Role of the human V1 vasopressin receptor COOH terminus in internalization and mitogenic signal transduction

Marc Thibonnier, Christine L. Plesnicher, Karim Berrada, and Liliana Berti-Mattera

Division of Clinical and Molecular Endocrinology, Department of Medicine, University Hospitals of Cleveland and Case Western Reserve University School of Medicine, Cleveland, Ohio, 44106


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We studied the role played by the intracellular COOH-terminal region of the human arginine vasopressin (AVP) V1-vascular receptor (V1R) in ligand binding, trafficking, and mitogenic signal transduction in Chinese hamster ovary cells stably transfected with the human AVP receptor cDNA clones that we had isolated previously. Truncations, mutations, or chimeric alterations of the V1R COOH terminus did not alter ligand binding, but agonist-induced V1R internalization and recycling were reduced in the absence of the proximal region of the V1R COOH terminus. Coupling to phospholipase C was altered as a function of the COOH-terminal length. Deletion of the proximal portion of the V1R COOH terminus or its replacement by the V2-renal receptor COOH terminus prevented AVP stimulation of DNA synthesis and progression through the cell cycle. Mutation of a kinase consensus motif in the proximal region of the V1R COOH terminus also abolished the mitogenic response. Thus the V1R cytoplasmic COOH terminus is not involved in ligand specificity but is instrumental in receptor trafficking and facilitates the interaction between the intracellular loops of the receptor, G protein, and phospholipase C. It is absolutely required for transmission of the mitogenic action of AVP, probably via a specific kinase phosphorylation site.

cellular proliferation; G protein-coupled receptors


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THE ANTIDIURETIC HORMONE 8-ARGININE VASOPRESSIN (AVP) belongs to the family of vasoactive and mitogenic peptides involved in physiological and pathological cell growth, differentiation, and proliferation (40). AVP exerts its actions through binding to specific V1-vascular (V1R), V2-renal (V2R), and V3-pituitary membrane receptor (V3R) subtypes that are coupled to distinct second messengers (34).

The human V1R is expressed in vascular smooth muscle cells, hepatocytes, blood platelets, adrenal cortex, kidney, reproductive organs, spleen, adipocytes, brain, and testis as the product of the same gene undergoing identical splicing (36). AVP binding to V1Rs leads to the activation of phospholipases C, D, and A2, the production of inositol 1,4,5-trisphosphate and diacylglycerol, the activation of calcium/calmodulin kinase, phosphatidylinositol 3-kinase (PI 3-kinase), protein kinase C (PKC) and p42/p44 mitogen-activated protein (MAP) kinases, the mobilization of intracellular calcium, the influx of extracellular calcium via receptor-operated Ca2+ channels, and the activation of the Na+-H+ exchanger (3, 30, 33). No stimulation of cAMP accumulation is noted after stimulation of V1Rs (32). The nuclear signals triggered by activation of V1Rs are the induction of immediate early-response gene expression and protein synthesis, leading to cellular hypertrophy and increased cell protein content (7). Indeed, activation of V1Rs leads to a mitogenic response in vascular smooth muscle cells, 3T3 cells, renal mesangial cells, hepatocytes, and adrenal glomerulosa cells. This response is specifically blocked by V1R antagonists of peptide and nonpeptide nature (29).

Agonist activation of G protein-coupled receptors (GPCRs) triggers a cascade of events, including G protein coupling, phosphorylation of intracellular domains, internalization of the occupied receptor, and activation of intracellular messengers. Indeed, AVP activation of V1R elicits a rapid internalization and phosphorylation of the receptor (11). The family of AVP receptors is interesting in that its various members are coupled to different G proteins and exert opposite effects on cell growth and proliferation: V1R is coupled to Gq and produces a mitogenic response, whereas V2R is coupled to Gs and produces an antimitogenic response (32). The structural elements of human V1R involved in the mitogenic properties of this receptor remain to be identified. Phosphorylation of intracytoplasmic residues of GPCRs plays an obligatory role in signal transmission (25). Examination of the amino acid composition of human V1R and V2R reveals the presence of several serine and threonine potential phosphorylation sites at the level of the intracytoplasmic domains of both receptors (Fig. 1). In contrast, the V1R COOH terminus contains one proximal and three distal PKC consensus motifs ([S/T] X [R/K]), whereas human V2R has no PKC motif in its COOH-terminal region. To examine the role played by the COOH terminus of the V1R in binding characteristics, internalization, recycling, and coupling to the mitogenic signal, we stably transfected Chinese hamster ovary (CHO) cells with a series of human V1Rs containing truncated or mutated portions of the COOH terminus. Also, chimeric constructs interchanging the human V1R and V2R COOH termini were designed. We observed that the human V1R COOH terminus is not involved in ligand binding affinity and specificity but modulates agonist-induced receptor internalization and recycling as well as transmission of the mitogenic signal. The V1R COOH terminus also alters the efficiency of coupling to phospholipase C. The first kinase motif present in the V1R COOH terminus plays a key role in the transmission of the mitogenic signal.


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Fig. 1.   Two-dimensional structure of the human V1-vascular and V2-renal arginine vasopressin (AVP) receptors (V1R and V2R). Cytoplasmic serine and threonine residues are shown in bold characters. The protein kinase C (PKC) consensus sites ([S/T] X [R/K]) are boxed, and the location of the HindIII restriction site (used for exchange of the COOH termini between the 2 receptors) is indicated by an arrow.


