold) the surface expression of a misfolded, nonfunctional and intracellularly localized vasopressin V(1a) receptor (V(1a)R) mutant (D148A). This rescue of surface expression (111 +/- 7%) was almost identical to wild type assessed by confocal microscopy and quantitative enzyme-linked immunosorbent assay-based techniques. Recovery was not specific to D148A, since other surface-impaired mutations, D148N and D148E, and wild type were also increased following SR49059 exposure. However, surface delivery was specific to SR49059, since V(1a)R-selective peptide ligands or unrelated ligands were unable to mimic this action, suggesting that SR49059 acts intracellularly. SR49059-mediated surface rescue was time-, mutant-, and concentration-dependent but not directly related to its binding affinity. Maximal recovery was achieved following 12 h of treatment and did not involve de novo receptor synthesis or a consequence of preventing endogenous constitutive activity and/or internalization. Once at the surface, all mutants displayed enhanced signaling ability, and D148A was able to undergo agonist-mediated internalization. SR49059 was not effectively removed from the receptor, since signaling (EC(50)) of both wild type and D148A was reduced approximately 40-fold. This is the first report of a pharmacological chaperone ligand to act on misfolded mutant V(1a) Rs. This work provides an excellent model to understand the mechanistic action of an important new class of drug that may have potential in the treatment of diseases caused by inherited mutations.
 ...
PMID:Pharmacological chaperone activity of SR49059 to functionally recover misfolded mutations of the vasopressin V1a receptor. 1656 83

Arginine vasopressin (AVP) is essential for maintaining body fluid homeostasis. The antidiuretic effects of AVP are initialized by binding of AVP to the type-2 vasopressin receptor (V2R) in the kidney collecting duct (CD), resulting in the exocytic insertion of aquaporin-2 (AQP-2) water channels into the apical plasma membrane. In this study, we describe the generation and characterization of a polyclonal antibody targeted against the NH2 terminus of the rat V2R. HEK-293 cells overexpressing the rat, mouse, or human V2R showed strong intracellular immunolabeling. Additionally, immunostaining of M-1 kidney cells expressing a V2R-green fluorescent protein (GFP) fusion construct showed colocalization between GFP and antibody-specific V2R labeling. Immunoblots of rat kidney showed 43- and 47-kDa proteins in all zones that were both reduced to 34-kDa by N-glycosidase F. Protein solubilization with nonionic detergents or the use of homobifunctional cross-linkers demonstrated that the rat V2R exists as a protein complex in native kidney. Immunohistochemistry of rat and mouse kidney revealed abundant labeling of the CD. Double-labeling confocal immunofluorescence microscopy [using distal convoluted tubule/connecting tubule (CNT)-specific marker calbindin and CNT/CD-specific marker AQP-2] showed V2R labeling in both CD and CNT. There was a complete absence of labeling in vascular structures and other renal tubules, including the thick ascending limb (TAL), although RT-PCR of microdissected tubules showed expression of V2R mRNA in TAL. Confocal microscopy demonstrated that at the subcellular level, V2R labeling was predominantly intracellular in normal kidneys, although some staining was apparent in basolateral membrane domains. Confocal microscopy of isolated inner medullary collecting duct tubules showed that the V2R is expressed both intracellularly and in basolateral membrane domains.
 ...
PMID:Cellular and subcellular distribution of the type-2 vasopressin receptor in the kidney. 1755 38

In this study, we identified the multifunctional protein GC1q-R as a novel vasopressin V(2) receptor (V(2)R) interacting protein. For this purpose, we have developed a proteomic approach combining pull-down assays using a cyclic peptide mimicking the third intracellular loop of V(2)R as a bait and mass spectrometry analyses of proteins isolated from either rat or human kidney tissues or the HEK 293 cell line. Co-immunoprecipitation of GC1q-R with the c-Myc-tagged h-V(2)R expressed in a HEK cell line confirmed the existence of a specific interaction between GC1q-R and the V(2) receptor. Then, construction of a mutant receptor in i3 loop allowed us to identify the i3 loop arginine cluster of the vasopressin V(2) receptor as the interacting determinant for GC1q-R interaction. Using purified receptor as a bait and recombinant (74-282) GC1q-R, we demonstrated a direct and specific interaction between these two proteins via the arginine cluster.
 ...
PMID:The multifunctional protein GC1q-R interacts specifically with the i3 loop arginine cluster of the vasopressin V2 receptor. 1835 46


<< Previous 1 2 3 Next >>