This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Thibonnier, M. Articles by Mattera, R. Search for Related Content PubMed PubMed Citation Articles by Thibonnier, M. Articles by Mattera, R.

The Human V₃ Pituitary Vasopressin Receptor: Ligand Binding Profile and Density-Dependent Signaling Pathways¹

Departments of Medicine and Physiology, Case Western Reserve University School of Medicine and University Hospitals of Cleveland, Cleveland, Ohio 44106-4951

Address all correspondence and requests for reprints to: Dr. Marc Thibonnier, Room BRB431, Division of Clinical and Molecular Endocrinology, Department of Medicine, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, Ohio 44106-4951. E-mail: mxt10{at}po.cwru.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The vasopressin (AVP) V₃ pituitary receptor (V₃R) is a G protein-coupled corticotropic phenotypic marker that is overexpressed in ACTH-hypersecreting tumors. Studies of the agonist/antagonist binding profile and signal transduction pathways linked to the human V₃R have been limited because of the scarcity of this protein. To define the signals activated by V₃Rs and the eventual changes triggered by developmental or pathological receptor regulation, we developed Chinese hamster ovary (CHO)-V₃ cells stably expressing low, medium, or high levels of human V₃Rs (binding capacity, <10, 10–25, and 25–100 pmol/mg, respectively).

The affinity of the V₃R for 21 peptide and nonpeptide AVP analogs was clearly distinct from that exhibited by the human V₁R and V₂R. AVP triggered stimulation of phospholipase C in CHO-V₃ cells (partially sensitive to treatment with pertussis toxin) with a potency directly proportional to receptor density. V₃R-mediated arachidonic acid release also was also sensitive to pertussis toxin and more efficacious in cells exhibiting medium than in those with high receptor density. AVP also stimulated the pertussis toxin-insensitive uptake of [³H]thymidine in CHO-V₃ cells. The concentration-response curves for this effect were monophasic in cells expressing low and medium levels of V₃Rs; on the contrary, a biphasic curve was observed in cells with high V₃R density. Coupling of V₃R to increased production of cAMP was only observed in CHOV₃ high cells, suggesting a negative relationship between increased cAMP production and DNA synthesis. Activation of mitogen-activated protein kinases by V₃R was pertussis toxin insensitive, but was dependent on activation of phospholipase C and protein kinase C; both the level and duration of activation were a function of the receptor density.

Thus, the human V₃R has a pharmacological profile clearly distinct from that of the human V₁R and V₂R and activates several signaling pathways via different G proteins, depending on the level of receptor expression. The increased synthesis of DNA and cAMP levels observed in cells expressing medium and high levels of V₃Rs, respectively, may represent important events in the tumorigenesis of corticotroph cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE NEUROHYPOPHYSIAL hormone vasopressin (AVP) is a cyclic nonapeptide whose actions are mediated by stimulation of tissue-specific G protein-coupled receptors (GPCRs) currently classified into V₁ vascular (V₁R), V₂ renal (V₂R), and V₃ pituitary (V₃R) subtypes (1, 2). The cardiovascular and renal effects of AVP are reasonably well characterized, but further documentation is required regarding the mechanisms of action of AVP at the level of the central nervous system, where it acts as a neurotransmitter or neuromodulator controlling blood pressure, memory, body temperature, brain development, and release of pituitary hormones.

AVP is an important physiological regulator of the hypothalamo-pituitary-adrenal axis. AVP stimulates ACTH secretion by binding to a unique anterior pituitary receptor, distinct from the V₁R and the V₂R, which has been named the V₃ or V_1b receptor (3, 4, 5, 6, 7, 8, 9). Studies of the binding characteristics and signal transduction pathways activated after binding of AVP to its V₃Rs have been hampered by the limited availability of this protein and were performed using either animal (rat, pig, and sheep) freshly isolated cells (3, 4, 10, 11, 12) or samples of human corticotroph adenomas (13). In these studies, occupancy of V₃Rs by agonists triggered the sequential activation of phospholipase C (PLC) and protein kinase C (PKC), the phosphorylation of the myristoylated alanine-rich C kinase substrate, and secretion of ACTH (13, 14, 15). Conflicting data regarding coupling of the V₃R to adenylyl cyclase have been also reported (11, 12, 16). No information is available regarding the nature of the G protein(s) and the kinases-phosphatases coupled to the V₃R as well as the eventual mitogenic role of this receptor. Recently, different groups cloned several members of the family of human and animal AVP/oxytocin (OT) receptors (7, 17, 18, 19, 20, 21). Stable expression of these cloned receptors in mammalian cells now allows detailed characterization of the signal transduction pathways coupled to activation of a given receptor subtype by AVP.