    MATERIAL AND METHODS
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Reagents. Standard reagents, unless stated otherwise, were from Sigma Chemical, St. Louis, MO. CHO-K1 cells were obtained from the American Type Culture Collection (Rockville, MD, CCL61), and cell culture media and geneticin were from GIBCO BRL (Grand Island, NY). Fetal bovine serum was from Hyclone (Logan, UT); restriction and modification enzymes were from Promega (Madison, WI) or NEN Biolabs, (Beverly, MA); and [3H]AVP (specific activity = 48 Ci/mmol), myo-[2-3H]inositol (specific activity = 20 Ci/mmol), and [3H]thymidine (specific activity = 20 Ci/mmol) were obtained from NEN-Du Pont (Wilmington, DE). The pBluescript II, phagemid KS, and XL2-Blue Escherichia coli strain were from Stratagene (La Jolla, CA); the pCI-neo mammalian expression vector was from Promega; and AVP and peptide analogs were from Bachem (Torrance, CA). The nonpeptide V1R antagonist SR-49059 (batch no. MY10-075) and V2R antagonist SR-121463A (batch no. DPL6.152.1) were provided by Dr. C. Serradeil-Le Gal, Sanofi Recherche, Toulouse, France.

Receptor constructs. The human V1R cDNA clone that we had isolated previously (31) was modified by PCR with the use of reverse primers hybridizing with the appropriate region of the COOH terminus and introducing an EcoRI restriction site downstream of the new stop codon to produce COOH-terminal regions of various lengths containing increasing numbers of kinase motifs (Fig. 2). The primers were synthesized with an Applied Biosystems oligonucleotide synthesizer and purified over OPC cartridges according to the manufacturer's recommendations (Applied Biosystems, Foster City, CA). The PCR reactions (final volume 100 µl) contained 20 pmol of each primer, 2.5 units of Taq polymerase, 1 µg of purified full-length wild-type V1R cDNA, 50 µM of each dNTPs in buffer of 10 mM Tris · HCl, 50 mM KCl, 1.5 mM MgCl2, 0.1% gelatin, pH 7.4. After initial denaturation at 95°C for 5 min, 35 cycles were run (at 95°C for 45 s, 56°C for 45 s, and 72°C for 1.5 min) with a final extension at 72°C for 7 min. The PCR products were purified (Wizard PCR DNA purification kit from Promega), digested with EcoRI, purified with the GeneClean II kit (Bio 101, Vista, CA), and ligated to the pCI-neo mammalian expression vector.


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Fig. 2.   Structure of the COOH termini of the mutated human V1R. Shown are the intracytoplasmic COOH termini of the truncated, chimeric, and mutated constructs of the receptor. The PKC consensus motifs ([S/T] X [R/K]) are underlined and in bold characters, and the portions of the chimeric constructs from the human V2R sequence are in shadow characters. The mutated kinase motif in the V1RS382G-R384G construct is in italics. Numbers refer to the amino acid respective positions in the human V1R and V2R sequences.

Also, a chimeric V1R/V2R construct made of the 5' part of the human V1R cDNA (Met1-Glu361) and the COOH terminus of the human V2R cDNA (Ser338-Ser371) was produced by digesting both clones with HindIII and ligating back in frame the 5' V1R fragment with the 3' V2R fragment before insertion in the pCI-neo vector. The V2R cDNA was produced by PCR as described before (32). A reverse strategy was implemented to produce a V2R/V1R chimera (Met1-Arg337 of V2R ligated to Ser362-Thr418 of the V1R) after digestion with HindIII.

The V1RS382G-R384G mutant, whose first kinase consensus sequence within the COOH terminus of the human V1R was inactivated, was produced by using the QuickChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's recommendations.

The amino acid COOH-terminal sequences of the truncated and mutated V1Rs, as well as the chimeric V1R/V2R and V2R/V1R clones, are presented in Fig. 2. The correct sequences of all of the constructs described were verified by double-strand sequencing (Taq Dye Deoxy Terminator cycle sequencing kit and a model 373A sequencer from Applied Biosystems) before CHO-K1 cell transfection.

Selection and stable expression in CHO cells. Stable transfection of CHO-K1 cells with the mutated V1R cDNAs was performed using the CaPO4 precipitation method (31). Pure clones were selected for their resistance to the antibiotic geneticin (400 µg/ml) and purified by the limiting dilution technique. Clones expressing the greatest number of mutated receptors in binding experiments were further explored by radioligand saturation and competition binding experiments as well as measurement of IP production, thymidine uptake, flow cytometry, and cell proliferation, as described next.

Radioligand binding assays. Control and transfected CHO-K1 cells were grown to confluence in 24-well dishes and washed twice with PBS + 10 mM MgCl2 + 0.2% BSA, pH 7.4. Competition binding experiments were performed in duplicate by incubating the cells (final volume 250 µl/well) in the same medium with one fixed concentration of [3H]AVP (2-5 nM) and increasing concentrations of unlabeled AVP or its analogs for 30 min at 30°C. The cells were washed three times with ice-cold PBS and lysed with 250 µl of 0.1 N NaOH-0.1% SDS. Cell-bound [3H]AVP was counted by liquid scintillation spectrometry (Beckman counter LS 5801; yield = 64%). IC50 values were derived from nonlinear least-squares analysis, and inhibition constant (Ki) values were calculated by the equation of Cheng and Prusoff: Ki = IC50/(1 + Lf/Kd).

