A vasopressin analog that binds but does not activate V1 or V2 vasopressin receptors is not internalized into cells that express V1 or V2 receptors.

To assess whether receptor binding is sufficient to initiate vasopressin receptor endocytosis in cells expressing the vasopressin V1 or V2 receptors, we synthesized a novel fluorescent-labeled vasopressin analog, [1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid), 2-(O-ethyl)-D-tyrosine, 4-valine, 8-lysine-N6-carboxytetramethylrhodamine] vasopressin (R-CLVP), that binds to vasopressin receptors but does not activate intracellular events such as the mobilization of intracellular calcium or the activation of adenylate cyclase. We compared the manner in which this analog was endocytosed in cells expressing V1 (A-10, rat smooth muscle cells) or V2 (LLC-PK1, porcine kidney cells) receptors with that of a full agonist, [1-(beta-mercaptopropionic acid), 8-lysine-N6-carboxytetramethylrhodamine] vasopressin (R-MLVP) [Lutz et al. (1990) J. Biol. Chem. 265, 4657-4663; Lutz et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,6507-6511]. We showed that R-CLVP bound to both types of receptors with good affinity. It failed to increase cyclic AMP concentrations in LLC-PK1 cells and did not increase the mobilization of intracellular calcium in A-10 cells. It bound to the surface of both these cell types in a diffuse manner and it did not undergo receptor endocytosis in either cell type. In contrast, R-MLVP, an agonist that bound to both receptor subtypes and elicited changes in intracellular cyclic AMP and calcium, bound to the surface of these cells in a diffuse manner at early times after exposure, and rapidly underwent endocytosis. We conclude that binding of vasopressin to its receptors alone is insufficient to cause receptor endocytosis, and other events distal to the receptor are required to initiate endocytosis. R-CLVP should be a useful analog in determining the factors responsible for initiating receptor endocytosis.     

Visualization of cell surface vasopressin V1a receptors in rat hepatocytes with a fluorescent linear antagonist.

To visualize cell surface V1a vasopressin receptors in rat hepatocytes in the absence of receptor-mediated endocytosis, we used a high-affinity fluorescent linear antagonist, Rhm8-PVA. Epifluorescence microscopy (3CCD camera) and fluorescence spectroscopy were used. Rhm8-PVA alone did not stimulate Ca2+ signals and competitively blocked Ca2+ signals (Kinact of 3.0 nM) evoked by arginine vasopressin (vasopressin). When rat hepatocytes were incubated with 10 nM of Rhm8-PVA for 30 min at 4C, the fluorescent antagonist bound to the surface of cells, presumably the plasma membrane. The V1a receptor specificity of Rhm8-PVA binding was confirmed by its displacement by the nonfluorescent antagonist V4253 and by the natural hormone vasopressin at 4C. Prior vasopressin-mediated endocytosis of V1a receptors at 37C abolished binding of the labeled antagonist, whereas in non-preincubated cells, Rhm8-PVA labeled the cell surface of rat hepatocytes. When cells labeled with Rhm8-PVA at 4C were warmed to 37C to initiate receptor-mediated internalization of the fluorescent complex, Rhm8-PVA remained at the cell surface. Incubation temperature at 4C or 37C had little effect on binding of Rhm8-PVA. We conclude that Rhm8-PVA is unable to evoke receptor-mediated endocytosis and can readily be used to visualize cell surface receptors in living cells.     

Functional rescue of the constitutively internalized V2 vasopressin receptor mutant R137H by the pharmacological chaperone action of SR49059

In most cases, nephrogenic diabetes insipidus results from mutations in the V2 vasopressin receptor (V2R) gene that cause intracellular retention of improperly folded receptors. We previously reported that cell permeable V2R antagonists act as pharmacological chaperones that rescue folding, trafficking, and function of several V2R mutants. More recently, the vasopressin antagonist, SR49059, was found to be therapeutically active in nephrogenic diabetes insipidus patients. Three of the patients with positive responses harbored the mutation R137H, previously reported to lead to constitutive endocytosis. This raises the possibility that, instead of acting as a pharmacological chaperone by favoring proper maturation of the receptors, SR49059 could mediate its action on R137H V2R by preventing its endocytosis. Here we report that the beta-arrestin-mediated constitutive endocytosis of R137H V2R is not affected by SR49059, indicating that the functional rescue observed does not result from a stabilization of the receptor at the cell surface. Moreover, metabolic labeling revealed that R137H V2R is also poorly processed to the mature form. SR49059 treatment significantly improved its maturation and cell surface targeting, indicating that the functional rescue of R137H V2Rs results from the pharmacological chaperone action of the antagonist.     

