Novel down-regulatory mechanism of the surface expression of the vasopressin V2 receptor by an alternative splice receptor variant.

In rat kidney, two alternatively spliced transcripts are generated from the V2 vasopressin receptor gene. The large transcript (1.2 kb) encodes the canonical V2 receptor, whereas the small transcript encodes a splice variant displaying a distinct sequence corresponding to the putative seventh transmembrane domain and the intracellular C terminus of the V2 receptor. This work showed that the small spliced transcript is translated in the rat kidney collecting tubules. However, the protein encoded by the small transcript (here called the V2b splice variant) is retained inside the cell, in contrast to the preferential surface distribution of the V2 receptor (here called the V2a receptor). Cells expressing the V2b splice variant do not exhibit binding to 3H-labeled vasopressin. Interestingly, we found that expression of the splice variant V2b down-regulates the surface expression of the V2a receptor, most likely via the formation of V2a.V2b heterodimers as demonstrated by co-immunoprecipitation and fluorescence resonance energy transfer experiments between the V2a receptor and the V2b splice variant. The V2b splice variant would then be acting as a dominant negative. The effect of the V2b splice variant is specific, as it does not affect the surface expression of the G protein-coupled interleukin-8 receptor (CXCR1). Furthermore, the sequence encompassing residues 242-339, corresponding to the C-terminal domain of the V2b splice variant, also down-regulates the surface expression of the V2a receptor. We suggest that some forms of nephrogenic diabetes insipidus are due to overexpression of the splice variant V2b, which could retain the wild-type V2a receptor inside the cell via the formation of V2a.V2b heterodimers.

In rat kidney, two alternatively spliced transcripts are generated from the V 2 vasopressin receptor gene. The large transcript (1.2 kb) encodes the canonical V 2 receptor, whereas the small transcript encodes a splice variant displaying a distinct sequence corresponding to the putative seventh transmembrane domain and the intracellular C terminus of the V 2 receptor. This work showed that the small spliced transcript is translated in the rat kidney collecting tubules. However, the protein encoded by the small transcript (here called the V 2b splice variant) is retained inside the cell, in contrast to the preferential surface distribution of the V 2 receptor (here called the V 2a receptor). Cells expressing the V 2b splice variant do not exhibit binding to 3 H-labeled vasopressin. Interestingly, we found that expression of the splice variant V 2b down-regulates the surface expression of the V 2a receptor, most likely via the formation of V 2a ⅐V 2b heterodimers as demonstrated by co-immunoprecipitation and fluorescence resonance energy transfer experiments between the V 2a receptor and the V 2b splice variant. The V 2b splice variant would then be acting as a dominant negative. The effect of the V 2b splice variant is specific, as it does not affect the surface expression of the G protein-coupled interleukin-8 receptor (CXCR1). Furthermore, the sequence encompassing residues 242-339, corresponding to the C-terminal domain of the V 2b splice variant, also down-regulates the surface expression of the V 2a receptor. We suggest that some forms of nephrogenic diabetes insipidus are due to overexpression of the splice variant V 2b , which could retain the wild-type V 2a receptor inside the cell via the formation of V 2a ⅐V 2b heterodimers.
Arginine vasopressin (AVP) 1 is a nonapeptide secreted by the pituitary gland and a major regulator of fluid and electrolyte balance and cardiovascular function (1,2). There are at least three vasopressin receptor subtypes, V 1 , V 2 , and V 3 , encoded by distinct genes (3). These receptors are expressed in a tissuespecific fashion; V 1 , V 2 , and V 3 receptors are preferentially expressed in vascular smooth muscle cells, kidney, and pituitary gland, respectively (4). The V 2 vasopressin receptor is coupled to adenylyl cyclase, whereas V 1 and V 3 receptors are preferentially coupled to phospholipase C. Activation of V 2 receptors increases the intracellular level of cAMP, which, in turn, triggers the recruitment of water channels (aquaporin 2) in the plasma membrane to stimulate water reabsorption (5). Importantly, mutations in the V 2 receptor are responsible for the X-linked nephrogenic diabetes insipidus (6).