Also, saturation binding experiments of AVP receptors of transfected CHO cells were performed in 24-well dishes in duplicate in 250 µl of PBS + 10 mM MgCl2 + 0.2% BSA, pH 7.4 with increasing concentrations of [3H]AVP ± 1 µM unlabeled AVP. Affinity (Kd) and capacity (Bmax) of the AVP receptors were calculated by a nonlinear least-squares analysis program (35). Protein concentration was measured with Pierce's bicinchoninic acid reagent with albumin as an internal standard.

Receptor internalization. Subconfluent monolayer cultures of CHO-K1 cells transfected with the wild-type or mutated V1Rs were grown in 24-well plates. The cells were washed twice and incubated at 37°C in 250 µl of PBS + 10 mM MgCl2 + 0.2% BSA, pH 7.4, with one concentration of [3H]AVP (5 nM) ± 1 µM unlabeled AVP for increasing times <= 60 min (n = 8 for each time point). Each well was washed three times with 0.5 ml of ice-cold PBS. Every other well was incubated with 0.5 M NaCl + 0.2 M acetic acid, pH 2.5, for 10 min on ice to displace surface-bound radioactivity and then was washed three times with 0.5 ml of ice-cold PBS. Radioactivity was counted as described (in Radioligand binding assays) to measure total specific binding and internalized specific binding. The percentage of internalized receptors was plotted against time and analyzed as one-phase exponential association to determine half-life of internalization (t1/2 in min) and maximum internalization percentage (Imax in %).

Receptor recycling. Subconfluent monolayer cultures of CHO-K1 cells transfected with the wild-type or mutated V1Rs were grown in 24-well plates. The cells were washed twice and incubated at 37°C in 250 µl of PBS + 10 mM MgCl2 + 0.2% BSA, pH 7.4, with unlabeled AVP (0.1 µM) for 20 min (n = 12 for each time point). Each well was washed three times with 1 ml of ice-cold PBS containing 50 mM EDTA to displace AVP bound to the membrane receptors and then washed again three times with 1 ml of ice-cold PBS. The cells were again incubated at 37°C for 0-60 min in 250 µl of PBS + 10 mM MgCl2 + 0.2% BSA, pH 7.4, to allow recycling of the receptors to the cell surface. Thereafter, medium was discarded and replaced by 250 µl of ice-cold PBS + 10 mM MgCl2 + 0.2% BSA, pH 7.4, containing [3H]AVP (5 nM) ± 1 µM unlabeled AVP for 2-h incubation on ice. The cells were washed three times with 0.5 ml of ice-cold PBS. Radioactivity was counted as described (in Radioligand binding assays) to measure cell-bound [3H]AVP. The percentage of receptors recycling to the cell surface was plotted against time as a percentage of the total number of surface receptors available before any agonist binding. The half-life of recycling (t1/2 in min) was calculated from the slope of the relationship.

IP production. Subconfluent monolayer cultures of CHO-K1 cells transfected with the wild-type or mutated V1Rs were grown for 48 h in 12-well dishes, washed with inositol-free DMEM, and labeled with myo-[2-3H]inositol (2-3 µCi/ml) in inositol-free DMEM for 20-24 h (8). Thereafter, the cells were preincubated for 1 h in DMEM, followed by 15-min preincubation in Hanks' balanced salt solution containing 10 mM glucose, 1.2 mM CaCl2, and 10 mM LiCl. Increasing concentrations of AVP were added, and the cells were incubated for 30 min at 37°C. The reaction was stopped by the addition of 0.5 ml of ice-cold methanol-HCl (100:1). The cells were scraped and transferred to glass tubes containing 1 ml of chloroform and 25 µg of phytic acid hydrolysate to obtain a final ratio of chloroform-methanol-H2O of 10:5:4. After phase separation, the upper phases were removed and loaded onto AG1-X8 Dowex columns (chloride form, 200-400 mesh). Inositol monophosphates, inositol bisphosphates, and inositol trisphosphate were eluted with 30 mM, 90 mM, and 0.5 M HCl, respectively, and quantitated by scintillation spectroscopy. The lower phases containing labeled phosphoinositides were washed with a theoric upper-phase solution containing 1 mM inositol and counted. The amount of IPs released was expressed as disintegrations per minute per well or as a fraction of total labeled phosphoinositides. Typical incorporation into phosphoinositides amounted to ~3.5-5 × 104 [3H]dpm/well. The efficacy (EC50) of AVP stimulation of IP production was calculated by a nonlinear least-squares analysis program.