Palmitoylation of the V2 vasopressin receptor carboxyl tail enhances beta-arrestin recruitment leading to efficient receptor endocytosis and ERK1/2 activation

A large number of G protein-coupled receptors are palmitoylated on cysteine residues located in their carboxyl tail, but the general role of this post-translational modification remains poorly understood. Here we show that preventing palmitoylation of the V2 vasopressin receptor, by site-directed mutagenesis of cysteines 341 and 342, significantly delayed and decreased both agonist-promoted receptor endocytosis and mitogen-activated protein kinase activation. Pharmacological blockade of receptor endocytosis is without effect on the vasopressin-stimulated mitogen-activated protein kinase activity, excluding the possibility that the reduced kinase activation mediated by the palmitoylation-less mutant could result from altered receptor endocytosis. In contrast, two dominant negative mutants of beta-arrestin which inhibit receptor endocytosis also attenuated vasopressin-stimulated mitogen-activated protein kinase activity, suggesting that the scaffolding protein, beta-arrestin, represents the common link among receptor palmitoylation, endocytosis, and kinase activation. Coimmunoprecipitation and bioluminescence resonance energy transfer experiments confirmed that inhibiting receptor palmitoylation considerably reduced the vasopressin-stimulated recruitment of beta-arrestin to the receptor. Interestingly, the changes in beta-arrestin recruitment kinetics were similar to those observed for vasopressin-stimulated receptor endocytosis and mitogen-activated protein kinase activation. Taken together the results indicate that palmitoylation enhances the recruitment of beta-arrestin to the activated V2 vasopressin receptor thus facilitating processes requiring the scaffolding action of beta-arrestin.     

Investigation of mechanism of desmopressin binding in vasopressin V2 receptor versus vasopressin V1a and oxytocin receptors: molecular dynamics simulation of the agonist-bound state in the membrane-aqueous system

The vasopressin V2 receptor (V2R) belongs to the Class A G protein-coupled receptors (GPCRs). V2R is expressed in the renal collecting duct (CD), where it mediates the antidiuretic action of the neurohypophyseal hormone arginine vasopressin (CYFQNCPRG-NH2, AVP). Desmopressin ([1-deamino, 8-D]AVP, dDAVP) is strong selective V2R agonist with negligible pressor and uterotonic activity. In this paper, the interactions responsible for binding of dDAVP to vasopressin V2 receptor versus vasopressin V1a and oxytocin receptors has been examined. Three-dimensional activated models of the receptors were constructed using the multiple sequence alignment and the complex of activated rhodopsin with Gt(alpha) C-terminal peptide of transducin MII-Gt(alpha) (338-350) prototype (Slusarz, R.; Ciarkowski, J. Acta Biochim Pol 2004 51, 129-136) as a template. The 1-ns unconstrained molecular dynamics (MD) of receptor-dDAVP complexes immersed in the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) membrane model was conducted in an Amber 7.0 force field. Highly conserved transmembrane residues have been proposed as being responsible for V2R activation and G protein coupling. Molecular mechanism of the dDAVP binding has been suggested. The internal water molecules involved in an intricate network of the hydrogen bonds inside the receptor cavity have been identified and their role in the stabilization of the agonist-bound state proposed.     