Functional characterization of GPCRs (e.g. ß₁- and ß₂-adrenergic receptors, LH receptor, and the AVP V₂-renal receptor) in mammalian cell lines indicates that a single receptor type can activate multiple second messenger pathways through interaction with one or more G proteins (22, 23, 24). In this context, the endogenous human TSH receptor activates not only G_s and G_q/11, but also members of the G_i and G₁₂ families, indicating a complex modulation of downstream pathways (25). This pluripotential appears to be modulated by receptor density (22, 23, 24), as shown by the direct correlation between increased expression of G_s-coupled receptors and their ability to stimulate PLC in stably transfected L fibroblasts (23). Thus, GPCRs have the potential to couple to multiple signaling pathways, with the activation of a specific pathway being dependent on both the receptor density and the agonist concentration. These multiple pathways may play an important role in regulating the effectiveness of the signals in different tissues.

ACTH-secreting tumors are associated with abnormal pharmacological responses to AVP (26) and exhibit increased expression of the V₃R gene, a marker of the corticotroph phenotype (8, 9). These findings highlight the relevance of understanding the role of V₃Rs in normal and abnormal growth and development of the corticotroph cell.

In this study, we compared the ligand-binding characteristics of the human V₃R with those of the human V₁R and V₂R and studied the cellular signals coupled to activation of different densities of human pituitary V₃Rs as a prerequisite to understanding the role played by this receptor in the regulation of corticotropic cell function.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Standard reagents, unless stated otherwise, were obtained from Sigma Chemical Co. (St. Louis, MO). Chinese hamster ovary (CHO) cells were obtained from the American Type Culture Collection (Rockville, MD). Cell culture media were purchased from Life Technologies (Grand Island, NY). FBS was obtained from HyClone (Logan, UT). Iodogen (1,3,4,6-tetrachloro-3-6-diphenylglycoluril) was purchased from Pierce Chemical Co. (Rockford, IL). Restriction and modification enzymes were obtained from Boehringer Mannheim (Indianapolis, IN) or New England Biolabs (Beverly, MA). [¹²⁵I]Na (SA, 131 mCi/ml), [³H]AVP (SA, 48 Ci/mmol), myo-[2-³H]inositol (SA, 20 Ci/mmol), [³H]arachidonic acid (SA, 1 mCi/ml), and [³H]thymidine (SA, 1 mCi/ml) were obtained from New England Nuclear-DuPont (Wilmington, DE). The pBluescript II phagemid KS and XL2-Blue Escherichia coli strain were obtained from Stratagene (La Jolla, CA). The expression vector pZeoSV and the antibiotic zeocin were purchased from Invitrogen (San Diego, CA). The DNA sequencing kit was obtained from U.S. Biochemical Corp. (Cleveland, OH). AVP, OT, arginine vasotocin, lysine vasopressin, and most of the peptide V₁, V₂, V₁/V₃, and OT agonists and antagonists were obtained from Bachem (Torrance, CA) unless indicated otherwise. The nonpeptide V₁ antagonist SR 49059 (batch MY10–075) was provided by Dr. C. Serradeil-Le Gal, Sanofi Recherche (Toulouse, France). The nonpeptide rat V₁ antagonist OPC 21268 (batch 93F92M) and the nonpeptide V₂ antagonist OPC 31260 (batch 93D96M) were provided by Dr. J. F. Liard, Otsuka America Pharmaceutical (Rockville, MD). The linear V₁ antagonists 4-hydroxyphenylacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-NH₂ (OHPhaaGln) and tert-butylacetyl-D-Tyr(Et)-Phe-Val-Asn-Lys-Pro-Arg-NH₂, the V₂ antagonist d(CH₂)₅[D-Ile²-Ile⁴-Ala-NH₂]AVP, and the linear V₁/V₃ antagonist phenylacetyl-D-Tyr-Phe-Val-Asn-Arg-Pro-Arg-Arg-NH₂ were provided by Dr. Maurice Manning, Medical College of Ohio (Toledo, OH). The linear V₁ AVP antagonist phenylacetyl-D-Tyr(Et)-Phe-Val-Asn-Lys-Pro-Tyr-NH₂ (TyrPhaa), custom synthesized by Bachem, was radioiodinated ([¹²⁵I]TyrPhaa) with the Iodogen technique and purified by HPLC as previously described (27).

