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• W1999532619 abstract "Inactivating mutations of the V2 vasopressin receptor (V2R) cause cross-linked congenital nephrogenic diabetes insipidus (NDI), resulting in renal resistance to the antidiuretic hormone AVP. In two families showing partial NDI, characterized by an apparently normal response to diagnostic tests and an increase in the basal ADH levels suggesting AVP resistance, we have identified two V2R mutations, Ser-333del and Y128S. Both mutant V2Rs, when expressed in COS-7 cells, show partial defects in vasopressin-stimulated cAMP accumulation and intracellular localization. The inhibition of internalization does not rescue their localization. In contrast, the non-peptide V2R antagonists OPC41061 and OPC31260 partially rescue the membrane localization and basal function of these V2R mutants, whereas they inhibit the basal activity of the wild-type V2R. These results indicate that a partial loss of function of Ser-333del and Y128S mutant V2Rs results from defective membrane trafficking. These findings further indicate that V2R antagonists can act as protean agonists, serving as pharmacological chaperones for inactivating V2R mutants and also as inverse agonists of wild-type receptors. We speculate that this protean agonism could underlie the possible dual beneficial effects of the V2R antagonist: improvement of hyponatremia with heart failure or polycystic kidney disease and potential rescue of NDI. Inactivating mutations of the V2 vasopressin receptor (V2R) cause cross-linked congenital nephrogenic diabetes insipidus (NDI), resulting in renal resistance to the antidiuretic hormone AVP. In two families showing partial NDI, characterized by an apparently normal response to diagnostic tests and an increase in the basal ADH levels suggesting AVP resistance, we have identified two V2R mutations, Ser-333del and Y128S. Both mutant V2Rs, when expressed in COS-7 cells, show partial defects in vasopressin-stimulated cAMP accumulation and intracellular localization. The inhibition of internalization does not rescue their localization. In contrast, the non-peptide V2R antagonists OPC41061 and OPC31260 partially rescue the membrane localization and basal function of these V2R mutants, whereas they inhibit the basal activity of the wild-type V2R. These results indicate that a partial loss of function of Ser-333del and Y128S mutant V2Rs results from defective membrane trafficking. These findings further indicate that V2R antagonists can act as protean agonists, serving as pharmacological chaperones for inactivating V2R mutants and also as inverse agonists of wild-type receptors. We speculate that this protean agonism could underlie the possible dual beneficial effects of the V2R antagonist: improvement of hyponatremia with heart failure or polycystic kidney disease and potential rescue of NDI. IntroductionLoss of function mutations of G-protein-coupled receptors (GPCRs) 5The abbreviations used are: GPCRG-protein-coupled receptorNDInephrogenic diabetes insipidusAVParginine vasopressinV2RV2 vasopressin receptorOPC4OPC41061OPC3OPC31260ERendoplasmic reticulumDDAVP1-desamino-8-arginine vasopressinADHantidiuretic hormoneNSIADneonatal syndrome of inappropriate antidiuresis. cause many endocrine and other diseases (1Tao Y.X. Inactivating mutations of G protein-coupled receptors and diseases. Structure-function insights and therapeutic implications.Pharmacol. Ther. 2006; 111: 949-973Crossref PubMed Scopus (106) Google Scholar, 2Spiegel A.M. Weinstein L.S. Inherited diseases involving G proteins and G protein-coupled receptors.Annu .Rev. Med. 2004; 55: 27-39Crossref PubMed Scopus (193) Google Scholar). Although many mutations cause the complete loss of expression of GPCR proteins, some