The ubiquitin-like protein PLIC-2 is a negative regulator of G protein-coupled receptor endocytosis

The activity of many signaling receptors is regulated by their endocytosis via clathrin-coated pits (CCPs). For G protein-coupled receptors (GPCRs), recruitment of the adaptor protein arrestin to activated receptors is thought to be sufficient to drive GPCR clustering in CCPs and subsequent endocytosis. We have identified an unprecedented role for the ubiquitin-like protein PLIC-2 as a negative regulator of GPCR endocytosis. Protein Linking IAP to Cytoskeleton (PLIC)-2 overexpression delayed ligand-induced endocytosis of two GPCRs: the V2 vasopressin receptor and β-2 adrenergic receptor, without affecting endocytosis of the transferrin or epidermal growth factor receptor. The closely related isoform PLIC-1 did not affect receptor endocytosis. PLIC-2 specifically inhibited GPCR concentration in CCPs, without affecting membrane recruitment of arrestin-3 to activated receptors or its cellular levels. Depletion of cellular PLIC-2 accelerated GPCR endocytosis, confirming its regulatory function at endogenous levels. The ubiquitin-like domain of PLIC-2, a ligand for ubiquitin-interacting motifs (UIMs), was required for endocytic inhibition. Interestingly, the UIM-containing endocytic adaptors epidermal growth factor receptor protein substrate 15 and Epsin exhibited preferential binding to PLIC-2 over PLIC-1. This differential interaction may underlie PLIC-2 specific effect on GPCR endocytosis. Identification of a negative regulator of GPCR clustering reveals a new function of ubiquitin-like proteins and highlights a cellular requirement for exquisite regulation of receptor dynamics.     

Analysis of interactions responsible for vasopressin binding to human neurohypophyseal hormone receptors-molecular dynamics study of the activated receptor-vasopressin-G(alpha) systems.

Vasopressin (CYFQNCPRG-NH(2), AVP) is a semicyclic endogenous peptide, which exerts a variety of biological effects in mammals. The main physiological roles of AVP are the regulation of water balance and the control of blood pressure and adrenocorticotropin hormone (ACTH) secretion, mediated via three different subtypes of vasopressin receptors: V1a, V1b and V2 receptors (V1aR, V1bR and V2R, respectively). They are the members of the class A, G-protein-coupled receptors (GPCRs). AVP also modulates several behavioral and social functions. In this study, the interactions responsible for AVP binding to vasopressin V1a and V2 receptors versus the closely related oxytocin ([I3,L8]AVP, OT) receptor (OTR) have been investigated. Three-dimensional models of the activated receptors were constructed using multiple sequence alignment, followed by homology modeling using the complex of activated rhodopsin with Gt(alpha) C-terminal peptide of transducin MII-Gt(338-350) prototype as a template. AVP was docked into the receptor-G(alpha) systems. The three lowest-energy pairs of receptor-AVP-G(alpha) (two complexes per each receptor) were selected. The 1-ns unconstrained molecular dynamics (MD) of complexes embedded into the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayer was conducted in the AMBER 7.0 force field. Six relaxed receptor-AVP-G(alpha) models were obtained. The residues responsible for AVP binding to vasopressin receptors have been identified and a different mechanism of AVP binding to V2R than to V1aR has been proposed.     

Heterologous inhibition of G protein-coupled receptor endocytosis mediated by receptor-specific trafficking of beta-arrestins

We have observed an unexpected type of nonreciprocal "cross-regulation" of the agonist-induced endocytosis of G protein-coupled receptors by clathrin-coated pits. Isoproterenol-dependent internalization of beta2-adrenergic receptors in stably transfected HEK293 cells was specifically blocked (>65% inhibition) by vasopressin-induced activation of V2 vasopressin receptors co-expressed at similar levels. In contrast, activation of beta2 receptors caused no detectable effect on V2 receptor internalization in the same cells. Several pieces of evidence suggest that this nonreciprocal inhibition of endocytosis is mediated by receptor-specific intracellular trafficking of beta-arrestins. First, previous studies showed that the activation of V2 but not beta2 receptors caused pronounced recruitment of beta-arrestins to endocytic membranes (Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S., and Caron, M. G. (1999) J. Biol. Chem. 274, 32248-32257). Second, overexpression of arrestin 2 or 3 (beta-arrestin 1 or 2) abolished the V2 receptor-mediated inhibition of beta2 receptor internalization. Third, mutations of the V2 receptor that block endomembrane recruitment of beta-arrestins eliminated the V2 receptor-dependent blockade of beta2 receptor internalization. These results identify a novel type of heterologous regulation of G protein-coupled receptors, define a new functional role of receptor-specific intracellular trafficking of beta-arrestins, and suggest an experimental method to rapidly modulate the functional activity of beta-arrestins in intact cells.     