Complementary DNA (cDNA) constructs
We have previously described the isolation of a V₃R cDNA clone from a human pituitary tumor (7). To rule out the possibility of mutations within the receptor sequence because of its tumoral origin, we generated by PCR the human V₃R cDNA from normal human pituitary gland cDNA (human pituitary gland QUICK-clone cDNA from Clontech, catalog no. 7173–1, lot 46910). The sense primer 5'-TGCTTGAAGTCCTCTGAACG-3' (nucleotides -167 to -148 in 24 and reverse primer 5'-AAGACAGCACCATCCTAGGC-3' (nucleotides 1578–1597 in Ref. 24, downstream from the stop codon and a native SpeI site) were prepared in a synthesizer (Applied Biosystems, Foster City, CA) and purified over oligonucleotide purification cartridges following the manufacturer’s recommendations. The PCR reaction (final volume, 100 µl) contained 20 pmol of each primer, 2.5 U Taq polymerase, 5 ng normal human pituitary gland cDNA, 25 µM of each deoxy-NTP in buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂, and 0.1% gelatin, pH 7.4). After initial denaturation at 95 C for 5 min, 30 cycles were run (95 C for 1 min, 55 C for 1 min, and 72 C for 2 min) with a final extension at 72 C for 15 min. The PCR product was purified with the GeneClean II system (Bio 101, Vista, CA). An SpeI restriction site was introduced 140 bp upstream of the initiation codon by PCR, and the SpeI-SpeI cDNA fragment was digested and purified. The V₃R SpeI-SpeI construct was ligated into pBluescript II phagemid vectors before transformation of XL2-Blue E. coli strain. Double stranded DNA sequencing was performed with the Taq Dye Deoxy Terminator cycle sequencing kit and a model 373A sequencer from Applied Biosystems. Nucleotide sequences were analyzed and compared with the computer package GeneWorks on a Macintosh computer (IntelliGenetics, Mountain View, CA).

We also generated a human V₂R clone by PCR from human kidney cDNA (Clontech human kidney QUICK-Clone cDNA, no. 7112–1, lot 48570) using the sense primer 5'-CATCATGGGCCCACCATGCTCATGGCG-3' (which introduces an ApaI restriction site upstream of the start codon and includes the first 12 nucleotides in the open reading frame) (23) and the antisense primer 5'-ACACCCAGCTCAGTGAGCTG-3' (downstream from the stop codon and a native ApaI site, including nucleotides 1347–1366 in 23 . The correct sequence of this 1381-nucleotide long clone was verified by sequencing both strands before cell transfection. The conditions of the PCR amplification were as indicated above for the V₃R clones. The ApaI-ApaI fragment was subcloned into pBluescript II as described above.

Selection and stable expression of AVP receptors in CHO cells
Stable transfection of CHO cells with the V₃R cDNA clone ligated into the eukaryotic expression vector pZeoSV was performed using the calcium phosphate precipitation method as described previously (17). Clones were grown in medium F-12 supplemented with 10% FCS, selected with the neomycin analog zeocin, and purified by the limiting dilution technique. Clones (CHO-V₃) expressing various densities of V₃Rs were studied by radioligand saturation and competition binding experiments as well as measurement of inositol phosphate production, arachidonic acid release, cAMP production, thymidine uptake, and mitogen-activated protein (MAP) kinase activation as described below. Similarly, CHO cells were also transfected with the pZeoSV expression vector containing the sequence of the human V₁R (CHO-V₁) (17) or V₂R (CHO-V₂) cDNAs.