mutations cause loss of function because of defects in protein localization, protein-protein interactions, and so on, and may therefore provide new insights into the physiological operations of the GPCRs. Partial loss of function mutations are good candidates in this regard because they show at least some protein expression.Nephrogenic diabetes insipidus (NDI) is caused by renal resistance to the antidiuretic effects of arginine vasopressin (AVP) and is associated with elevated levels of this hormone (3Robben J.H. Knoers N.V. Deen P.M. Cell biological aspects of the vasopressin type-2 receptor and aquaporin 2 water channel in nephrogenic diabetes insipidus.Am. J. Physiol. Renal Physiol. 2006; 291: F257-F270Crossref PubMed Scopus (149) Google Scholar, 4Bichet D.G. Nephrogenic diabetes insipidus.Adv. Chronic Kidney Dis. 2006; 13: 96-104Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Inactivating mutations of V2 receptor (V2R) are responsible for about 90% of congenital NDI cases and are transmitted in a cross-linked manner, thus manifesting as a complete loss of function phenotype in most instances. Among the more than 200 V2R mutations identified to date, however, only seven have been reported to cause a partial congenital NDI phenotype in clinical cases (5Ala Y. Morin D. Mouillac B. Sabatier N. Vargas R. Cotte N. Déchaux M. Antignac C. Arthus M.F. Lonergan M. Turner M.S. Balestre M.N. Alonso G. Hibert M. Barberis C. Hendy G.N. Bichet D.G. Jard S. Functional studies of twelve mutant V2 vasopressin receptors related to nephrogenic diabetes insipidus. Molecular basis of a mild clinical phenotype.J. Am. Soc. Nephrol. 1998; 9: 1861-1872Crossref PubMed Google Scholar, 6Chen C.H. Chen W.Y. Liu H.L. Liu T.T. Tsou A.P. Lin C.Y. Chao T. Qi Y. Hsiao K.J. Identification of mutations in the arginine vasopressin receptor 2 gene causing nephrogenic diabetes insipidus in Chinese patients.J. Hum. Genet. 2002; 47: 66-73Crossref PubMed Scopus (26) Google Scholar, 7Faerch M. Christensen J.H. Corydon T.J. Kamperis K. de Zegher F. Gregersen N. Robertson G.L. Rittig S. Clin. Endocrinol. 2008; 68: 395-403Crossref PubMed Scopus (27) Google Scholar, 8Inaba S. Hatakeyama H. Taniguchi N. Miyamori I. The property of a novel v2 receptor mutant in a patient with nephrogenic diabetes insipidus.J. Clin. Endocrinol. Metab. 2001; 86: 381-385Crossref PubMed Scopus (24) Google Scholar, 9Pasel K. Schulz A. Timmermann K. Linnemann K. Hoeltzenbein M. Jääskeläinen J. Grüters A. Filler G. Schöneberg T. Functional characterization of the molecular defects causing nephrogenic diabetes insipidus in eight families.J. Clin. Endocrinol. Metab. 2000; 85: 1703-1710PubMed Google Scholar, 10Sadeghi H. Robertson G.L. Bichet D.G. Innamorati G. Birnbaumer M. Biochemical basis of partial nephrogenic diabetes insipidus phenotypes.Mol. Endocrinol. 1997; 11: 1806-1813Crossref PubMed Scopus (0) Google Scholar, 11Faerch M. Christensen J.H. Rittig S. Johansson J.O. Gregersen N. de Zegher F. Corydon T.J. Diverse vasopressin V2 receptor functionality underlying partial congenital nephrogenic diabetes insipidus.Am. J. Physiol. Renal Physiol. 2009; 297: F1518-F1525Crossref PubMed Scopus (15) Google Scholar).The classic mode of GPCR activation is the two-state model (12Gether U. Uncovering molecular mechanisms involved in activation of G protein-coupled receptors.Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1097) Google Scholar, 13Kenakin T. Functional selectivity through protean and biased agonism. Who steers the ship?.Mol. Pharmacol. 