Renal vasopressin receptor expression and function in rats following spaceflight.

It has been suggested there is a decreased renal responsiveness to vasopressin following spaceflight and that this may be the mechanism for the increased urine flow that is observed following return to normal gravity. In the present study, we have therefore measured vasopressin receptor expression and activity in kidneys taken from rats 1 and 14 days following spaceflight of 15 days duration. Measurements of renal vasopressin V(2) and V(1a) receptor mRNA expression by quantitative RT-PCR demonstrated little difference at either 1 day or at 14 days following return from space. Evaluation of (3)H-labeled arginine vasopressin binding to membranes prepared from kidneys indicated that the majority of the vasopressin receptors were V(2) receptors. Furthermore, the data suggested that binding to vasopressin V(2) or V(1a) receptors was unaltered at 1 day and 14 days following spaceflight. Similarly, the ability of vasopressin to stimulate adenylate cyclase suggested no change in vasopressin V(2) receptor activity in these animals. These data suggest that, whatever changes in fluid and electrolyte metabolism are observed following spaceflight, they are not mediated by changes in vasopressin receptor number or vasopressin-induced stimulation of adenylate cyclase.     

Association of beta-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization.

Resensitization of G protein-coupled receptors (GPCRs) following agonist-mediated desensitization is a necessary step for maintaining physiological responsiveness. However, the molecular mechanisms governing the nature of GPCR resensitization are poorly understood. Here, we examine the role of beta-arrestin in the resensitization of the beta(2) adrenergic receptor (beta(2)AR), known to recycle and resensitize rapidly, and the vasopressin V2 receptor (V2R), known to recycle and resensitize slowly. Upon agonist activation, both receptors recruit beta-arrestin to the plasma membrane and internalize in a beta-arrestin- and clathrin-dependent manner. However, whereas beta-arrestin dissociates from the beta(2)AR at the plasma membrane, it internalizes with the V2R into endosomes. The differential trafficking of beta-arrestin and the ability of these two receptors to dephosphorylate, recycle, and resensitize is completely reversed when the carboxyl-terminal tails of these two receptors are switched. Moreover, the ability of beta-arrestin to remain associated with desensitized GPCRs during clathrin-mediated endocytosis is mediated by a specific cluster of phosphorylated serine residues in the receptor carboxyl-terminal tail. These results demonstrate that the interaction of beta-arrestin with a specific motif in the GPCR carboxyl-terminal tail dictates the rate of receptor dephosphorylation, recycling, and resensitization, and thus provide direct evidence for a novel mechanism by which beta-arrestins regulate the reestablishment of GPCR responsiveness.     

Internalization of V2-vasopressin receptors in LLC-PK1-cells: evidence for receptor-mediated endocytosis.

The mechanism of internalization of the vasopressin-receptor (V2-subtype) of LLC-PK1-cells, a pig renal tubular cell line, is unknown. We studied internalization utilizing a novel, highly specific vasopressin analogue ((125I)-[8-p(OH)-phenylpropionyl]-LVP, 2000 Ci/mmol). Scatchard analysis performed with membranes of LLC-PK1-cells revealed a Kd of 0.8 +/- 0.2 nM and a Bmax of 366 +/- 41 fmol/mg of protein. Degradation of the ligand was excluded by RP-HPLC-analysis. Internalization was proven by the acid-wash technique, quantitative light-microscopic autoradiography and electron microscopy. The ligand was internalized in a time- and temperature-dependent manner. At 4 degrees C, no uptake was found; at 22 degrees C, after 30 min of incubation, more than 50% of the radioligand was found inside the cell. Electron microscopy demonstrated that plasma-membrane bound vasopressin receptors are internalized by receptor-mediated endocytosis via coated pits.     