Several studies have suggested that G protein-coupled receptors can form homodimers or heterodimers between related receptors, which may expand their functional diversity (7). For example, ␥-aminobutyric acid B receptor subtypes appear to form heterodimers inside the cells. Similar findings have been also reported with regard to taste receptor subtypes, suggesting that heterodimerization of these receptors is required for their targeting to the cell surface. Vasopressin receptors of different subtypes form homodimers and heterodimers in the endoplasmic reticulum, indicating that dimerization takes place during receptor synthesis (8). The functional significance of the dimerization of vasopressin receptors is unknown, but it seems that homodimerization or heterodimerization of these receptors does not affect their signaling mechanisms, although recent studies indicate that heterodimerization of V 1a and V 2 vasopressin receptors regulate their trafficking profiles (9). The V 2 vasopressin receptor gene consists of three exons and two introns ( Fig 1A). The first exon encodes the first nine amino acids of the N terminus, the second exon encodes transmembrane domains I-VI, and the third exon encodes transmembrane domain VII and the C-terminal domain (10). Reverse transcription PCR of RNA from isolated kidney tubules showed two transcripts, a major 1.2-kb transcript corresponding to the sequence encoding the V 2 receptor and a splice variant of 1.1 kb. (Fig 1B) This variant encodes an identical amino acid sequence to the V 2 receptor up to residue 303; however, the downstream sequence of the splice variant encodes a distinct amino acid sequence from the seventh transmembrane domain to the C terminus of the V 2 receptor (Fig 1C). The splice variant was generated by an alternative splicing at a site 76 bp downstream of the V 2 receptor splice site, resulting in a frameshift in the 3Ј-end coding region. The mRNA of the splice variant is expressed in the collecting tubules at ϳ15% that of the major V 2 receptor (11). In this work, we showed that the splice variant mRNA is translated into a protein (here called V 2b ) but is retained inside the cell. Most importantly, we showed that the V 2b protein forms heterodimers with the wild-type V 2 receptor (here called V 2a ) and acts as a dominant negative by sequestering V 2a receptors inside the cells.

EXPERIMENTAL PROCEDURES
Reagents-AVP was purchased from Bachem (Torrance, CA), [ 3 H]AVP came from PerkinElmer Life Sciences, and the monoclonal anti-GFP antibody was from Molecular Probes (Eugene, OR). Rat monoclonal anti-HA (3F10) and FuGENE 6 were from Roche Diagnostics, and Ultraspec RNA came from Biotecx (Houston, TX). Enhanced chemiluminescence solutions and the secondary anti-rabbit peroxidase-labeled antibody were from Pierce. The reporter vectors ECFP-N1, EGFP-N1, and EYFP-N1 were from BD Biosciences Clontech. Dulbecco's modified Eagle's medium, Ham's medium, penicillin G, streptomycin sulfate, and Fungizone were from Invitrogen.
The cDNAs encoding the wild-type V 2a vasopressin receptor and the splice variant were synthesized by reverse transcription PCR using rat kidney RNA along with a sense primer (5Ј-ggaattcggtgtgttaggtcatcatcaa-3Ј) and an antisense primer (5Ј-gctctagacagttgagctacagagggttt-3Ј). Both cDNAs were cloned into the expression vector pcDNA 3.1. To create expression plasmids encoding fusion proteins, the wild-type V 2a receptor cDNA was amplified with a sense primer (5Ј-ggaattcggtgtgttaggtcatcatcaa-3Ј) and an antisense primer (5Ј-acgcgtcgacgtggagggtgtatcc-3Ј); the splice variant cDNA was amplified with the same sense primer and the antisense primer 5Ј-acgcgtcgacgtaagaggagctgg-3Ј. These amplified products were subcloned in-frame to the cDNAs encoding the enhanced cyan fluorescent protein or the enhanced yellow fluorescent protein of the reporter vectors.