2007; 72: 1393-1401Crossref PubMed Scopus (238) Google Scholar, 14Leach K. Sexton P.M. Christopoulos A. Allosteric GPCR modulators: taking advantage of permissive receptor pharmacology.Trends Pharmacol. Sci. 2007; 28: 382-389Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar), in which the receptor exists in equilibrium between an inactive (R) and active (R*) conformation. Recently, however, accumulating evidence has suggested that GPCRs can adopt multiple conformational states (12Gether U. Uncovering molecular mechanisms involved in activation of G protein-coupled receptors.Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1097) Google Scholar, 13Kenakin T. Functional selectivity through protean and biased agonism. Who steers the ship?.Mol. Pharmacol. 2007; 72: 1393-1401Crossref PubMed Scopus (238) Google Scholar, 14Leach K. Sexton P.M. Christopoulos A. Allosteric GPCR modulators: taking advantage of permissive receptor pharmacology.Trends Pharmacol. Sci. 2007; 28: 382-389Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 15Hill S.J. G-protein-coupled receptors. Past, present and future.Br. J. Pharmacol. 2006; 147: S27-S37Crossref PubMed Scopus (135) Google Scholar). In this multistate model, the receptor is proposed to alternate spontaneously between multiple active and inactive conformations, and each ligand is postulated to stabilize a specific conformation of the GPCR, which then leads to a set of specific biological effects. This type of unique ligand behavior has been termed functional selectivity or biased agonism (15Hill S.J. G-protein-coupled receptors. Past, present and future.Br. J. Pharmacol. 2006; 147: S27-S37Crossref PubMed Scopus (135) Google Scholar, 16Urban J.D. Clarke W.P. von Zastrow M. Nichols D.E. Kobilka B. Weinstein H. Javitch J.A. Roth B.L. Christopoulos A. Sexton P.M. Miller K.J. Spedding M. Mailman R.B. Functional selectivity and classical concepts of quantitative pharmacology.J. Pharmacol. Exp. Ther. 2007; 320: 1-13Crossref PubMed Scopus (891) Google Scholar). Strikingly, some β2 adrenergic receptor ligands, such as ICI 118551 and propranolol, are inverse agonists of G protein-mediated signaling and thereby decrease cAMP levels while at the same time functioning as partial agonists of the MAPK cascade (17Azzi M. Charest P.G. Angers S. Rousseau G. Kohout T. Bouvier M. Piñeyro G. β-arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 11406-11411Crossref PubMed Scopus (417) Google Scholar). Furthermore, our recent identification of a unique autoantibody against calcium-sensing receptor in an acquired hypocalciuric hypercalcemia patient (18Makita N. Sato J. Manaka K. Shoji Y. Oishi A. Hashimoto M. Fujita T. Iiri T. An acquired hypocalciuric hypercalcemia autoantibody induces allosteric transition among active human Ca-sensing receptor conformations.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 5443-5448Crossref PubMed Scopus (85) Google Scholar, 19Makita N. Iiri T. AfCS Nature Molecule Pages. 2008; 10.1038/mp.a004000.01Google Scholar) suggests that the allosteric modulation and functional selectivity of GPCRs are a pathophysiological mode of operation and may possibly, therefore, be physiological processes also.A special form of GPCR-based functional selectivity is “protean” agonism, in which certain agonists may change or even reverse their effects depending on the states or systems adopted (13Kenakin T. Functional selectivity through protean and biased agonism. Who steers the ship?.Mol. Pharmacol. 2007; 72: 1393-1401Crossref PubMed Scopus (238) Google Scholar, 15Hill S.J. G-protein-coupled receptors. Past, present and future.Br. J. Pharmacol. 