[3H]thymidime uptake. Subconfluent monolayer cultures of CHO-K1 cells transfected with the wild-type or mutated V1Rs were grown in F12 medium + 10% FBS at 37°C for 48 h in 24-well plates to measure thymidine uptake in the presence of AVP, as described before (34). Cells were washed in 500 µl of F12 medium and grown for 72 h in 500 µl of F12 medium + 25 mM HEPES + 0.1% BSA. Increasing concentrations of AVP (10-12-10-6 M final concentrations) were added to the wells, and 18 h later, 10 µCi of [3H]thymidine were added for 45 min. The cells were transferred on ice, washed twice with 0.5 ml of ice-cold PBS, fixed with 1 ml of ice-cold 10% trichloroacetic acid (TCA) for 30 min at 4°C, washed twice with 1 ml of ice-cold 5% TCA solution, and solubilized with 250 µl of 0.1 N NaOH-0.1% SDS. Aliquots were collected for radioactivity determination.

Flow cytometric analysis. Subconfluent cultures of CHO-K1 cells transfected with the wild-type or mutated V1Rs were grown in F12 medium + 15 mM HEPES and 10% FBS at 37°C for 48 h in 100-mm dishes to assess cell cycle progression in the presence of AVP. Cells were serum starved for 5 days in F12 medium + 15 mM HEPES and 0.1% BSA. Thereafter, AVP (10-8 M final concentration) was added alone or in the presence of various inhibitors, and 24 h later, the cells were harvested. Flow cytometric analysis (FACS) was performed after the nuclei were labeled with propidium iodide (PI). PI was excited with an air-cooled argon-ion laser operating at 15 mW at 488 nm (EPICS XL-MCL, Coulter, Miami, FL). Linear and peak PI fluorescence was collected with a 620-nm band pass optical filter. Linear and peak fluorescence was plotted as bivariate parameters to form a doublet discriminator that was used to estimate the single cell, cell aggregate, and apoptotic cells in the sample. System II software was used to acquire the data, and ModFIT 5.2 (Verity Software House, Topsham, ME) was used to model cell cycle phases.

Data analysis. Nucleotide and amino acid sequences were analyzed with the computer package MacVector (Oxford Molecular, Oxford, UK). Nonlinear least-squares analysis was performed with the Kaleidagraph software package from Synergy Software (Reading, PA). Data are expressed as means ± SE.


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Radioligand binding characteristics of the mutated AVP receptors. To test the influence of the human V1R COOH-terminal alterations on the receptor's intracellular processing and binding profile, radioligand binding characteristics of the mutated V1Rs were explored in intact CHO cells stably transfected with these mutants. Thibonnier et al. (31) have previously shown that control CHO cells did not express endogenous AVP receptors that may have interfered with the binding assays. Nonspecific binding was negligible in transfected CHO cells; moreover, no specific binding was observed in cells transfected with the pCI-neo vector alone (data not shown).

Saturation binding experiments performed with [3H]AVP in intact CHO cells transfected with the mutated V1R cDNAs revealed a single class of high-affinity binding sites in all mutated clones (Table 1). Hence, the various truncations, mutations, and/or chimeric alterations of the V1R COOH terminus did not hamper the receptor insertion inside the cell membrane. The affinity of the mutated V1R clones for the native ligand AVP (expressed as Kd value) was similar to that of the wild-type receptor, except for the V2R/V1R chimera, whose Kd was slightly, but significantly, increased to 1.61 nM. Interestingly, this V2R/V1R chimera displayed the lowest level of expression among the various constructs developed for this study.

                              
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Table 1.   Binding characteristics of the mutated forms of the human AVP receptor

Competition binding experiments confirmed that all of the truncated and mutated V1Rs still displayed a typical V1R subtype binding profile (data not shown). More importantly, the affinity of the V1R/V2R chimeric construct (whose COOH terminus was exclusively made of the human V2R subtype sequence) for AVP and the nonpeptide V1R antagonist SR-49059 remained high (Ki = 0.85 ± 0.09 and 0.76 ± 0.14 nM, respectively), whereas the affinity for the nonpeptide V2R antagonist SR-121463A was low (Ki = 2,314 ± 157 nM; Fig. 3A). Conversely, the affinity of the V2R/V1R chimeric construct (whose COOH terminus was exclusively made of the human V1R subtype sequence) for AVP and the nonpeptide V2R antagonist SR-121463A was high (Ki = 1.61 ± 0.06 and 1.08 ± 0.08 nM, respectively), whereas the affinity for the nonpeptide V1R antagonist SR-49059 was low (Ki = 228 ± 9 nM; Fig. 3B). These observations suggest that the COOH-terminal portion of the human V1R is not required for proper folding and cell surface expression of the receptor. Furthermore, it is not involved in the determination of ligand receptor subtype selectivity.


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Fig. 3.   Ligand selectivity of the human V1R/V2R and V2R/V1R chimeric receptors. Stably transfected Chinese hamster ovary (CHO) cells prepared as described in MATERIALS AND METHODS were grown to subconfluence in 24-well dishes. Shown is the mean of competition binding experiments performed with 1 concentration of [3H]AVP and increasing concentrations of AVP or the nonpeptide V1R antagonist SR-49059 or the nonpeptide V2R antagonist SR-121463A (n = 4-8 experiments for each analog carried out in duplicate).

