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• W1990398484 abstract "To understand the mechanisms of G protein-coupled receptor delivery and steady state localization, we examined the trafficking itineraries of wild type (WT) and mutant V2 vasopressin receptors (V2Rs) in polarized Madin-Darby canine kidney II (MDCK II) cells and in COS M6 cells; the mutant V2Rs represent selected alleles responsible for X-linked nephrogenic diabetes insipidus. The WT V2R is localized on the plasma membrane and mediates arginine vasopressin (AVP)-stimulated cAMP accumulation, whereas the clinically relevant V2R mutants, L292P V2R, ΔV278 V2R, and R337X V2R, are retained intracellularly, are insensitive to extracellularly added AVP, and are not processed beyond initial immature glycosylation, manifest by their endoglycosidase H sensitivity. Reduced temperature and pharmacological, but not chemical, strategies rescue mutant V2Rs to the cell surface of COS M6 cells; surface rescue of L292P V2R and R337X V2R, but not of ΔV278 V2R, parallels acquisition of AVP-stimulated cAMP production. Pharmacological rescue of the L292P or R337X V2R by incubation with the membrane-permeant V2R antagonist, SR121463B, leads to a mature glycosylated form of the receptor that achieves localization on the basolateral surface of polarized MDCK II cells indistinguishable from that of the WT V2R. Surprisingly, however, the immature form of the mutant L292P V2R escapes to the apical, but not basolateral, surface of polarized MDCK II cells, even in the absence of SR121463B. These findings are consistent with the interpretation that the receptor conformation that allows appropriate processing through the N-linked glycosylation pathway is also essential for V2R targeting to the appropriate surface of polarized epithelial cells. To understand the mechanisms of G protein-coupled receptor delivery and steady state localization, we examined the trafficking itineraries of wild type (WT) and mutant V2 vasopressin receptors (V2Rs) in polarized Madin-Darby canine kidney II (MDCK II) cells and in COS M6 cells; the mutant V2Rs represent selected alleles responsible for X-linked nephrogenic diabetes insipidus. The WT V2R is localized on the plasma membrane and mediates arginine vasopressin (AVP)-stimulated cAMP accumulation, whereas the clinically relevant V2R mutants, L292P V2R, ΔV278 V2R, and R337X V2R, are retained intracellularly, are insensitive to extracellularly added AVP, and are not processed beyond initial immature glycosylation, manifest by their endoglycosidase H sensitivity. Reduced temperature and pharmacological, but not chemical, strategies rescue mutant V2Rs to the cell surface of COS M6 cells; surface rescue of L292P V2R and R337X V2R, but not of ΔV278 V2R, parallels acquisition of AVP-stimulated cAMP production. Pharmacological rescue of the L292P or R337X V2R by incubation with the membrane-permeant V2R antagonist, SR121463B, leads to a mature glycosylated form of the receptor that achieves localization on the basolateral surface of polarized MDCK II cells indistinguishable from that of the WT V2R. Surprisingly, however, the immature form of the mutant L292P V2R escapes to the apical, but not basolateral, surface of polarized MDCK II cells, even in the absence of SR121463B. These findings are consistent with the interpretation that the receptor conformation that allows appropriate processing through the N-linked glycosylation pathway is also essential for V2R targeting to the appropriate surface of polarized epithelial cells. Extensive investigation has revealed several mechanisms that modulate G protein-coupled receptor (GPCR) 1The abbreviations used are: GPCR, G protein-coupled receptor; α2-AR, α2-adrenergic receptor; AVP, arginine vasopressin; Δ, deletion (as in ΔV278, deletion of Val278); Endo H, endoglycosidase H; HA, hemagglutinin; HERG, human ether-a-go-go-related gene; MDCK, Madin-Darby canine kidney; NDI, nephrogenic diabetes insipidus; NHS, sulfo-N-hydroxysuccinimide; PBS, phosphate-buffered saline; PNGase F, peptide:N-glycosidase F; X, Stop (as in R337X, Arg337 → Stop); V2R, V2 vasopressin receptor; WT, wild type; DMEM, Dulbecco's modified Eagle's medium; TEMED, N,N,N′,N′-tetramethylethylenediamine. responsiveness following agonist occupancy, including receptor relocalization (reviewed in Refs. 1Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar and 2Tsao P. Zastrow M. Curr. Opin. Neurobiol. 