Radioligand binding assays
Control and transfected CHO cells were grown to confluence in 24-well dishes and washed twice with PBS, 10 mM MgCl₂, and 0.2% BSA, pH 7.4. Competition-binding experiments were performed as described previously (17), using one fixed concentration of [³H]AVP for CHO-V₂ cells and CHO-V₃ cells or [¹²⁵I]TyrPhaa for CHO-V₁ cells, in the presence or absence of increasing concentrations of unlabeled AVP or peptide and nonpeptide analogs (n = 3–5 for each analog) for 30 min at 30 C. IC₅₀ values were derived from nonlinear least square analysis, and K_i values were calculated by the equation of Cheng and Prusoff: K_i = IC₅₀/(1 + L_f/K_d), where L_f and K_d represent the concentration and dissociation constant of the radioligand, respectively.

Also, saturation binding experiments of AVP receptors of transfected CHO cells were performed in 24-well dishes in duplicate with increasing concentrations of [³H]AVP with or without 1 µM unlabeled AVP (17). The affinity (K_d) and binding capacity (B_max) of the AVP receptors were calculated by a nonlinear least square analysis program (28, 29). The protein concentration was measured with Pierce’s BCA reagent using albumin as an internal standard.

Inositol phosphate production
Subconfluent monolayer cultures of CHO-V₃ cells were grown for 48 h in 12-well dishes, washed with serum- and inositol-free medium (Life Technologies, no. 88–5090 EC) and labeled overnight with myo-[2-³H]inositol (1 µCi/well) in same medium. The effect of pertussis toxin (PTX) on inositol phosphate production was assessed by treatment with 0.5 µg/ml toxin during the overnight labeling period. Thereafter, the cells were preincubated for 1 h in serum-free, inositol-containing medium, followed by 15-min preincubation in HBSS containing 10 mM glucose, 1.2 mM CaCl₂, and 10 mM LiCl. Incubations were carried out for 30 min at 37 C in the presence of increasing concentrations of AVP. The reaction was stopped by the addition of 1 ml ice-cold methanol-HCl (100:1), followed by 400 µl H₂O and chloroform, to obtain a ratio of chloroform/methanol/HCl of 200:100:1. Agonist-stimulated release of inositol monophosphate was determined as described previously (17, 30). The amount of inositol phosphate released was expressed as a percentage of total labeled phosphoinositides. Typical incorporation into phosphoinositides amounted to approximately 3.5–5 x 10^{4 3}H dpm/well.

[³H]Arachidonic acid release
Subconfluent monolayer cultures of transfected CHO cells grown in 12-well plates were labeled overnight with 0.5 µCi/well [³H]arachidonic acid in serum-free medium containing 0.1% BSA. In some experiments, PTX (0.5 µg/ml) was also added during the labeling period. After labeling, the cells were washed five times with serum-free medium containing 0.1% BSA. Incubation at 37 C for 10 min was started by the addition of 1 ml medium/well containing 0.1% BSA, 100 µM unlabeled arachidonic acid, and increasing concentrations of AVP. At the end of this period, 700-µl samples of incubation medium were centrifuged at 14,000 x g for 2 min at room temperature. The release of [³H]arachidonic acid was measured by counting 500-µl samples of the supernatants.

cAMP production
Subconfluent monolayer cultures of control and transfected CHO cells were grown for 48 h in 12-well dishes and serum depleted in F-12-HEPES medium for 4 h, followed by labeling in medium containing 1 µCi/ml [³H]adenine for 2 h. Cells were washed and incubated in medium containing 0.5 mM isobutylmethylxanthine in the absence or presence of 10 µM forskolin and/or different amounts of AVP for 10 min. The reaction was stopped by the removal of medium and the addition of 0.5 ml ice-cold 5% trichloroacetic acid (TCA) containing 1.5 mM cAMP. Separation of [³H]cAMP was carried out as described by Evans et al. (31). The amount of [³H]cAMP synthesized during the incubation with agonists was expressed as a fraction of the total labeled nucleotides present in the cell extracts (10^-4 x cAMP dpm/total dpm in acid extract).

