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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ABSTRACT |
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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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INTRODUCTION |
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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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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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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 (1012-10
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 (108 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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RESULTS |
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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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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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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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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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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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DISCUSSION |
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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 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 -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.
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ACKNOWLEDGEMENTS |
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We thank Judy A. Preston, Doreen M. Conarty, and Siobhan McKenna for excellent technical expertise.
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FOOTNOTES |
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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.
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