2000; 10: 365-369Crossref PubMed Scopus (135) Google Scholar). However, less attention has focused on the molecular mechanisms accounting for how receptors achieve localization in the agonist-naive state. A key determinant governing the specificity of GPCR signaling entails appropriate receptor localization on the cell surface, permitting access to requisite ligands and signal transduction machinery. Cell surface localization is governed by two predominant mechanisms: 1) receptor delivery to a particular site and 2) retention at that site. The functional importance of GPCR localization is emphasized by diseases that result from receptor mislocalization, such as retinitis pigmentosa, X-linked nephrogenic diabetes insipidus (NDI), and hypogonadotropic hypogonadism (3Leanos-Miranda A. Janovick J.A. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 4825-4828Crossref PubMed Scopus (109) Google Scholar), which result from intracellular accumulation of mutant rhodopsin, the V2 vasopressin receptor (V2R), or the gonadotropin-releasing hormone receptor, respectively. Our previous studies have exploited α2-AR subtypes as a model for characterization of GPCR trafficking and localization because of their different trafficking itineraries in polarized cells and in response to agonist (reviewed in Ref. 4Saunders C. Limbird L.E. Pharmacol. Ther. 1999; 84: 193-205Crossref PubMed Scopus (111) Google Scholar). All three α2-AR subtypes (α2A-, α2B-, and α2C-AR) are located, at steady state, at the basolateral surface in polarized Madin-Darby canine kidney (MDCK) II cells, analogous to their localization in vivo (5Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar, 6Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). However, they achieve this basolateral localization via different trafficking itineraries. Whereas the α2A-AR and α2C-AR are directly targeted to the basolateral surface, the α2B-AR is randomly distributed to both the apical and basolateral surface and then selectively retained at the lateral subdomain (6Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Truncations of the α2A-AR and chimeras with the apically targeted A1 adenosine receptor reveal that α2A-AR targeting to the basolateral surface relies upon multiple, non-contiguous, membrane-embedded sequences within or near the lipid bilayer (7Keefer J.R. Kennedy M.E. Limbird L.E. J. Biol. Chem. 1994; 269: 16425-16432Abstract Full Text PDF PubMed Google Scholar, 8Saunders C. Keefer J.R. Bonner C.A. Limbird L.E. J. Biol. Chem. 1998; 273: 24196-24206Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), suggesting that a three-dimensional surface provides the basis for interaction with trafficking molecules. Consequently, it can be reasoned that no single linear sequence can be exploited to identify receptor targeting machinery. Thus, to elucidate the mechanisms involved in basolateral delivery of GPCRs, we explored the human V2R and naturally occurring point mutations in the V2R responsible for the pathogenesis of X-linked NDI. Many of the >160 human mutations described to date cause the receptor to be retained intracellularly (9Oksche A. Rosenthal W. J. Mol. Med. 1998; 76: 326-337Crossref PubMed Scopus (117) Google Scholar), unresponsive to its physiological ligand arginine vasopressin (AVP). We examined the trafficking and localization of the wild type V2R and three intracellularly retained V2R mutants to assess whether these mutants could be spatially and functionally rescued and whether cell surface rescue was also paralleled by localization on the appropriate surface of polarized MDCK II cells. Materials—Human wild type (WT) and mutant V2R cDNAs were graciously provided by Dr. Jürgen Wess or were constructed by overlap extension PCR mutagenesis. EASYTAG [35S]-EXPRE35SS protein labeling mix (1175 Ci/mmol), [2,8-3H]adenine (30.4 Ci/mmol), 8-arginine [phenylalanyl-3,4,5-3H]vasopressin (68.5 Ci/mmol), [8-14C]adenosine 3′,5′-cyclic phosphate ammonium salt (51.2 mCi/mmol), [methoxy-3H]inulin-methoxy (430 mCi/g), EN3HANCE autoradiography enhancer, and Entensify™ autoradiography enhancer were purchased from PerkinElmer Life Sciences. DEAE-dextran