2006; 147: S27-S37Crossref PubMed Scopus (135) Google Scholar). These agonists are thus termed protean agonists. Kenakin (20Kenakin T. Inverse, protean, and ligand-selective agonism: matters of receptor conformation.FASEB J. 2001; 15: 598-611Crossref PubMed Scopus (353) Google Scholar) was the first to describe this phenomenon, in which it was demonstrated that although some β2-adrenergic receptor ligands produce positive agonism in whole cells, the same ligands produce inverse agonism when the cAMP assay is performed for cell membranes. In this model, a partial agonist of a GPCR that is mildly activating a GPCR in a quiescent system, may operate as an inverse agonist in a constitutively active system, for example in the presence of a constitutive mutant of the same GPCR (13Kenakin T. Functional selectivity through protean and biased agonism. Who steers the ship?.Mol. Pharmacol. 2007; 72: 1393-1401Crossref PubMed Scopus (238) Google Scholar, 15Hill S.J. G-protein-coupled receptors. Past, present and future.Br. J. Pharmacol. 2006; 147: S27-S37Crossref PubMed Scopus (135) Google Scholar). Thus, the agonist, inverse agonist, or neutral antagonist activities of each GPCR ligand do not seem to be intrinsically decided by the characteristic of each ligand but by the system adopted.In this study, we have identified two mutations in the V2R gene (AVPR2) in patients with partial NDI who had both been initially diagnosed with psychogenic polydipsia. One is a novel mutation, Ser-333del, which causes a serine deletion at the 333 position of the C terminus of the V2R. The other is a previously reported mutation Y128S (21Bichet D.G. Arthus M.F. Lonergan M. Hendy G.N. Paradis A.J. Fujiwara T.M. Morgan K. Gregory M.C. Rosenthal W. Didwania A. X-linked nephrogenic diabetes insipidus mutations in North America and the Hopewell hypothesis.J. Clin. Invest. 1993; 92: 1262-1268Crossref PubMed Scopus (128) Google Scholar, 22Bichet D.G. Birnbaumer M. Lonergan M. Arthus M.F. Rosenthal W. Goodyer P. Nivet H. Benoit S. Giampietro P. Simonetti S. Nature and recurrence of AVPR2 mutations in X-linked nephrogenic diabetes insipidus.Am. J. Hum. Genet. 1994; 55: 278-286PubMed Google Scholar, 23Arthus M.F. Lonergan M. Crumley M.J. Naumova A.K. Morin D. De Marco L.A. Kaplan B.S. Robertson G.L. Sasaki S. Morgan K. Bichet D.G. Fujiwara T.M. Report of 33 novel AVPR2 mutations and analysis of 117 families with X-linked nephrogenic diabetes insipidus.J. Am. Soc. Nephrol. 2000; 11: 1044-1054Crossref PubMed Google Scholar), but an association with partial NDI has not been identified for this amino acid substitution. We further analyzed the functional properties of these two mutations. To clarify the mechanisms underlying the loss of function of V2R in each case, we also evaluated other V2R mutants: R137H (21Bichet D.G. Arthus M.F. Lonergan M. Hendy G.N. Paradis A.J. Fujiwara T.M. Morgan K. Gregory M.C. Rosenthal W. Didwania A. X-linked nephrogenic diabetes insipidus mutations in North America and the Hopewell hypothesis.J. Clin. Invest. 1993; 92: 1262-1268Crossref PubMed Scopus (128) Google Scholar, 22Bichet D.G. Birnbaumer M. Lonergan M. Arthus M.F. Rosenthal W. Goodyer P. Nivet H. Benoit S. Giampietro P. Simonetti S. Nature and recurrence of AVPR2 mutations in X-linked nephrogenic diabetes insipidus.Am. J. Hum. Genet. 1994; 55: 278-286PubMed Google Scholar, 23Arthus M.F. Lonergan M. Crumley M.J. Naumova A.K. Morin D. De Marco L.A. Kaplan B.S. Robertson G.L. Sasaki S. Morgan K. Bichet D.G. Fujiwara T.M. Report of 33 novel AVPR2 mutations and analysis of 117 families with X-linked nephrogenic diabetes insipidus.J. Am. Soc. Nephrol. 