Receptor activity-independent recruitment of betaarrestin2 reveals specific signalling modes

The roles of betaarrestins in regulating G protein coupling and receptor endocytosis following agonist stimulation of G protein-coupled receptors are well characterised. However, their ability to act on their own as direct modulators or activators of signalling remains poorly characterised. Here, betaarrestin2 intrinsic signalling properties were assessed by forcing the recruitment of this accessory protein to vasopressin V1a or V2 receptors independently of agonist-promoted activation of the receptors. Such induction of a stable interaction with betaarrestin2 initiated receptor endocytosis leading to intracellular accumulation of the betaarrestin/receptor complexes. Interestingly, betaarrestin2 association to a single receptor protomer was sufficient to elicit receptor dimer internalisation. In addition to recapitulating betaarrestin2 classical actions on receptor trafficking, the receptor activity-independent recruitment of betaarrestin2 activated the extracellular signal-regulated kinases. In the latter case, recruitment to the receptor itself was not required since kinase activation could be mediated by betaarrestin2 translocation to the plasma membrane in the absence of any interacting receptor. These data demonstrate that betaarrestin2 can act as a 'bonafide' signalling molecule even in the absence of activated receptor.     

Endocytosis of the vasopressin receptor by anterior pituitary cells is increased by corticotropin-releasing factor (CRF)

Endocytosis of certain receptors such as the transferrin receptor and the EGF-receptor appears to be influenced by second messengers. If second messengers are involved in modulation of endocytosis, not only endocytosis of the stimulated receptor itself but also of receptors for other ligands on the cell surface may be influenced by receptor occupancy. Corticotropin-releasing factor (CRF) and vasopressin act synergistically on secretion of ACTH from the anterior pituitary. The results presented here demonstrate that CRF increases retrieval of the vasopressin receptor in anterior pituitary cells in primary culture without influencing the surface binding of vasopressin. This is not a function of an increased membrane turnover since endocytosis of the transferrin receptor is not influenced by CRF.     

c-Src-mediated phosphorylation of AP-2 reveals a general mechanism for receptors internalizing through the clathrin pathway

Clathrin-mediated endocytosis is a complex process regulated at many different levels. We showed previously that activation of the angiotensin type 1 receptor (AT1R), which belongs to the G protein-coupled receptor (GPCR) family, leads to c-Src-dependent tyrosine phosphorylation of beta2-adaptin, a subunit of the clathrin adaptor AP-2. The phosphorylation of beta2-adaptin on tyrosine residue 737 (Y737) negatively regulates its interaction with betaarrestin, another important clathrin adaptor for GPCR internalization. Here we sought to determine whether AP-2 phosphorylation represents a general mechanism for different receptors internalizing through the clathrin pathway. Using a specifically designed antibody against the phosphorylated form of Y737 on beta2-adaptin, we demonstrate that this residue is phosphorylated by AT1R in different cell types like HEK293, COS-7 and vascular smooth muscle cells. Using RNA interference approaches, we reveal that this agonist-mediated event is both betaarrestin- and c-Src-dependent, and that it occurs at the plasma membrane in clathrin-coated vesicles (CCVs). We further show that this is not only a common event employed by other GPCRs like the beta2-adrenergic, vasopressin V2, bradykinin type 2, platelet-activating factor and endothelin A receptors but that the epidermal growth factor receptor is capable of eliciting the phosphorylation of AP-2 in CCVs. Our results imply that tyrosine phosphorylation of Y737 on beta2-adaptin is a common regulatory mechanism employed by different receptors undergoing clathrin-dependent endocytosis, and suggest a wider function for this event than originally anticipated.     