was from Amersham Biosciences. Paraformaldehyde (16% solution, EM grade) was from Electron Microscopy Sciences (Washington, PA). Polyvinylidene difluoride nylon membranes were from Millipore Corp. (Bedford, MA). Dowex AG50 W-X4 resin, 40% acrylamide, TEMED, and ammonium persulfate were from Bio-Rad. Adenosine 3′,5′-cyclic monophosphate (sodium salt), alumina, aprotinin, [Arg8]vasopressin (acetate salt), bacitracin, bovine serum albumin, chloroquine (diphosphate salt), fetal calf serum, 3-isobutyl-1-methylxanthine, leupeptin, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and Triton X-100 were from Sigma. The protein A-purified mouse 12CA5 monoclonal antibody (1 μg/μl) and mouse HA.11 monoclonal antibody (5 μg/μl) directed against the hemagglutinin (HA) epitope tag engineered into the amino terminus of the various V2R structures was obtained from Babco, and the Cy3-conjugated donkey anti-mouse IgG (2 μg/μl) was from Jackson Immunochemicals (West Grove, PA). Rat anti-HA monoclonal antibody (100 μg/ml; clone 3F10) against the HA epitope tag was obtained from Roche Applied Science, and the Alexafluor-488-conjugated goat anti-rat IgG (2 μg/μl) was from Molecular Probes, Inc. (Eugene, OR). Protein A-agarose beads were from Vector (Burlingame, CA). EZ-link™ sulfo-N-hydroxysuccinimide (NHS)-Biotin and Immunopure™ immobilized streptavidin-agarose were from Pierce. The 12- and 24.5-mm polycarbonate membrane filters (Transwell chambers; 0.4-μm pore size) were obtained from Costar (Cambridge, MA). Aqua-Poly/Mount was from PolySciences Inc. (Warrington, PA). Dulbecco's modified Eagle's medium (DMEM) and trypsin/EDTA were prepared by the cell culture core facility sponsored by the Diabetes Research and Training Center at Vanderbilt University Medical Center. All other chemicals were reagent grade. Cell Lines—Permanent clonal MDCK II cell lines expressing HA epitope-tagged WT and mutant V2Rs were developed using the CaPO4 method as described previously (7Keefer J.R. Kennedy M.E. Limbird L.E. J. Biol. Chem. 1994; 269: 16425-16432Abstract Full Text PDF PubMed Google Scholar). Briefly, 10 μg of V2pcD-N-HA, pCMV4N-V2R-L292P, pCMV4N-V2R-R337X, or pCMV4N-V2R-ΔV278 (individual cDNAs encoding HA epitope-tagged human wild type (WT) or mutant (L292P, R337X, and ΔV278) V2Rs, respectively) were each co-transfected with 2 μg of pRSVneo (cDNA encoding neomycin resistance) into MDCK II cells. Colonies were selected based on resistance to G418, a neomycin analog, and isolated as described previously (7Keefer J.R. Kennedy M.E. Limbird L.E. J. Biol. Chem. 1994; 269: 16425-16432Abstract Full Text PDF PubMed Google Scholar). G418-resistant colonies were screened for WT V2R expression by assaying binding of the radioligand [3H]AVP. V2R mutant-expressing cell lines were screened via immunofluorescence against the HA epitope, using either the mouse 12CA5 monoclonal antibody or the rat anti-HA monoclonal antibody. Parental and stably expressing WT V2R MDCK II cells were maintained in DMEM supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C, 5% CO2. Simian kidney fibroblast (COS M6) cells were maintained in supplemented DMEM plus 20 mm HEPES. The studies presented were obtained in the WT V2R clonal cell line number 61, which expresses the HA epitope-tagged WT V2R at a density of 12.6 pmol of specific [3H]AVP binding/mg of protein, estimated in saturation binding studies. Binding density could not be determined for the mutant V2R cell lines because a decreased V2R affinity inherent to the L292P V2R and R337X V2R means that [3H]AVP binding is untrappable for these receptors. Further, we have no evidence that the ΔV278 V2R binds AVP, since AVP-stimulated cAMP production is not observed following temperature or pharmacological rescue of this mutant receptor to the surface of COS M6 cells. However, relative V2R density could be qualitatively obtained by immunoblotting for the HA epitope in resolved lysates derived from cells expressing the heterologous V2Rs. If WT V2R total cell content under control conditions is defined as 1.0, then L292P (clone 30F) is 0.28 of the density of WT-expressing MDCK II cells in the absence of SR121463B rescue and 0.31 of this density after pharmacological rescue. The R337X V2R is 0.55 of control V2R expression but increases to 