Transient and Stable Transfections-COS-1 and MDCK cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. CHO K1 cells were grown in F12 Ham's medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 g/ml streptomycin, and 0.25 g/ml Fungizone. CHO-K1 cells seeded in 24-well plates at 6 ϫ 10 4 cells/well were transiently transfected with 0.2 g of V 2a ⅐GFP cDNA and/or V 2b ⅐GFP cDNA per well, 0.4 g of CXCR1 cDNA, 0.4 g of CXCR1 plus V 2b cDNA (3:1 ratio), 0.4 g of V 2b or the V 2a 242 tail (the cDNA encoding from residue 242 to residue 371 of the V 2a receptor), or the V 2b 242 tail (the cDNA encoding from residue 242 to 329 of the V 2b splice variant) plus pcDNA3 vector (1:3 ratio) per well using FuGENE 6 at a 2:3 (w/w) ratio. MDCK cells were stably transfected with V 2a ⅐GFP cDNA and/or V 2b ⅐GFP cDNA using FuGENE 6, and cell clones were selected with 700 g/ml Geneticin and by fluorescence microscopy.
Immunocytochemistry-Rat kidney tissues were embedded in paraffin, sectioned, deparaffinized, dehydrated, and treated with 1% hydrogen peroxide. Sections were incubated with anti-peptide antibodies directed against the V 2 a C-terminal peptide (QRHTTHSLGPQDES-CATASSSLMKDTPS) or with antibodies directed against the V 2 b Cterminal peptide (HTAWVLKMNPVPQP). Bound antibodies were detected using an avidin-biotin kit (LSABϩ; Dako, Carpinteria, CA) following the manufacturer's instructions. Peroxidase activity was detected with 0.1% (w/v) 3-3Ј-diaminobenzidine and 0.03% (v/v) hydrogen peroxide for 5 min at room temperature. For the immunofluorescence studies, cells were transfected with cDNAs encoding V 2a tagged with an HA epitope at its N terminus and V 2b tagged with cyan fluorescence protein (CFP) at its C terminus. After 48 h of transfection, cells were fixed with 4% paraformaldehyde and permeabilized with cold methanol. Cells were stained with anti-HA monoclonal antibody to detect the expression of V 2a . Fluorescence in the cells was analyzed in a Zeiss fluorescence microscope.
Subcellular Fractionation-Stably transfected MDCK cells were washed with cold phosphate-buffered saline, harvested, and disrupted with a tight fitting Dounce homogenizer in 10 mM HEPES (pH 7.5) buffer containing 0.25 M sucrose and the protease inhibitors leupeptin and aprotinin (10 g/ml each). The cell homogenates were centrifuged for 10 min at 6,000 ϫ g. The post-nuclear supernatant was adjusted to 1.3 M sucrose and overlaid on a discontinuous sucrose gradient (2 ml each of 1.2, 1.15, 0.86, and 0.25 M sucrose) in 10 mM HEPES (pH 7.5) and centrifuged for 18 h at 24,000 rpm in a SW 40 Ti rotor. Onemilliliter fractions were collected from the bottom, diluted four times, and centrifuged for 45 min at 150,000 ϫ g av . Subcellular distribution of each fraction was analyzed by Western blot analysis using rabbit polyclonal anti-Na ϩ /K ϩ -ATPase (Rockland Immunochemicals), anti-calnexin (Abcam, Cambridge, United Kingdom), and anti Golgi-97 (Molecular Probes) antibodies to identify plasma membrane, ER, and Golgienriched membranes, respectively.
Co-immunoprecipitation-COS-1 cells were co-transfected with two plasmids, one containing the cDNA encoding the V 2a receptor tagged with GFP and the other containing the cDNA encoding the V 2b splice variant tagged with HA. After 48 h, the cells were washed with phosphate-buffered saline and homogenized with a Dounce homogenizer in a 5 mM Tris-HCl (pH 7.4) buffer containing 15 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 g/ml leupeptin and aprotinin (each). The homogenate was centrifuged for 10 min at 6,000 ϫ g, and the post-nuclear supernatant was centrifuged for 45 min at 150,000 ϫ g av . The membrane pellet was solubilized in radioimmune precipitation assay buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P40, 10 mM N-ethylmaleimide, 0.1 mM phenylmethylsulfonyl fluoride, 5 mg/ml soybean trypsin inhibitor, and 1 g/ml leupeptin) and centrifuged for 45 min at 16,000 ϫ g. The supernatant was incubated with a polyclonal anti-GFP (2.5 g) and 20 l of agarose-protein A slurry at 4°C for 16 h. The immunoabsorbent was washed three times with radioimmune precipitation assay buffer and collected by centrifugation for 2 min at 1,000 ϫ g. The immune complexes were resuspended in sample buffer and subjected to SDSpolyacrylamide gel electrophoresis and Western blot. Co-immunoprecipitation of HAV 2b was detected using a rat monoclonal anti-HA antibody and a peroxidase-labeled donkey anti-rat antibody (Jackson Laboratories).