[³H]Thymidime uptake
Subconfluent monolayer cultures of transfected CHO cells were grown in 24-well plates to measure thymidine uptake in the presence of AVP as described previously (32). In some experiments, the effect of treatment with either PTX (0.5 µg/ml) or forskolin (10 µM) was assessed by adding these reagents during the last 24 h of culture in serum-free medium. Cells were washed with 500 µl F-12 medium and grown for 72 h in 500 µl F-12 medium supplemented with 25 mM HEPES and 0.1% BSA. Cells were treated with increasing concentrations of AVP (ranging from 10^-12–10^-6 M) for 18 h, followed by incubation with 0.5 µCi [³H]thymidine for 45 min. The cells were subsequently transferred on ice, washed twice with 0.5 ml ice-cold PBS, fixed with 1 ml ice-cold 10% TCA for 30 min at 4 C, washed twice with 1 ml ice-cold 5% TCA solution, and solubilized with 250 µl 0.1 N NaOH–0.1% SDS. Radioactivity present in samples of the extracts was measured using a scintillation counter. Similar experiments were conducted with a [³H]thymidine pulse for 6 h.

Cell proliferation assay
Subconfluent monolayer cultures of transfected CHO cells were grown in 96-well plates to measure cell proliferation in the presence of AVP using the CellTiter 96 cell proliferation assay from Promega (Madison, WI). Cells were washed with F-12 medium and grown for 72 h in 100 µl F-12 medium supplemented with 25 mM HEPES and 0.1% BSA. Cells were treated with 10% FBS or AVP for 18 h, followed by incubation with 15 µl dye solution for 4 h according to the manufacturer’s instructions. Subsequently, 100 µl solubilization solution were added, and after mixing for 1 h at room temperature, absorbance was recorded at 570-nm wavelength using an enzyme-linked immunosorbent assay plate reader (650-nm reference wavelength). Values recorded for positive control (cells not stimulated by AVP or FCS) were subtracted.

Phosphorylation of MAP kinase (MAPK)
Phosphorylation of p42 and p44 MAPKs by AVP was measured in subconfluent monolayer cultures of transfected CHO cells grown in 12-well plates. Cells were serum starved for 48 h before agonist stimulation. In some experiments, pretreatment with PTX (0.5 µg/ml) took place during the last 18 h of culture in serum-free medium. After stimulation with the various compounds described in Results, cells were washed twice with ice-cold PBS and lysed in buffer containing 50 mM HEPES-Tris (pH 7.5), 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml leupeptin, 10 µg/ml pepstatin, 50 µg/ml bestatin, 200 µM Na₃PO₄, and 1 mM NaF. The lysed cells were scraped and centrifuged at 14,000 x g for 20 min. The supernatants were subjected to SDS-PAGE. After electrophoresis, proteins were transferred to Immobilon-P membrane (Millipore, Bedford, MA) and immunoblotted overnight at 4 C with the phospho-specific p44/42 MAPK (Tyr²⁰⁴) antibody (rabbit polyclonal IgG affinity-purified antibody from New England Biolabs, Beverly, MA). Immunodetection was carried out with the ECL kit (Amersham, Arlington Heights, IL) following the manufacturer’s recommendations. Quantification of MAPK phosphorylation was performed by scanning densitometry of the autoradiograms with a U.S. Biochemical Corp. SciScan 5000 automated scanning system (Cleveland, OH).