1.18 of control V2R expression with SR121463B treatment. For comparison, the WT V2R increases its total cell content from 1.0 (as defined) under control conditions to 1.06 following overnight SR121463B treatment. Transient Expression Studies—COS M6 cells were seeded the day prior to transfection at a density of 3.5 × 105 (35-mm dish), 2.0 × 106 (100-mm dish), or 4.5 × 106 (150-mm dish). On the day of transfection, cells were rinsed in PBS prior to incubation with a mixture containing 0.67, 4, or 9 μg of plasmid DNA, respectively, with 500 μg/ml DEAE-dextran for 20 min at 37 °C, 5% CO2. The DNA/DEAE-dextran mixture was aspirated and replaced with fresh DMEM supplemented with 20 mm HEPES and 100 μm chloroquine and maintained at 37 °C, 5% CO2 for 2 h. At this time, the medium was aspirated, and the cells were subjected to an Me2SO shock (10% Me2SO in DMEM, 3 min at 37 °C) before replacing with 10% serum-supplemented DMEM plus 20 mm HEPES prior to assessment 72 h post-transfection. Sulfo-NHS-Biotin Surface Labeling Strategy—Assessment of steady-state localization of the WT V2R, R337X V2R, and L292P V2R in stably expressing MDCK II cell lines was accomplished via covalent labeling of the apical or basolateral surface of polarized MDCK II cells with sulfo-NHS-biotin exactly as described previously. Integrity of the cell monolayer was determined via [3H]methoxyinulin leak assays (7Keefer J.R. Kennedy M.E. Limbird L.E. J. Biol. Chem. 1994; 269: 16425-16432Abstract Full Text PDF PubMed Google Scholar). Delivery to Cell Surfaces of Polarized MDCK II Cells—Delivery of nascent V2R to the cell surface was examined by metabolic labeling and surface biotinylation strategies, essentially as described previously (6Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 7Keefer J.R. Kennedy M.E. Limbird L.E. J. Biol. Chem. 1994; 269: 16425-16432Abstract Full Text PDF PubMed Google Scholar). Polarized MDCK II cells expressing WT V2R were incubated in cysteine/methionine-free medium for 2 h prior to a 90-min pulse in Cys/Met-free medium supplemented with 1 μCi/μl [35S]-EXPRE35SS protein labeling mix. At the end of the pulse phase, Transwells were biotinylated at either the apical or basolateral surface with two sequential rounds of sulfo-NHS-Biotin (1 mg/ml). Cells were extracted into ice-cold dodecyl-β-d-maltoside/cholesteryl hemisuccinate buffer (4 and 0.8 mg/ml, respectively, containing 20% glycerol, 25 mm glycylglycine, 20 mm Hepes, 100 mm NaCl, 5 mm EGTA, 100 μm phenylmethylsulfonyl fluoride, 1 μg/ml soybean trypsin inhibitor, 1 μg/ml leupeptin, pH 7.4) and centrifuged at 100,000 × g for 60 min, and the supernatant was sequentially incubated with mouse 12CA5 anti-HA antibody (1:50) and adsorbed to protein A and streptavidin-agarose (10Wilson M.H. Limbird L.E. Biochemistry. 2000; 39: 693-700Crossref PubMed Scopus (32) Google Scholar). The washed resin was eluted with radioimmune precipitation assay buffer (150 mm NaCl, 50 mm Tris, pH 8.0, 5 mm EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) and the above protease inhibitors at 90 °C. The biotinylated protein eluate was subjected to SDS-PAGE on 7.5% polyacrylamide gels. The gels were incubated for 60 min in EN3HANCE prior to drying and exposure to Eastman Kodak Co. X-Omat film. Data shown are from autoradiograms. Surface Localization of V2R—Biotinylation strategies also can allow quantitation of the relative amount of surface versus internalized receptors (MDCK II; COS M6), or apical versus basolateral receptors (MDCK II), even without metabolic labeling. For these studies, one 60-mm dish of COS M6 cells or the appropriate number of 24.5-mm Transwells of polarized (7 days of growth) WT or mutant V2R in MDCK II cells were biotinylated with sulfo-NHS-biotin (1 mg/ml) on the apical or basolateral surface as described previously (6Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 7Keefer J.R. Kennedy M.E. Limbird L.E. J. Biol. Chem. 1994; 269: 16425-16432Abstract Full Text PDF PubMed Google Scholar). MDCK II cells plated in Transwells were pretreated overnight with 10 μm SR121463B to achieve pharmacological rescue or not (control) prior to surface biotinylation. The cells were scraped into radioimmune precipitation assay buffer with protease inhibitors present, and the extracts were centrifuged at 100,000 × g. The supernatant (detergent-solubilized preparation) was incubated overnight at 4 °C with streptavidin-agarose. The streptavidin-agarose resin was washed and eluted as above (10Wilson M.H. Limbird L.E. Biochemistry. 