2000; 11: 1044-1054Crossref PubMed Google Scholar, 24Boson W.L. Della Manna T. Damiani D. Miranda D.M. Gadelha M.R. Liberman B. Correa H. Romano-Silva M.A. Friedman E. Silva F.F. Ribeiro P.A. De Marco L. Novel vasopressin type 2 (AVPR2) gene mutations in Brazilian nephrogenic diabetes insipidus patients.Genet. Test. 2006; 10: 157-162Crossref PubMed Scopus (11) Google Scholar, 25Carroll P. Al-Mojalli H. Al-Abbad A. Al-Hassoun I. Al-Hamed M. Al-Amr R. Butt A.I. Meyer B.F. Novel mutations underlying nephrogenic diabetes insipidus in Arab families.Genet. Med. 2006; 8: 443-447Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 26Kalenga K. Persu A. Goffin E. Lavenne-Pardonge E. van Cangh P.J. Bichet D.G. Devuyst O. Intrafamilial phenotype variability in nephrogenic diabetes insipidus.Am. J. Kidney Dis. 2002; 39: 737-743Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 27Schoneberg T. Schulz A. Biebermann H. Gruters A. Grimm T. Hubschmann K. Filler G. Gudermann T. Schultz G. V2 vasopressin receptor dysfunction in nephrogenic diabetes insipidus caused by different molecular mechanisms.Hum. Mutat. 1998; 12: 196-205Crossref PubMed Scopus (63) Google Scholar, 28Shoji Y. Takahashi T. Suzuki Y. Suzuki T. Komatsu K. Hirono H. Yokoyama T. Kito H. Takada G. Hum. Mutat. 1998; 1: S278-S283Crossref PubMed Scopus (10) Google Scholar), which causes complete NDI; R137L (29Feldman B.J. Rosenthal S.M. Vargas G.A. Fenwick R.G. Huang E.A. Matsuda-Abedini M. Lustig R.H. Mathias R.S. Portale A.A. Miller W.L. Gitelman S.E. Nephrogenic syndrome of inappropriate antidiuresis.N. Engl. J. Med. 2005; 352: 1884-1890Crossref PubMed Scopus (266) Google Scholar), which causes the neonatal syndrome of inappropriate antidiuresis (NSIAD), and S329R (7Faerch M. Christensen J.H. Corydon T.J. Kamperis K. de Zegher F. Gregersen N. Robertson G.L. Rittig S. Clin. Endocrinol. 2008; 68: 395-403Crossref PubMed Scopus (27) Google Scholar), which is located near to Ser-333del and causes partial NDI. The characteristics of these V2R mutants and the effects of V2R antagonists OPC41061 (OPC4) and OPC31260 (OPC3) (30Macion-Dazard R. Callahan N. Xu Z. Wu N. Thibonnier M. Shoham M. Mapping the binding site of six nonpeptide antagonists to the human V2-renal vasopressin receptor.J. Pharmacol. Exp. Ther. 2006; 316: 564-571Crossref PubMed Scopus (49) Google Scholar, 31Robben J.H. Sze M. Knoers N.V. Deen P.M. Functional rescue of vasopressin V2 receptor mutants in MDCK cells by pharmacochaperones. Relevance to therapy of nephrogenic diabetes insipidus.Am. J. Physiol. Renal Physiol. 2007; 292: F253-F260Crossref PubMed Scopus (77) Google Scholar) enabled us to elucidate the molecular defects underlying partial NDI and reveal a unique example of protean agonism.EXPERIMENTAL PROCEDURESPatient ProfilesPatient 1A 7-year-old boy who had been diagnosed with psychogenic polydipsia was admitted to the department of pediatrics in our hospital to evaluate the underlying cause of his polydipsia and polyuria using a water deprivation test followed by a DDAVP loading test (Fig. 1A), which showed an apparently normal response. On the basis of the effectiveness of the DDAVP treatment and the high levels of ADH, his V2 receptor was genotyped, and a Y128S mutation was identified (Fig. 1B).FIGURE 2cAMP accumulation and immunofluorescence microscopy analysis of V2R-WT and V2R mutant-expressing cells. A, cAMP accumulation associated with the partial NDI mutant, the control mutant, and WT V2R. COS-7 cells (1 × 106 cells) were transiently transfected with V2R-WT, control V2R mutants (V2R-R137L, V2R-R137H), and partial NDI mutants (V2R-Ser-333del, V2R-Y128S, and V2R-S329R) (0.2 μg of pcDNA3 containing each myc-tagged V2R was used). 24 h later, the cells were reseeded into 24-well plates at 0.75 × 105 cells/well to which [3H]adenine (2 μCi/ml, Amersham Biosciences) was added. 