Internalization and recycling of the agonist-occupied mutated receptors. Platelet and hepatic V1 AVP receptors internalize and recycle once they are occupied by AVP (6, 30). The molecular determinants underlying the internalization, sequestration, and recycling of the AVP/oxytocin (OT) receptor family remain largely unexplored, but the involvement of the third intracytoplasmic loop and the COOH terminus has been suggested for other G protein-coupled receptor (GPCR) families (9, 20). We explored the possible interference of truncations of the V1R COOH terminus with the internalization and recycling of the AVP receptors in the stably transfected CHO cells expressing the mutated clones. As shown in Fig. 4, the wild-type V1R was quickly internalized at 37°C with an internalization half-life of 5 min and a maximal amount of internalization of 89%. For the V1R359X mutant, the extent of internalization was reduced to 54%, and the internalization half-life was slightly slower (6 min). The internalization pattern for the V1R399X, V1R406X, V1R409X, and V1RS382G-R384G mutants was similar to that of the wild-type V1R (internalization half-life between 4 and 5 min, maximal level of internalization between 82 and 94%), thus suggesting that only the NH2-terminal portion of the V1R COOH terminus plays a significant role in its internalization. Like other investigators (12), we observed that the internalization rate of the V2R was slower than that of the V1R: the internalization half-life of the wild-type V2R in CHO cells was 13 min, and the maximal level of internalization was 91%. The internalization rate of the V1R/V2R chimera (internalization half-life of 4 min) was similar to that of the wild-type V1R, whereas the internalization of the V2R/V1R chimeric receptor was slower than that of the wild-type receptors (internalization half-life of 19 min).


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Fig. 4.   Internalization of the agonist-occupied wild-type and mutated AVP receptors. Stably transfected CHO cells were grown to confluence in 24-well dishes before binding assay with [3H]AVP at 37°C for increasing times <= 1 h. Surface-bound radioactivity and internalized radioactivity were measured as described in MATERIALS AND METHODS (n = 3 experiments for each clone carried out in quadruplicate).

Agonist-promoted internalization of AVP receptors is followed by rapid recycling of the receptors to the cell surface after removal of the hormone from the medium (13). Thus we looked at the role played by the V1R COOH terminus in the recycling process of the receptor. As shown in Fig. 5, only 10% of the wild-type V1Rs remained at the cell surface after a 20-min exposure to 100 nM AVP at 37°C. After removal of the agonist, the wild-type V1Rs recycled quickly to the cell surface at 37°C (recycling half-life of 19 min). At variance, 50% of the V1R359X mutant still remained at the cell surface after a 20-min exposure to 100 nM AVP at 37°C, and recycling of the V1R359X mutant to the cell surface occurred at a slower pace (recycling half-life of 35 min). Recycling of the V1R399X, V1R406X, V1R409X, and V1RS382G-R384G mutants was similar to that of the wild-type V1R (data not shown).


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Fig. 5.   Recycling of wild-type and mutated AVP receptors. Stably transfected CHO cells grown to confluence in 24-well dishes were treated for 20 min at 37°C with 100 nM AVP, chilled, washed with EDTA-containing buffer, reincubated at 37°C for 0-60 min before binding assay with [3H]AVP at 4°C. The number of binding sites present at the cell surface is expressed as the percentage of the initial number of sites present before any agonist binding (n = 3 experiments for each clone performed in octoplicate).

AVP-induced IP production in CHO cells expressing mutated receptors. V1Rs are coupled to phospholipase C with a subsequent increased production of IPs. The signal transduction of the mutated human V1Rs was explored by measuring AVP-induced IP production in CHO cells stably transfected with the wild-type or mutated V1Rs (Table 2). In CHO-K1 cells transfected with the wild-type V1R, AVP produced a dose-dependent increase in the formation of IP that was blocked by the nonpeptide V1R antagonist SR-49059 but not by the nonpeptide V2R antagonist SR-121643A (data not shown). AVP could still stimulate IP production in cells expressing the truncated V1Rs. The maximum response was similar to that noted for the wild-type receptor (57- to 60-fold increase), but there was a shift in the dose-response curve to the right that was related to the length of the COOH terminus of the various mutants and, presumably, the number of available PKC sites. AVP stimulation of IP production in the V1RS382G-R384G mutant receptor was normal. The V1R/V2R chimera was also able to stimulate IP production with a normal efficacy but a slightly reduced maximum stimulation (92% that of the wild type). The V2R/V1R chimera, like the wild-type V2R, was unable to stimulate IP production. These data suggest that the COOH terminus of the V1R participates in the stimulation of IP production but cannot elicit such a response by itself.

                              
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Table 2.   Stimulation of IP production by AVP in CHO cells transfected with mutated forms of the human V1 receptor

AVP-induced [3H]thymidine uptake in CHO cells expressing the mutated receptors. One major cellular event resulting from the stimulation of V1Rs is cell growth and proliferation, which can be assessed by measuring nucleic acid synthesis through [3H]thymidine uptake. Thibonnier et al. (34) have previously shown that AVP induces a dose-dependent increase in [3H]thymidine uptake in CHO cells transfected with the wild-type V1R. To explore the role of the V1R COOH terminus in the transmission of this mitogenic signal, AVP-induced [3H]thymidine uptake was measured in CHO cells transfected with the wild-type or the mutated form of the human V1R. AVP stimulation of DNA synthesis required the involvement of the receptor COOH-terminal proximal portion as shown in Fig. 6. There was a progressive restoration of DNA synthesis with the lengthening of the COOH terminus. The proximal PKC consensus site of the V1R COOH terminus seemed to play a major role in the mitogenic signal, because AVP effect on thymidine uptake was lost in the V1RS382G-R384G mutant receptor. Introduction of the V2R COOH terminus into the V1R sequence led to a dramatic reduction of AVP stimulation of DNA synthesis as found in the V1R/V2R chimeric clone. Of interest is the fact that introduction of the V1R COOH terminus into the V2R sequence was not sufficient to stimulate DNA synthesis by the V2R/V1R chimera. These data suggest that, as for IP production, the COOH terminus of the V1R participates in the stimulation of DNA synthesis but cannot elicit such a response by itself.