2000; 39: 693-700Crossref PubMed Scopus (32) Google Scholar), and the eluate was resolved by SDS-PAGE on 12% gels. The resolved proteins were transferred to polyvinylidene difluoride as described previously, and the biotinylated, epitope-tagged V2R was identified by Western blot analysis using mouse HA.11 monoclonal antibody against the HA epitope. Digestion of V2R with Endoglycosidases—Proteins were eluted from protein A-agarose (for Fig. 10) or from streptavidin agarose (data not shown) using a buffer consisting of 0.5% SDS and 1% β-mercaptoethanol. Endoglycosidase treatment was according to the protocol provided by New England Biolabs. Briefly, protein eluates were treated with Endoglycosidase H (1000 units) in G5 enzyme buffer (0.05 m sodium citrate, pH 5.5) or treated with peptide:N-glycosidase F (PNGase F) (1000 units) in G7 enzyme buffer (0.05 m sodium phosphate, pH 7.5, plus 1% Nonidet P-40). Incubations were for 30–60 min at 37 °C. Immunolocalization of the Wild Type and Mutant V2R—Stably expressing WT V2R MDCK II cells or transiently transfected COS M6 cells were grown on 12-mm glass coverslips (COS M6 cells) or in 12-mm Transwell chambers (MDCK II cells) and maintained for 2 days (coverslips) or 5 days (Transwells) prior to fixing with 4% paraformaldehyde for 20 min. Paraformaldehyde-fixed cells were washed in PBS, rinsed with 50 mm NH4Cl in PBS for 15 min, and permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature. Intact and permeabilized cells were washed once with PBS and once with PBS containing 2% bovine serum albumin prior to incubation with a 1:50 dilution of mouse 12CA5 monoclonal primary antibody for 1 h at room temperature. After rinsing cells three times in PBS (10 min/wash), a 1:1000 dilution of Cy3-conjugated donkey anti-mouse IgG in PBS with 2% bovine serum albumin was added as the secondary antibody to the cells and incubated in the dark for 1 h at room temperature. In some studies, a 1:1000 dilution of rat anti-HA monoclonal primary antibody and a 1:1000 dilution of secondary Alexafluor-488-conjugated goat anti-rat IgG were employed. Cells were then washed prior to mounting on glass slides with Aqua-Poly/Mount. The figure legends indicate whether mouse 12CA5 or rat anti-HA was used as the primary antibody. Slides were stored in the dark until examining on a Leitz fluorescent microscope using a 40× oil immersion lens at 1.5× magnification or on a Zeiss LSM 410 confocal laser-scanning inverted microscope in the Vanderbilt Cell Imaging Core Facility. cAMP Accumulation—Basal or AVP-mediated cAMP accumulation in intact cells was measured by assessing the conversion of [3H]ATP into [3H]cAMP. COS M6 cells in 150-mm dishes were trypsinized the day following transfection and seeded into 24-well plates. Twenty-four hours later, the cell culture medium was aspirated, and wells were labeled overnight (12–16 h at 37 °C) with 3 μCi/ml [3H]adenine in DMEM, DMEM plus 10% glycerol, or DMEM plus 10 μm SR121463B. To assess the ability of reduction in culture temperature to enhance surface expression of mutant V2R, [3H]adenine-labeled cells were maintained for 12–16 h at 28 °C; wild type cells were treated similarly and served as controls. Functional studies of AVP-stimulated cAMP production before and after rescue were restricted to COS M6 cells. Although we could achieve surface rescue with SR121463B in MDCK II cells, we were not able to observe AVP-stimulated cAMP accumulation in polarized (Transwell) or unpolarized (plated) MDCK II cultures following overnight treatment with SR121463B due to inadequate wash-out of this membranepermeant antagonist. Rescue by lowering culture temperature is not efficient in MDCK II cells, in contrast to COS M6 cells. Using the concentration response for AVP-stimulated cAMP accumulation in WT cells as a monitor of residual SR121463B, we found that wash protocols that had successfully removed SR121463B in COS M6 cell experiments to allow subsequent detection of AVP-stimulated cAMP accumulation were not sufficient in removing the V2R antagonist from MDCK II