48 h later, after washing in assay medium with 1 mm isobutylmethylxanthine, the cells were incubated for 30 min at 37 °C in serum-free medium with or without increasing concentrations of AVP (from 1 nm to 10 μm). cAMP accumulation was measured as described under “Experimental Procedures.” Data are mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01. B, immunofluorescence microscopy analysis of COS-7 cells transiently expressing myc-tagged V2R-WT or the indicated V2R mutant was performed as described under “Experimental Procedures.” C, colocalization of myc-tagged V2R-Ser-333del or V2R-Y128S with ER marker. Immunofluorescence microscopy analysis of COS-7 cells transiently expressing myc-tagged V2R-WT or the indicated V2R mutant was performed using confocal microscopy, and fluorescence images were collected either with anti-myc antibody or with anti-protein disulfide isomerase (ER marker) antibody as described under “Experimental Procedures.” Each set of results is representative of at least two additional experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Patient 2A 3-year-old boy who had been diagnosed with psychogenic polydipsia was admitted to our hospital for an evaluation of the polydipsia and polyuria using water deprivation and DDAVP loading tests (Fig. 1A). Although laboratory testing data appeared to be compatible with psychogenic polydipsia, his basal ADH was high (Fig. 1A). His V2R was therefore sequenced and revealed a Ser-333 deletion (Ser-333del, Fig. 1B).Pharmacological ChaperonesThe V2R antagonists OPC4 and OPC3 (30Macion-Dazard R. Callahan N. Xu Z. Wu N. Thibonnier M. Shoham M. Mapping the binding site of six nonpeptide antagonists to the human V2-renal vasopressin receptor.J. Pharmacol. Exp. Ther. 2006; 316: 564-571Crossref PubMed Scopus (49) Google Scholar, 31Robben J.H. Sze M. Knoers N.V. Deen P.M. Functional rescue of vasopressin V2 receptor mutants in MDCK cells by pharmacochaperones. Relevance to therapy of nephrogenic diabetes insipidus.Am. J. Physiol. Renal Physiol. 2007; 292: F253-F260Crossref PubMed Scopus (77) Google Scholar), were kindly provided by Otsuka Pharmaceutical Company.Expression Constructs, Cell Culture, and TransfectionAn expression construct encoding a myc-tagged wild-type V2R was kindly provided by M. Bouvier (Université de Montreal, Canada), and each mutant causative for diabetes insipidus was generated using a manual procedure (Stratagene, La Jolla, CA). COS-7 cells, maintained in DMEM containing 10% (v/v) fetal bovine serum, were transfected with constructs encoding myc-tagged WT-V2R or myc-tagged mutant V2R using Lipofectamine 2000 (Invitrogen).cAMP AssaycAMP accumulation in COS-7 cells transiently expressing WT-V2R and each V2R mutant was assayed as described previously (32Iiri T. Herzmark P. Nakamoto J.M. van Dop C. Bourne H.R. Rapid GDP release from Gs α in patients with gain and loss of endocrine function.Nature. 1994; 371: 164-168Crossref PubMed Scopus (243) Google Scholar, 33Iiri T. Farfel Z. Bourne H.R. Conditional activation defect of a human Gs α mutant.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 5656-5661Crossref PubMed Scopus (48) Google Scholar, 34Iiri T. Bell S.M. Baranski T.J. Fujita T. Bourne H.R. A Gs α mutant designed to inhibit receptor signaling through Gs.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 499-504Crossref PubMed Scopus (57) Google Scholar, 35Makita N. Sato J. Rondard P. Fukamachi H. Yuasa Y. Aldred M.A. Hashimoto M. Fujita T. Iiri T. Human G(sα) mutant causes pseudohypoparathyroidism type Ia/neonatal diarrhea, a potential cell-specific role of the palmitoylation cycle.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 17424-17429Crossref PubMed Scopus (21) Google Scholar). Briefly, 1 day after transfection, cells were reseeded into 24-well plates to which [3H]adenine (2 μCi/ml) was added. 16 h before performing the assay, the cells were pretreated with OPC3 or OPC4 at the indicated concentrations. 