Data analysis
Nucleotide and amino acid sequences were analyzed with the computer package GeneWorks on a Macintosh computer (IntelliGenetics). Binding parameters (K_d and B_max) of AVP receptors and the pharmacodynamic profile of stimulation of intracellular signals by AVP (EC₅₀ and E_max) were calculated using a nonlinear least square analysis program (28). Data were expressed as the mean ± SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Radioligand-binding characteristics of the human V₃R
The sequence of the V₃R cDNA amplified by PCR from normal human pituitary gland was identical to that present in the clone we obtained previously from a large ACTH-secreting pituitary tumor (7) and to that isolated by Sugimoto et al. from a normal pituitary gland cDNA library (33).

The binding profile of the human V₃R clone expressed in CHO cells (CHO-V₃) is clearly distinct from those of the human V₁ vascular and V₂ renal AVP receptor subtypes expressed in the same CHO cell line (Table 1). None of the 20 AVP analogs tested in our experiments had a higher affinity for the human V₃R than the endogenous ligand AVP. OT, the other endogenous ligand in humans, has a very low affinity for the V₃R (1782 nM). Interestingly, the two V₃ antagonists we tested exhibited a significantly higher affinity for the V₁R than for the V₃R, thus confirming the current lack of potent and selective antagonists for the V₃ receptor. The V₁ antagonist 4-hydroxyphenylacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-NH₂, which displays an excellent affinity for the V₁R (K_d = 0.45 nM) also binds to the V₃R with a good affinity (K_d = 2.22 nM). Although the two V₂ agonists tested in these experiments (dDAVP and dVDAVP) indeed show a good affinity for the human V₂R (2.7 and 0.8 nM, respectively), they are not very selective, as they also bind to the human V₁R and V₃R with dissociation constants in the 10–20 nM range. Along the same line, the two peptide V₂ antagonists tested in these experiments (d(CH₂)₅[D-Ile²-Ile⁴-Ala-NH₂]AVP and d(CH₂)₅[D-Ile²-Ile⁴-Arg⁸-Ala⁹-NH₂]AVP) show a rather low affinity for the human V₂R (88 and 76 nM), which is even weaker than that for the human V₁R (53 and 30 nM). This lack of selectivity for the human receptors may explain some of the discrepancies with the subtype of AVP receptor involved in the vasodilatory action of dDAVP or dVDAVP and the inhibitory properties of the antagonists tested (34). The two peptide OT antagonists tested in our experiments (d(CH₂)₅[O-Me-Tyr²-Thr⁴-Orn⁸]vasotocin and d(CH₂)₅[O-Me-Tyr²-Thr⁴-Orn⁸-Tyr⁹-NH₂]vasotocin) also display high affinity for the human V₁R, but bind poorly to the human V₃R. Finally, the three nonpeptide V₁R or V₂R antagonists tested in our cell lines (SR49059, OPC-21268, and OPC-31260) have a low affinity for the human V₃R. These results underscore the need for developing specific and potent analogs, interacting with the various human AVP receptor subtypes.

View larger version (33K): [in this window] [in a new window]   Figure 1. AVP-dependent stimulation of PLC in CHO cells transfected with the human V3R. A, Production of inositol monophosphate in response to increasing concentrations of AVP. B, Effect of PTX (0.5 µg/ml; overnight pretreatment) on the production of inositol monophosphate in response to increasing concentrations of AVP. CHO-V3 cells expressing high (V3High), medium (V3Medium), or low (V3Low) densities of V3 pituitary receptors were grown to confluence in 12-well dishes and incubated in inositol-free DMEM buffer, pH 7.4, with myo-[2-3H]inositol. Formation of [3H]inositol phosphates was measured after the addition of increasing concentrations of AVP for 30 min. Results are expressed as the percentage of [3H]inositol incorporated into phosphoinositides and represent the mean ± SEM of two independent experiments carried out in triplicate.