cells. Residual antagonist masked AVP-stimulated cAMP accumulation, even in WT V2R-expressing cells, which are highly sensitive to AVP. Extended wash protocols with or without bovine serum albumin and/or serum also did not successfully remove residual SR121463B from MDCK II cells after overnight incubation. We do not have an explanation for this observation other than the realization that the density of V2Rs in MDCK II cells (∼12 pmol/mg for WT V2R) represents the density of the receptor in all cells, since this is a clonal cell line, whereas the apparent density (also ∼12 pmol/mg for WT V2R) in transiently expressing COS M6 cells represents a mean of all densities for cells overexpressing the receptor (i.e. ≫ 12 pmol/mg) and cells expressing no receptor. Perhaps the high receptor expression per cell in COS M6 cells fosters receptor sensitivity to AVP even in the presence of some residual SR121463B. Forskolin does elevate cAMP in WT and mutant V2R-expressing MDCK II cells before and after SR121463B treatment, indicating that the adenylyl cyclase system is operative in the cells and that our assay is able to detect cAMP changes in MDCK II cells if they occur. AVP also readily stimulates cAMP production in WT V2R-expressing MDCK II cells when not pretreated with SR121463B as a “rescue” treatment. On the day of the assay of cAMP accumulation, COS M6 cells were incubated with prewarmed cell culture medium containing 0.1% bovine serum albumin followed by a single wash with medium. Cells were then washed with prewarmed PBS and incubated with PBS plus 500 μm 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, for 5 min. Following aspiration, cells were treated with selected concentrations of AVP for 15 min in a final assay volume of 200 μl. Reactions were terminated by adding 750 μl of an ice-cold solution containing 12% trichloroacetic acid, 2 mm cAMP, 2 mm ATP, and [14C]cAMP (∼1600 cpm/ml) to permit an assessment of recovery of [3H]cAMP in subsequent purification steps. Cells were then placed on ice for 10 min, after which the reactions were transferred to glass test tubes using disposable transfer pipettes. Each well was washed with 1.05 ml of H2O and combined with its respective harvested sample. After neutralization with 120 μl of 5 n NaOH, cellular debris was pelleted by centrifugation (3000 rpm, 10 min at 4 °C). The supernatants were subjected to sequential Dowex and alumina column chromatography to isolate [3H]cAMP. Data are presented as [3H]cAMP/([3H]ATP + [3H]ADP). The value of [3H]ATP + [3H]ADP was estimated from the 3H cpm eluted from the Dowex column as pass-through (11Johnson R.A. Salomon Y. Methods Enzymol. 1991; 195: 3-21Crossref PubMed Scopus (122) Google Scholar). To compare trafficking of the HA epitope-tagged WT V2R (referred to as WT V2R throughout) with clinically relevant V2R mutations, we examined the localization of these structures following transient expression in COS M6 cells. Immunostaining of intact cells (no Triton X-100 in the incubation with rat monoclonal anti-HA primary antibody) revealed that the WT V2R was readily detected on the cell surface (Fig. 1A), although it also could be visualized at intracellular sites when cells were permeabilized with Triton X-100 (Fig. 1E). This intracellular as well as surface expression of the WT V2R in COS M6 cells is probably a result of the considerable overexpression of the V2R in these cells, rendering receptor processing rate-limiting, as described previously (12Petaja-Repo U.E. Hogue M. Laperriere A. Walker P. Bouvier M. J. Biol. Chem. 2000; 275: 13727-13736Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). In contrast, immunostaining of intact cells expressing the selected V2R mutants L292P V2R, ΔV278 V2R, and R337X V2R (Fig. 1, B–D, respectively) demonstrated no cell surface immunoreactivity (i.e. no immunofluorescence above control cells not expressing epitope-tagged V2R when primary antibody was incubated with cells in the absence of Triton X-100). These three mutant receptors are expressed, however, since they are immediately detectable when the rat anti-HA antibody is introduced in the presence of 0.2% Triton X-100, revealing that each of these V2R mutants is synthesized but is retained intracellularly (Fig. 1, compare F–H with B–D). Mutant receptor expression