24 h later, the cells were washed and incubated in 0.5 ml of assay medium containing 1 mm isobutylmethylxanthine with or without the indicated concentrations of vasopressin, OPC3, or OPC4. Reactions were terminated after 30 min by removing the medium and lysing the cells in 5% trichloroacetic acid containing ATP and cAMP (1 mm each). [3H]cAMP and [3H]ATP were separated on AG 50W-X4 Dowex and alumina columns, and the data are presented as the ratio of [3H]cAMP to [3H]cAMP plus [3H]ATP, as described previously (32Iiri T. Herzmark P. Nakamoto J.M. van Dop C. Bourne H.R. Rapid GDP release from Gs α in patients with gain and loss of endocrine function.Nature. 1994; 371: 164-168Crossref PubMed Scopus (243) Google Scholar, 33Iiri T. Farfel Z. Bourne H.R. Conditional activation defect of a human Gs α mutant.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 5656-5661Crossref PubMed Scopus (48) Google Scholar, 34Iiri T. Bell S.M. Baranski T.J. Fujita T. Bourne H.R. A Gs α mutant designed to inhibit receptor signaling through Gs.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 499-504Crossref PubMed Scopus (57) Google Scholar, 35Makita N. Sato J. Rondard P. Fukamachi H. Yuasa Y. Aldred M.A. Hashimoto M. Fujita T. Iiri T. Human G(sα) mutant causes pseudohypoparathyroidism type Ia/neonatal diarrhea, a potential cell-specific role of the palmitoylation cycle.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 17424-17429Crossref PubMed Scopus (21) Google Scholar).Cell Surface ELISAThe cell surface expression of exogenous receptors in COS-7 cells transiently transfected with myc-tagged WT-V2R or each V2R mutant was quantified by ELISA (36Bernier V. Lagacé M. Lonergan M. Arthus M.F. Bichet D.G. Bouvier M. Functional rescue of the constitutively internalized V2 vasopressin receptor mutant R137H by the pharmacological chaperone action of SR49059.Mol. Endocrinol. 2004; 18: 2074-2084Crossref PubMed Scopus (129) Google Scholar, 37Bernier V. Morello J.P. Zarruk A. Debrand N. Salahpour A. Lonergan M. Arthus M.F. Laperrière A. Brouard R. Bouvier M. Bichet D.G. Pharmacologic chaperones as a potential treatment for X-linked nephrogenic diabetes insipidus.J. Am. Soc. Nephrol. 2006; 17: 232-243Crossref PubMed Scopus (186) Google Scholar). Briefly, 1 day after transfection, cells were reseeded into 12-well plates. 16 h before performing the assay, cells were pretreated with OPC3 or OPC4 at the indicated concentrations. After 24 h, the cells were then washed twice with ice-cold 1% BSA/PBS twice, placed on ice for 5 min, and incubated in an anti-myc antibody solution (1:4000) diluted in 1% BSA/PBS for 1 h at 4 °C. This was followed by two further washes in 1% BSA/PBS. The cells were next fixed at 4 °C for 15 min in 4% paraformaldehyde/PBS and again washed twice with 1% BSA/PBS and incubated in anti-mouse HRP-conjugated secondary antibody solution (1:12000) diluted in 1% BSA/PBS at room temperature for 1 h. This was followed by two washes in 1% BSA/PBS for 20 min and a final wash in PBS. Finally, the cells were treated with substrate (o-phenylenediamine dihydrochloride, Sigma) for 5 min at room temperature. This reaction was stopped by the addition of an equivalent volume of 2.5 n HCl. The absorption levels were then read at 492 nm using a plate reader (Bio-Rad).Immunofluorescence MicroscopyImmunofluorescence staining was performed as described previously (18Makita N. Sato J. Manaka K. Shoji Y. Oishi A. Hashimoto M. Fujita T. Iiri T. An acquired hypocalciuric hypercalcemia autoantibody induces allosteric transition among active human Ca-sensing receptor conformations.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 5443-5448Crossref PubMed Scopus (85) Google Scholar, 37Bernier V. Morello J.P. Zarruk A. Debrand N. Salahpour A. Lonergan M. Arthus M.F. Laperrière A. Brouard R. Bouvier M. Bichet D.G. Pharmacologic chaperones as a potential treatment for X-linked nephrogenic diabetes insipidus.J. Am. Soc. Nephrol. 