Confocal Microscopy-Stably transfected MDCK cells were grown on coverslips for 2 weeks to achieve cell polarization. After washing the cells twice with Krebs-Ringer HEPES (136 mM NaCl, 10 mM HEPES, 4.7 mM KCl, 1.25 mM CaCl 2 , and 1.25 mM glucose), the coverslips were mounted in a camera to examine the cells in an LSM 510 Meta confocal laser-scanning microscope (Zeiss) in the Optical Imaging Laboratory at the University of Texas Medical Branch. The cells were excited with the laser at 488 nM, and the light emitted was detected using an LP 514 filter.
Fluorescence Resonance Energy Transfer (FRET)-COS-1 cells seeded at 5 ϫ 10 5 cells per 60-mm dish were transiently co-transfected with a pair of plasmids (2 g of each plasmid), V 2a ⅐CFP or V 2b ⅐CFP and either V 2a ⅐YFP or V 2b ⅐YFP. After 48 h, the coverslips were washed twice with Krebs-Ringer Hepes (136 mM NaCl, 10 mM HEPES, 4.7 mM KCl, 1.25 mM CaCl 2 , and 1.25 mM glucose) and mounted in the camera of a LSM 510 Zeiss confocal laser-scanning microscope. FRET was monitored by the three filter set procedure described by Gordon et al. (12). The donor filter set (D) is composed of a laser for excitation (458 nm), a main beam splitter (HFT 458/514), and secondary beam splitters (NFT 545 and NFT 515). The light emitted by the donor was detected with a 475-525-nm bandpass filter. The acceptor filter set (A) consisted of a laser for excitation (514 nm), a main beam splitter (HFT 458/514), and secondary beam splitters (NFT 570 and NFT 515). The light emitted by the acceptor was detected with a LP 530 filter. The FRET filter set (F) consisted of a laser for excitation (458 nm), corresponding to the absorption spectrum of the donor and the beam splitters described above. The FRET signal was obtained recorded using an LP 530 filter.
To normalize FRET (FRETN), the background given by the images from non-transfected cells was subtracted from the images from transfected cells, and the resulting images were processed by the ImageJ software (National Institutes of Health) according to the formula shown in Equation 1, in which we use the two-letter symbols proposed by Gordon et al. in 1998 (12). Aa and Fa represent images obtained with the Acceptor and FRET filter sets, respectively, when only the acceptor fluorophore is present in the sample, whereas Dd and Fd represent images obtained with the Donor and FRET filter sets, respectively, with the donor fluorophore. Ff, Af, and Df represent images obtained with the FRET, Acceptor, and Donor filter sets, respectively, when both fluorophores are present. G is a factor that relates the loss of donor emission due to FRET in the Donor filter set to the gain of acceptor emission due to FRET in the FRET filter set.