A comparative study of plasma vasopressin levels and V1 and V2 vasopressin receptor properties in congenital hypothyroid rat under thyroxine or vasopressin therapy

The effects of propylthiouracil (PTU) treatment on the plasma vasopressin level, on the number of hepatic (V1) or renal (V2) vasopressin receptors and on the hormone-sensitive adenylate cyclase activity in the kidney of developing rats were studied in parallel. In addition, we investigated the corrective effects of thyroxine therapy on the plasma vasopressin level and parameters related to the liver, and the effects of vasopressin therapy on the parameters related to the kidney. As already reported in the case of the number of V2 receptors and adenylate cyclase activity in the kidney, the deficient plasma vasopressin level in hypothyroid rats was completely corrected by two daily physiological doses of thyroxine given from birth to the age of sacrifice (1 month). Unlike the V1 receptors, the V2 receptors are known to be highly dependent on their specific circulating ligand. Since, first of all, the deficit was similar in the numbers of V1 and V2 receptors in hypothyroid rats, and, secondly, the treatment of hypothyroid rats by two daily physiological doses of long lasting vasopressin was found ineffective to recover the deficit in the number of V2 receptors, it can be concluded that thyroid deficiency directly alters vasopressin receptor biosynthesis in both liver and kidney, instead of acting via the depressed plasma vasopressin level.     

Hyaluronate-hydrolases system and hydroosmotic effect of vasopressin]

Involvement of enzymes catabolizing hyaluronic acid (hyaluronidase, beta-glucuronidase, N-acetyl-beta-D-hexosaminidase) in the hydroosmotic action of vasopressin on the amphibian urinary bladder Rana Ridibunda was studied. It was found that vasopressin (50 nM), agonist of V2 receptors dDAVP (1.5 mcM) and forscolin (30 mcM) induce an activation of enzymes and its release into the Ringer solution at the mucosal surface simultaneously with the increase in the osmotic water flow. Maximal effect was observed 10 min later than hydroosmotic response. Release of enzymes under vasopressin effect was found in the absence of osmotic gradient and water flow through the epithelium. The repeated substitution of the outer Ringer solution for the fresh one resulted in the increase in the both the water permeability and the release of enzymes through the mucosal surface. We suggested that involvement of hyaluronate-hydrolases in the vasopressin effect is mediated by the cAMP-dependent mechanism. It is supposed that this effect creates conditions for the increase in the permeability of glycosaminoglycan structures covering adjacent to the apical cell surface.     

Microfilament network is needed for the endocytosis of water channels and not for apical membrane insertion upon vasopressin action

In the current study, a novel role for the microfilaments in vasopressin-induced water transport in toad urinary bladders, a popular model for the mammalian collecting duct, was established. Vasopressin-induced water transport was not affected by cytochalasin D (CD, 20 microM) or latrunculin B (Lat B, 0.5-2 microM), microfilament-disrupting reagents, suggesting that the initial trafficking of vesicles containing water channels and insertion of membranes into the apical membrane are microfilament-independent. After the removal of vasopressin, bladders treated with CD or Lat B continued to transport water at least 2-3-fold greater than those that received the vehicle. Furthermore, the enhanced water transport was inhibited by HgCl2 (1 mM), a potent inhibitor of water channel-mediated water flow, suggesting that the enhanced water flow was through water channels. In addition, Lat B and CD inhibited vasopressin-induced endocytosis of horseradish peroxidase (HRP), a fluid endocytotic marker. These results suggested that although microfilaments are not needed for the initial trafficking of water channels to the apical side, the microfilament network is essential for the retrieval of water channels following their insertion into apical membranes.     

Similarity of vasopressin receptors in seminal vesicles and renal medulla of pigs

To test the hypothesis that the vasopressin receptors found in seminal vesicles are similar to those present in the renal tubules competition experiments were performed with vasopressin and several analogues with different specificities for the V1 and V2 subtypes of vasopressin receptor. Autoradiographic studies were carried out on sections from seminal vesicles and kidney to identify the cellular target of vasopressin. Vasopressin receptors in renal medulla and seminal vesicles of pigs shared the same rank order of potency for vasopressin and its analogues and were localized in the epithelium of the seminal vesicles and in collecting tubules of renal medulla. These results strongly suggest that the vasopressin receptors present in kidney and seminal vesicles belong to the same subtype, V2, of vasopressin receptor.