2006; 17: 232-243Crossref PubMed Scopus (186) Google Scholar). Briefly, transiently transfected COS-7 cells were placed onto glass coverslips. 16 h before performing the assay, cells were pretreated with OPC3 or OPC4 at the indicated concentrations. Cells were then washed with PBS twice, fixed with ice-cold 100% methanol, and incubated at −20 °C for 2 min to evaluate whole cell expression (with permeabilization). To evaluate cell surface expression (without permeabilization), the cells were fixed at 4 °C for 15 min in 4% paraformaldehyde/PBS. After two further washes in PBS, the cells were blocked using Block Ace at room temperature for 20 min and sequentially incubated with anti-myc antibody (1:4000) or anti PDI (protein disulfide isomerase, an ER marker) antibody (1:4000) diluted in 20% Block Ace/PBS at 4 °C overnight. The cells were then further washed twice in 20% Block Ace/PBS and incubated with anti-mouse antibody 488 (for anti-myc antibody) or anti-rabbit antibody 594 (for anti-PDI antibody) diluted in 20% Block Ace at room temperature for 1 h. After two additional washes in 0.1% Tween 20/PBS and once in PBS alone, the cells were mounted on glass slides for analysis.Genetic Analysis of the V2 Receptor Gene (AVPR2) in Two PatientsGenetic analysis of the V2 receptor gene was performed under approval of the Institutional Review Board of the University of Tokyo. After written informed consent was obtained from the parents of the patients, genomic DNA was prepared from the peripheral blood leukocytes with the use of a DNA isolation kit, and all exons and exon-intron boundaries of AVPR2 were analyzed using a PCR direct sequencing method.Water Deprivation Test and DDAVP Administration TestWater deprivation tests were performed as follows. Patients were allowed to drink water freely prior to commencing the test. After the final urination, the test was started. Urine samples were collected at hourly intervals until 6 h, accompanied by the measurement of body weight, serum osmolarity, plasma concentration of ADH, and urine volume. One day after the fluid deprivation test, DDAVP (5 units/m2) was administered subcutaneously. Samples were collected before the test and at 30, 60, and 120 min after DDAVP administration. The urine volume, gradation, osmolarity, and concentration of cAMP with serum osmolarity were then measured.Statistical Method and AnalysisThe statistical method used was the Dunnett's multiple comparison procedure. To evaluate the dose response, Williams multiple comparison was used. Averaged data of three independent experiments are shown, and error bars represent mean ± S.D. These analyses were performed using Microsoft Excel software. *, p < 0.05; **, p < 0.01.DISCUSSIONIn this study, we have identified two mutations in the V2R gene in patients who had been initially diagnosed with psychogenic polydipsia but were finally diagnosed with partial NDI. On the basis of the clinical phenotypes associated with the partial NDI in these cases, we suspected that the receptor mutant proteins must be expressed and that an analysis of their functional properties may provide further clues to the mechanisms underlying normal V2R functions. Our intact cell experiments showed that both mutant proteins were indeed expressed but did not localize at the cell surface. Pr" @default.
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• W1999532619 date "2012-01-01" @default.
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• W1999532619 title "V2 Vasopressin Receptor (V2R) Mutations in Partial Nephrogenic Diabetes Insipidus Highlight Protean Agonism of V2R Antagonists" @default.
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• W1999532619 doi "https://doi.org/10.1074/jbc.m111.268797" @default.
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