Subcellular Localization of V 2a
Receptor and the Splice Variant V 2b -As described previously (11), reverse transcription PCR with total RNA of rat kidney showed co-expression of two transcripts encoding two potential V 2 receptor subtypes (Fig.  1B). The large transcript of 1.2 kb encodes the classical V 2 receptor (V 2a ), whereas the small transcript of 1.1 kb encodes the splice variant V 2b . The latter displays an identical sequence to the V 2a receptor up to residue 303. However, because of the frameshift, the splice variant displays a short and distinct sequence from the corresponding seventh transmembrane and C terminus domains of the wild-type V 2a receptor (Fig. 1C). Here, we showed by the immunostaining of rat kidney cells with subtype-specific antibodies that V 2a receptors are preferentially expressed in the plasma membrane ( Fig. 2A), whereas the splice variant V 2b is broadly distributed in the cell (Fig. 2B). Interestingly, MDCK cells expressing the V 2a receptor⅐GFP fusion protein exhibited significant fluorescence in both the perinuclear region and the plasma membrane (Fig. 3A). In contrast, cells expressing the splice variant V 2b ⅐GFP fusion protein revealed fluorescence only in the cytoplasmic region without any significant labeling in the plasma membrane (Fig.  3B). Consistent with these findings, CHO cells expressing V 2a receptors exhibited cell surface [ 3 H]AVP high affinity binding, whereas cells expressing the splice variant V 2b displayed negligible high affinity binding to [ 3 H]AVP (Fig. 3C). This finding is in good agreement with the intense fluorescence displayed by permeabilized cells expressing the V 2b splice variant tagged with HA at its N terminus (Fig 4A), which is in contrast to the negligible fluorescence displayed by the corresponding nonpermeabilized cells (Fig 4B). As expected, non-permeabilized cells expressing the HA-tagged V2a receptor showed intense fluorescence (Fig 4C). These results indicate that the splice variant is retained inside the cells. To identify the location of the splice variant inside the cell, we performed subcellular fractionation. The V 2b splice variant preferentially localizes in the ER/Golgi enriched fractions, as monitored with the specific markers for ER (calnexin) and Golgi (Golgin-97). On the other hand, V 2a localized in all the fractions, including the plasma membrane-enriched fraction, as monitored by the Na ϩ /K ϩ -ATPase as a marker (Fig. 5). These data indicates that V 2b is unable to traffic to the plasma membrane because it is retained in the ER/Golgi compartment. It is likely that the V 2b splice lacks the sorting signals to traffic to the plasma membrane. Indeed, the seventh transmembrane domain and the C-terminal region of the V 2 receptor have been shown to play key roles in the trafficking of this receptor (13), as truncated V 2 receptor mutants (Trp 293 3 Stop and Leu312 3 Stop) found in patients with nephrogenic diabetes insipidus are retained inside the cell (14,15). To determine whether the V 2b splice variant still retains the ability to bind AVP, we performed binding assays in cell homogenates. As shown in Fig. 6, AVP exhibits negligible binding to homogenates from cells expressing the splice variant, whereas high affinity AVP binding is observed in homogenates from cell expressing the V 2a receptor.  (Fig. 7A). Interestingly, the V 2a receptor and the splice variant form heterodimers inside the cell (Fig. 7B). Only a low FRET signal was detected in cells expressing the unfused (control) fluorescence proteins CFP and YFP (Fig. 7C), indicating little interaction between these proteins. Quantitative fluorescence analysis showed that cells expressing V 2a ⅐CFP/ V 2a ⅐YFP and V 2a ⅐CFP/V 2b ⅐YFP pairs had a ϳ6-fold higher FRET signal than cells expressing CFP and YFP (Fig. 7D). We also demonstrated by co-immunoprecipitation that the V 2a receptor and the V 2b splice isoform oligomerize. As shown in Fig.  8, a HA-tagged V 2b splice variant can be immunoprecipitated with anti-GFP antibodies from extracts of cells expressing both the splice variant and the GFP-tagged V 2a receptor.

Heterodimerization of V 2a and V 2b Vasopressin Receptors-To
Splice Variant V 2b Down-regulates the Surface Expression of the V 2a Receptor-Because the splice variant V 2b forms heterodimers with V 2a receptors inside the cell, we investigated whether this splice variant regulates the trafficking of V 2a receptors. The surface expression of V 2a receptors was monitored by measuring [ 3 H]AVP binding in CHO cells transfected with cDNAs encoding the V 2a receptor and the splice variant V 2b . We found that the surface expression of V 2a receptors was abrogated by increasing the expression of the splice variant V 2b (Fig. 9). Interestingly, the total cell expression of the V 2a receptors was unaffected by the expression of the splice variant as demonstrated by Western blotting of the V 2a ⅐GFP fusion Binding assays were carried out three times; this result is from a representative experiment. protein using anti-GFP antibodies (Fig. 9, inset). Further analysis of the binding data indicates that the dissociation constant (K d ) of the binding of [ 3 H]AVP to the