also was verified by Western analysis (cf. “Experimental Procedures”). Localization—To determine whether the intracellularly trapped V2R mutants could be rescued to the cell surface, where their functionality could be assessed, cells expressing mutant V2R were incubated with either chemical or pharmacological chaperones or grown at reduced (28 °C) temperature. Subjecting cells to 10% glycerol, a so-called “chemical chaperone” that can rescue some intracellularly trapped proteins (presumably by protein stabilization (13Zhou Z. Gong Q. January C.T. J. Biol. Chem. 1999; 274: 31123-31126Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar)), had no effect on the redistribution of mutant V2Rs nor on the WT V2R (Fig. 2, compare E–H with A–D). The lack of surface expression in glycerol-treated cells was not due to differences in protein expression level, since each of the V2R mutants was synthesized but was trapped intracellularly when examined in permeabilized cells (data not shown). In contrast, treatment with the membrane-permeant V2R antagonist SR121463B dramatically increased detectable V2R expression at the surface for all mutant structures examined (Fig. 2, J–L). It had been previously demonstrated that application of SR121463B increased cell surface expression of other selected V2R mutants, presumably due to promotion or stabilization of properly folded receptors during their maturation in intracellular compartments (14Morello J. Bouvier M. Petaja-Repo U.E. Bichet D.G. Trends Pharmacol. Sci. 2000; 21: 466-469Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 15Morello J.P. Salahpour A. Laperriere A. Bernier V. Arthus M.F. Lonergan M. Petaja-Repo U. Angers S. Morin D. Bichet D.G. Bouvier M. J. Clin. Invest. 2000; 105: 887-895Crossref PubMed Scopus (479) Google Scholar). Moreover, subjecting COS M6 cells to reduced temperature during culture enhanced cell surface localization of the V2R mutants to an extent comparable with pharmacological rescue (Fig. 2, N–P), suggesting that 28 °C is permissive for appropriate folding and trafficking events for these structures in COS M6 cells. We utilized cell surface biotinylation strategies in COS M6 cells to provide quantitative information regarding the delivery of mutant V2R to the surface following rescue with temperature or SR121463B, using WT V2R as a comparator, as shown in Fig. 3. For these calculations, we defined the amount of WT V2R on the surface as 100%; in our studies, we observed that 38% of the total WT V2R expressed in COS M6 cells was on the surface in the absence of SR121463B. SR121463B treatment leads to rescue of the L292P V2R from 8.0 to 38.2% of WT V2R surface expression; the R337X V2R from 22.0 to 88.9% of WT V2R surface expression; and the ΔV278 V2R from 5.3 to 21.2% of WT V2R surface expression (Fig. 3). Interestingly, of the receptor expressed at the surface following exposure to SR121463B, virtually all of the WT V2R, R337X V2R, and ΔV278 V2R was in the mature glycosylated form, whereas none of the L292P V2R was detected in the mature glycosylated form (data not shown). For V2R expressed in COS M6 cells and exposed to 28 °C culture, quantitatively less mutant V2R was expressed at the cell surface overall. Furthermore, in contrast to findings for rescue by SR121463B, only ∼50% of the R337X and ΔV278 V2R that reaches the cell surface achieves the mature glycosylated form; none of the L292P V2R achieves the mature glycosylated form. These data suggest that occupancy of the R337X and ΔV278 V2R by SR121463B stabilizes a single or limited set of V2R conformations that successfully achieve mature glycosylation, whereas incubation of cells at 28 °C does not. In neither circumstance is L292P co" @default.
• W1990398484 created "2016-06-24" @default.
• W1990398484 creator A5081127970 @default.
• W1990398484 creator A5086900416 @default.
• W1990398484 creator A5089716121 @default.
• W1990398484 date "2003-09-01" @default.
• W1990398484 modified "2023-10-16" @default.
• W1990398484 title "Appropriate Polarization following Pharmacological Rescue of V2 Vasopressin Receptors Encoded by X-linked Nephrogenic Diabetes Insipidus Alleles Involves a Conformation of the Receptor That Also Attains Mature Glycosylation" @default.
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