View larger version (33K): [in this window] [in a new window]   Figure 2. AVP-dependent release of arachidonic acid in CHO-V3 cells. A, Release of arachidonic acid in response to increasing concentrations of AVP. B, Effect of PTX (0.5 µg/ml; overnight pretreatment) on the release of arachidonic acid in response to increasing concentrations of AVP. Transfected CHO-V3 cells expressing high (V3High), medium (V3Medium), or low (V3Low) densities of V3 pituitary receptors were grown to confluence in 12-well dishes and prelabeled for 12–15 h in DMEM buffer, pH 7.4, with 0.5 µCi/well [3H]arachidonic acid. The release of [3H]arachidonic acid was measured after incubation for 10 min in the presence of increasing concentrations of AVP. Results represent the mean ± SEM of two independent experiments carried out in triplicate.

View larger version (20K): [in this window] [in a new window]   Figure 3. Stimulation of DNA synthesis by AVP in CHO cells stably expressing high (CHO-V3High), medium (CHO-V3Medium), or low (CHO-V3Low) densities of V3 pituitary receptors. Transfected CHO cells prepared as described in Materials and Methods were grown to subconfluence in 24-well dishes and incubated in serum-free F-12 medium, pH 7.4, for 72 h. The cells were stimulated by increasing concentrations of AVP overnight, followed by incubation with [3H]thymidine for 45 min, DNA precipitation, and liquid scintillation counting. Results represent the average of three independent experiments carried out in octoplicate. Similar basal values were measured in the different V3-expressing clones (range, 2000–2500 cpm/well).

View larger version (19K): [in this window] [in a new window]   Figure 4. Stimulation of DNA synthesis by AVP in stably transfected CHO cells expressing medium range densities of either V1, V2, or V3 receptors. Transfected CHO cells prepared as described in Materials and Methods were grown to subconfluence in 24-well dishes and incubated in serum-free F-12 medium, pH 7.4, for 72 h. The cells were subsequently stimulated by increasing concentrations of AVP overnight, followed by incubation with [3H]thymidine for 45 min, DNA precipitation, and liquid scintillation counting. Similar basal incorporations were measured in the different clones (range, 2000–2500 cpm/well). Results shown are the average of three independent experiments carried out in octoplicate.

View larger version (26K): [in this window] [in a new window]   Figure 5. Effects of forskolin and PTX on AVP-dependent stimulation of DNA synthesis in CHO-V3Medium cells. CHO-V3R cells prepared as described in Materials and Methods were grown to subconfluence in 24-well dishes and incubated in serum-free F-12 medium, pH 7.4, for 72 h. Treatment with either PTX (0.5 µg/ml) or forskolin (10 µM) was performed during the last 24 h of serum depletion. The cells were stimulated overnight by increasing concentrations of AVP, followed by the addition of [3H]thymidine for 45 min, DNA precipitation, and radioactivity counting. Results are representative of one experiment of three performed with similar results (all carried out in octoplicate).

View larger version (20K): [in this window] [in a new window]   Figure 6. AVP-induced phosphorylation of p42 and p44 MAPKs in CHO cells stably expressing human V3 pituitary receptors. Transfected CHO-V3High cells prepared as described in Materials and Methods were grown to subconfluence in 12-well dishes and incubated in serum-free F-12 medium, pH 7.4, for 72 h. The cells were subsequently incubated for 10 min with AVP (0.1 µM), alone or in the presence of V1, V2, or V3 antagonists (0.1 µM). Immunoblotting was performed with a phospho-specific p44/42 MAPK (Tyr204) antibody. A and B depict the signal from the ECL reaction of the blot and its corresponding densitometry, respectively. Results are representative of two similar experiments. For details, see text.

View larger version (22K): [in this window] [in a new window]   Figure 7. Concentration-response curves corresponding to AVP-induced phosphorylation of p42 and p44 MAPKs in CHO cells stably expressing high (CHO-V3High), medium (CHO-V3Medium), or low (CHO-V3Low) densities of V3 pituitary receptors. Transfected CHO cells prepared as described in Materials and Methods were grown to subconfluence in 12-well dishes, followed by incubation in serum-free F-12 medium, pH 7.4, for 72 h. The cells were stimulated for 10 min with AVP (10-12–10-6 M), and immunoblotting was performed with a phospho-specific p44/42 MAPK (Tyr204) antibody. The results shown in A and B represent the densitometry of p44 and p42 bands, respectively, in the immunoblot. Results are from a single experiment representative of two independent experiments.