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Fig. 6.   AVP stimulation of DNA synthesis in CHO cells transfected with wild-type and mutated AVP receptors. Transfected CHO cells were grown to subconfluence in 24-well dishes and incubated in serum-free F12 medium, pH 7.4, for 72 h. The cells were subsequently stimulated by 1 µM AVP overnight, followed by incubation with [3H]thymidine for 45 min, DNA precipitation, and liquid scintillation counting. [3H]thymidine uptake results are expressed as a percentage of 10% FBS stimulation. *P < 0.05, **P < 0.01, (n = 3-6 experiments for each clone in octuplicate).

AVP-induced progression through the cell cycle in cells expressing mutated AVP receptors. Thibonnier et al. (33) recently showed that, in CHO cells transfected with the wild-type V1R, AVP induced a progression through both the S and G2-M phases of the cell cycle. We explored here the effect of AVP on cell cycle progression in the V1R mutated clones. Compared with the wild-type V1R, AVP no longer produced progression through the cell cycle in the V1R359X truncated mutant (Fig. 7). AVP produced a normal progression through the cell cycle in the mutants displaying the proximal portion of the V1R COOH terminus (V1R399X, V1R406X, and V1R409X clones; data not shown). AVP stimulation of the V1RS382G-R384G mutant receptor produced a progression from the G0-G1 phase to the S phase but no further progression to the G2-M phase (Fig. 7). Study of the cell cycle progression in the chimeric V1R/V2R and V2R/V1R clones revealed patterns similar to those of the corresponding wild-type receptors, i.e., cell cycle progression for the V1R/V2R clone but no cell cycle progression for the V2R/V1R chimera (data not shown). These data suggest that, as for IP production and DNA synthesis, the COOH terminus of the V1R participates in the effect of AVP on cell cycle progression but cannot elicit such a response by itself.


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Fig. 7.   AVP effect on cell cycle progression in CHO cells transfected with wild-type and mutated AVP receptors. CHO cells transfected with the wild-type V1R, the V1R359X, or the V1RS382G-R384G mutants were grown to subconfluence in 100-mm dishes and incubated in serum-free F12 medium for 5 days. Thereafter, the cells were stimulated by 10 nM AVP for 24 h, followed by flow cytometry analysis. The x-axis represents relative fluorescence intensity, which is proportional to DNA content; the y-axis represents forward light scatter, which is proportional to cell number. The positions of apoptotic, G0-G1, S, and G2-M DNA contents are indicated. A: resting cells; B: AVP stimulation.


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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

The superfamily of GPCRs is structurally defined by an extracellular NH2 terminus, seven transmembrane domains, and an intracellular COOH terminus. Interaction with G proteins and intracellular messengers takes place at the level of the intracytoplasmic regions of the receptors. The length of the COOH terminus of GPCRs is highly variable, from being absent in the gonadotropin-releasing hormone receptor to measuring up to 162 amino acids (1). Comparison of the family of AVP/OT receptors reveals variable COOH-terminal lengths (arbitrarily counted after the conserved cysteine-cysteine motif located after the seventh transmembrane domain): 51 amino acids for the V1R, 29 for the V2R, 68 for the V3R, and 42 for the OTR. The V1R and V2R COOH termini differ not only in their length but also in their amino acid composition. The V1R COOH terminus contains 16 serine and threonine residues and four PKC consensus sites ([S/T] X [R/K]), whereas the V2R COOH terminus contains 12 serine and threonine residues but no PKC consensus site. Because of these structural differences between the V1R and V2R COOH termini and the opposite effects of these two structurally related receptors on cell proliferation, we studied the role of the receptor COOH termini in binding characteristics and signal transduction patterns in mutated, truncated, and chimeric forms of human V1R and V2R.

Truncations, mutations, and chimeric alterations of the human V1R COOH terminus do not hinder the expression, processing, or normal insertion of the receptor inside the cell membrane, as shown by our ligand-binding experiments performed in intact cells. In addition, structural alterations of the human V1R COOH terminus did not modify the ligand-binding characteristics of the receptor. Affinity for the native ligand AVP remained excellent; moreover, the insertion of the V2R COOH terminus into the V1R sequence did not modify the V1R subtype ligand selectivity. Likewise, insertion of the V1R COOH terminus into the V2R sequence did not modify the V2R subtype ligand selectivity.