V 2a receptor was unaffected by the expression of the splice variant; however, B max decreased by almost 3-fold (Fig 10A). These findings indicate that the splice variant V 2b down-regulates the surface expression of the V 2a receptor, which is retained inside the cells, probably as V 2a ⅐V 2b receptor heterodimers. We also tested whether the receptor C-terminal domain also regulates V 2 re-ceptor trafficking, as that domain rescued V 2 receptor mutants (14,15). We found that the sequence encompassing residues 242-339 of the splice variant V 2b down-regulates the surface expression of the V 2a receptor (Fig. 10B), but not with the corresponding sequence 242-371 of the V 2a receptor (Fig. 10C). These results suggest that the region comprising the sixth transmembrane domain is part of the dimer interface, whereas the seventh transmembrane domain and the C terminus contain the sorting motifs for translocation of the receptor to the plasma membrane. We also demonstrate that the down-regulation of the V 2a receptor by the splice variant is specific, as the surface expression of the G protein-coupled receptor CXCR1 (interleukin-8 receptor A, a chemokine receptor) was unaffected on CHO cells co-transfected with cDNAs encoding CXCR1 and the splice variant V 2b (Fig. 10D). DISCUSSION In contrast to the preferential distribution of the V 2a receptor to the cell surface, we demonstrate that the translated splice variant V 2b transcript is retained in ER/Golgi compartments, as shown by subcellular fractionation analysis. Although the V 2b splice variant is normally expressed in the kidney, its functional significance does not appear to be related to its signaling or binding to AVP, as this splice variant did not exhibit high affinity binding to [ 3 H]AVP. However, we found that the expression of the splice variant down-regulates the surface expression of the V 2a receptor. V 2a receptors form both homodimers and heterodimers with V 2b , as demonstrated by FRET experiments and co-immunoprecipitation studies between V 2a receptor and the V 2b splice variant. These findings are consistent with the view that the V 2b splice variant downregulates the surface expression of the V 2a receptor by forming V 2a ⅐V 2b heterodimers, which are then retained inside the cells. The splice variant V 2b would be then acting as a dominant negative. The effect of V 2b is specific, as it does not affect the surface expression of CXCR1. Furthermore, the sequence encompassing residues 242-339 of the V 2b receptor mimics the down-regulation of the V 2a receptor by the full-length splice variant, suggesting that the sixth transmembrane segment of these receptors is part of the dimerization interface. Similar effects have been reported in other G protein-coupled receptors, e.g. a splice variant of the calcitonin receptor lacking 14-residues of the seventh transmembrane segment prevented the surface expression of the wild-type receptor (16). The precise mechanism underlying the retention of the V 2a receptor by the splice variant V 2b is unknown. On the basis of current information about the folding and trafficking of proteins, we reasoned that the V 2a ⅐V 2b heterodimer complex may be improperly folded to be processed and transported to the plasma membrane. Indeed, misfolded mutants of V 2 receptors that cause nephrogenic diabetes insipidus accumulate inside the cells but can be rescued by non-peptide antagonists of the V 2 receptor (17). It is argued that the binding of the V 2 receptor antagonist stabilizes the folded conformation of the receptor and primes it for processing and transport to the cell surface. Also, misfolded rhodopsin mutants causing retinal degeneration interfere with the processing and transport of the wild-type rhodopsin (18). The best-studied system is the improperly folded ⌬F508 mutant of the cystic fibrosis transmembrane conductance regula- tor, which is retained inside the cell (19). However this misfolded mutant can be rescued by organic solutes that stabilize its folded conformation (20). All of these disorders belong to a group of misfolding diseases that include Alzheimer's disease, prion encephalopathies, Parkinson's disease, and some cancers (21)(22)(23)(24)(25)(26). This model of retention of the misfolded receptor complex inside the cell would be consistent with the down-regulation of the wild-type V 2a receptor by a misfolded splice variant V 2b (27)(28)(29). We suggest that the fragment encompassing residues 242-339 of the splice variant is misfolded, as expression of this fragment is sufficient to down-regulate the surface expression of the V 2a receptor. Our findings support the idea that dimerization of V 2a receptors inside the cell stabilizes the folded structure of the receptor for processing and transport to the plasma membrane, whereas heterodimerization of the splice variant V 2b with the wild-type V 2a receptor gives rise to a misfolded V 2a ⅐V 2b complex, which is retained inside the cell. It is possible that some forms of nephrogenic diabetes insipidus may be due to the overexpression of the splice variant V 2b , which retains the wild-type V 2a receptor inside the cell via the formation of V 2a ⅐V 2b heterodimers.