However, the V2R/V1R chimera's affinity for [3H]AVP was slightly but significantly reduced, and the level of expression of this particular clone was not as high as that of the other constructs. Site-directed mutagenesis experiments conducted by several investigators with the V1R, V2R, and OTR from different species indicate that the ligand-binding pocket and the molecular determinants of AVP/OT receptor subtype ligand specificity are located in the extracellular loops and the upper part of the transmembrane domains of the receptors (4, 14, 21, 26, 39). In agreement with our findings, Liu and Wess (16) observed that chimeric constructs exchanging the rat V1R and the human V2R COOH termini did not alter high-affinity ligand binding. We observed that the deletion of the V1R COOH terminus did not prevent the receptor from being properly folded and expressed at the cell surface. These observations are different from those reported for the V2R. Truncation of the last 14 and 27 amino acids of the human V2R (V2R345X and V2R358X mutants) did not alter membrane insertion and binding affinity of the mutant receptors (12). However, complete deletion of the COOH terminus of the human V2R (V2R337X mutant) reduced the membrane integration of the V2R, which was retained inside the endoplasmic reticulum and no longer coupled to adenylyl cyclase (22). Increasing the number of COOH-terminal residues (truncations after codons 348, 354, and 356) restored G protein coupling but produced a length-dependent reduction of cell surface expression. These data suggest that residues in the NH2-terminal part of the human V2R COOH terminus are necessary for correct folding and that COOH-terminal residues are important for efficient cell surface expression. Moreover, a chimeric V2R with a beta 2-adrenergic receptor COOH terminus also did not bind AVP and was retained inside the cells. Thus it appears that the COOH termini of the V1R and the V2R play different roles in terms of receptor folding, membrane insertion, and, consequently, ligand binding; the V1R receptor's COOH terminus is not involved in these processes, whereas specific amino acids in the proximal portion of V2R receptor COOH terminus are required.

The amino acid residues of the proximal portion of the V2R receptor COOH terminus involved in proper membrane insertion remain to be identified. On the basis of the work done by Sadeghi et al. (28) with the human V2R, the two adjacent cysteines that are conserved in the COOH terminus of the AVP/OT receptor family are palmitoylated but are not required for AVP binding, as their replacement did not alter ligand-binding affinity, AVP-stimulation of adenylyl cyclase, receptor internalization, or desensitization. Similarly, mutations of the two adjacent cysteines of the rat V1R did not alter ligand-binding affinity or AVP stimulation of intracellular calcium release, desensitization, and internalization (27).

Cell surface receptors internalize through clathrin-coated pits and vesicles, a phenomenon that is usually triggered by agonist ligand occupancy of the receptor ligand-binding pocket. Various segments of the COOH-terminal region of several GPCRs are involved in signal transduction, endocytosis, and desensitization. For instance, endocytosis of the angiotensin II type 1 receptor (AT1a) involves two distinct regions of the COOH terminus, one that is rich in hydrophobic and aromatic residues (residues 315-329) and another distal to Lys333 that contains several serine and threonine residues (37). Maximal endocytosis of the AT1a receptor requires phosphorylation of this serine/threonine-rich segment of the COOH terminus (38). Our study indicates that the proximal portion of the V1R COOH terminus between residues 360 and 399 modulates the extent of receptor internalization, whereas the distal portion of the V1R COOH terminus between residues 399 and 418 does not seem to be involved in the process. These findings are unique to the human V1R, as a study revealed that deletion of the last 51 amino acids of the rat V1R did not affect internalization of the receptor (27). However, the deletion of 14 additional amino acids in the rat V1R sequence did impair internalization of the receptor. Mutation of Cys371 and Cys372 of the rat V1R sequence (which are also missing in our V1R359X mutant) decreased endocytosis by 26%. Substitution of Leu361 and Leu362 of the rat V1R sequence (two residues that are present in our V1R359X mutant in positions 355 and 356) reduced endocytosis by 56% without affecting calcium signaling.

Krause et al. (15) recently reported that the conserved glutamate-dileucine motif present in the COOH terminus of the human V2R functions as a small, linear vesicular transport signal that is essential for receptor transport from the endoplasmic reticulum to the Golgi apparatus. Three-dimensional modeling predicted that this glutamate-dileucine motif forms a U-like loop within the intracellular COOH terminus. In addition, Leu339 constitutes a close hydrophobic interaction with Ala61 and Leu62 of the first cytoplasmic loop. The determination of the crystal structure of rhodopsin at 2.8-Å resolution confirmed the existence of a fourth intracellular loop that forms a helical structure lying perpendicular to the seventh transmembrane domain (23). This fourth intracellular loop facilitates the stabilization of rhodopsin by a number of intramolecular interactions. From these studies, one may conclude that the two adjacent cysteines present in the proximal portion of the COOH terminus are involved in the internalization of both the human and rat V1Rs, but the upstream dileucine motif is instrumental in the internalization of the rat V1R only. Our data also suggest that the same segment of the human V1R COOH terminus is involved in receptor recycling. The COOH terminus of the V2R is also instrumental in its internalization, but it appears that it is the distal portion that plays a significant role, because a deletion of the last 14 amino acids of the V2R impaired internalization to a greater extent than the deletion of the last 27 amino acids (12). The mechanisms that underlie the internalization of GPCRs are quite complex and in some cases require receptor phosphorylation. For instance, PKC activation of the sst2A somatostatin receptor potentiates internalization of the receptor via clathrin-coated pits (10). At variance, our data suggest that PKC activation of the V1R, which is required for signal transduction, is not involved in receptor internalization, because the mutation of the first kinase consensus motif present in the V1R COOH terminus did not alter its internalization pattern. Similarly, the absence of PKC consensus motifs in the V2R COOH terminus excludes a role for this kinase in the internalization process of the family of AVP/OT receptors. The molecular determinants of the proximal portion of the V1R COOH terminus playing a role in its internalization will have to be determined by further mutational experiments. A recent work done by Preisser et al. (27), who measured by ELISA the internalization of the rat V1R, suggests that removal of the last 51 amino acids did not significantly alter the kinetics or amplitude of internalization, whereas deletion of the last 65 amino acids reduced receptor internalization by 80%.

Our data indicate that the COOH terminus of the human V1R is involved in signal transduction. As for the internalization pattern, the proximal portion of the V1R seems to be the critical segment involved. The systematic exchange of the V1R and V2R intracellular domains performed by Liu and Wess (16) revealed that the V1R's second intracellular loop was required for activation of the phosphatidylinositol pathway, whereas the V2R's third intracellular loop was required for activation of the adenylyl cyclase pathway. In a subsequent article, Erlenbach and Wess (5) reported that other AVP receptor domains besides the intracellular loops were also critical for optimum G protein-coupling efficiency. Substitution of the 21 amino acids of the proximal portion of the V2R COOH terminus in the V1R sequence resulted in a chimeric receptor that gained the ability to stimulate cAMP production to a significant extent (29% of maximum effect of the wild-type V2R). Insertion into the V1R sequence of both the third intracellular loop and the proximal portion of the V2R COOH terminus led to an agonist-induced production of cAMP similar to that of the wild-type V2R. These observations suggest that different segments of the V2R cooperate to produce a maximal activation of adenylyl cyclase, presumably through Gs coupling. Similarly, our own data with the V1R indicate that the proximal portion of the V1R COOH terminus contributes to the receptor-coupling selectivity and optimizes the efficiency of IP production. However, this segment must work in concert with other fragments of the receptor (presumably the second intracellular loop) to produce efficient Gq/11 coupling and phospholipase C activation, because the V1R COOH terminus by itself does not generate such signals and its replacement by the V2R COOH terminus does not result in dramatic alterations of IP production. Similarly, introduction of the V1R COOH terminus into the V2R sequence is not sufficient to elicit Gq/11 coupling. Moreover, the proximal portion of the V1R is absolutely required to observe a mitogenic signal. This mitogenic signal seems to necessitate the intervention of a kinase, because the inactivation of the first kinase consensus site in the proximal region of the V1R COOH terminus led to the loss of the mitogenic response. Indeed, Berrada et al. (2) recently reported that the inactivation of the first kinase consensus motif within the COOH terminus of the human V1R led to a dramatic reduction of the receptor phosphorylation. Nevertheless, the proximal region of the V1R COOH terminus is not sufficient to elicit such a signal by itself, because the introduction of the V1R COOH terminus into the V2R sequence could not produce a mitogenic signal. From these findings, one may conclude that the COOH terminus of the V1R is an active component of the scaffolding role of the receptor that coordinates protein interactions with the receptor (17). Agonist-induced activation of GPCRs leads to the successive phosphorylation by G protein-coupled receptor kinase, binding of beta -arrestins, and recruitment of several kinases including c-Src, extracellular factor-regulated kinase (ERK)1, ERK2, and c-Jun kinase 3 (18, 19). The proximal portion of the V1R is presumably involved in the formation of such scaffolding. In its absence, coupling to phospholipase C is still present, albeit with a reduced efficiency, but the further addition of downstream kinases of the scaffolding is hampered, with subsequent loss of the mitogenic signal.

Hormones modulate cellular phenotype through interaction with the cell cycle machinery by regulating cell cycle control proteins through various pathways. Several members of the GPCR family that alter cellular differentiation, proliferation, and apoptosis do so by modifying the transition between different cell cycle states (24). For instance, angiotensin II stimulates G1 phase progression via induction of cyclin D1 (CD1) mRNA and CD1K activity, presumably through a ras-ERK-AP1 pathway (41). We observed that AVP activation of the V1R led to a progression through the cell cycle all the way to the M phase, in agreement with previous assessment of cell proliferation (34). Thibonnier et al. (33) have previously shown that this progression was mediated by the simultaneous and parallel activation of several kinase pathways, mainly calcium/calmodulin kinase II, PI 3-kinase, and PKC-p42/p44 MAP kinase. Our data with the V1RS382G-R384G mutant receptor suggest that AVP stimulation may activate not only the CD1 complex controlling the restriction point of the G1 phase but also the cyclin B-cdc2 complex that controls mitogenic entry. This point will be investigated in the near future.

In conclusion, we have shown that the COOH terminus of the human V1R plays an important role in the trafficking of the receptor and works in concert with other intracytoplasmic segments of the receptor to elicit a mitogenic signal.


    ACKNOWLEDGEMENTS

We thank Judy A. Preston, Doreen M. Conarty, and Siobhan McKenna for excellent technical expertise.


    FOOTNOTES

This work was supported by National Institutes of Health Grants RO1 HL-39757 and PO1 HL-41618.

Address for reprint requests and other correspondence: M. Thibonnier, Rm. BRB431, Div. of Clinical and Molecular Endocrinology, Dept. of Medicine, Case Western Reserve Univ. School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4951 (E-mail: mxt10{at}po.cwru.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 July 2000; accepted in final form 15 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 281(1):E81-E92
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