View larger version (45K): [in this window] [in a new window]   Figure 8. Time courses of AVP-induced phosphorylation of p42 and p44 MAPKs in CHO cells stably expressing high (CHO-V3High), medium (CHO-V3Medium), or low (CHO-V3Low) densities of V3 pituitary receptors. Transfected CHO cells prepared as described in Materials and Methods were grown to subconfluence in 12-well dishes, followed by incubation in serum-free F-12 medium, pH 7.4, for 72 h. The cells were stimulated with AVP (10-7 M) for varying times, and immunoblotting was performed with a phospho-specific p44/42 MAPK (Tyr204) antibody. Results shown are representative of two independent experiments.

View larger version (28K): [in this window] [in a new window]   Figure 9. Pathways involved in activation of p42 and p44 MAPKs by AVP in CHO cells stably expressing AVP V3 pituitary receptors. Transfected CHO-V3High cells prepared as described in Materials and Methods were grown to subconfluence in 12-well dishes, followed by incubation in serum-free F-12 medium, pH 7.4, for 72 h. The cells were treated with vehicle, PTX (0.5 µg/ml), or TPA (1 µM) during the last 24 h of culture in serum-free medium. Cells were subsequently stimulated for 10 min with 10-8 M AVP in the presence or absence of the indicated compounds. Immunoblotting was performed with a phospho-specific p44/42 MAPK (Tyr204) antibody. A depicts exposure after ECL reaction; B and C represent the densitometry of p44 and p42 bands, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Before the cloning and functional expression of the human pituitary V₃R, the pharmacological characterization of this receptor has been very limited, relying upon experiments conducted in primary cultures of pituitary or corticotroph cells of human or animal origin. Anterior pituitary cells present in dispersed primary cultures constitute a heterogeneous mixture of well differentiated secretory cell types, including somatotropes, lactotropes, thyrotropes, gonadotropes, and corticotropes. Because of this cellular heterogeneity and the demonstrated presence of several subtypes of AVP/OT receptors in the anterior pituitary gland, development of a homogeneous cell line expressing a defined receptor subtype was central to exploring its signaling properties. No information was available in the literature regarding coupling of V₃Rs to G protein coupling, MAPK activation, and cell proliferation in normal and tumorous human corticotroph cells. Although there has been no direct assessment of the level of V₃R density in normal and tumorous human corticotroph cells, Dahia et al. recently reported a 2- to 5-fold increase in V₃R messenger RNA levels in human ACTH-secreting tumors (8). Although it is possible to infer from the level of expression of human endogenous vascular V₁Rs and renal V₂Rs that CHO-V₃Low clones express endogenous levels of V₃Rs presumably present in normal human corticotroph cells, the exact relationship of the expression level in CHO-V₃Medium and CHO-V₃High to human pathophysiology remains to be established.

Stable expression of the human pituitary V₃R in CHO cells allowed us to carry out a complete characterization of the ligand-binding properties of this AVP receptor subtype in comparison to the other two subtypes, i.e. the vascular V₁R and the renal V₂R. Our studies show that the linear V₁/V₃ antagonist phenylacetyl-D-Tyr-Phe-Val-Asn-Arg-Pro-Arg-Arg-NH₂, which displayed the highest affinity for the porcine V₃R (K_i = 37.2 nM) among AVP analogs recently available, also exhibits a reasonable affinity for the human V₃R in our studies (K_i = 15.9 nM); however, it behaves as a more potent V₁R analog in both species. In this context, it appears that the V₁R antagonist 4-OH-Phaa, the most potent V₁R compound tested in our series of experiments, also displays a high affinity for the human V₃R (K_i = 2.2 nM). As this compound can be radioiodinated, it may replace tritiated AVP as a radioligand with high specific activity to carry out further ligand binding characterization of the human V₃R. Recently, Barberis et al. (35) reported that the same compound displays a high affinity for the rat V₃R (K_i = 4.5 nM) and significantly blocked AVP-induced ACTH release in cultured rat pituitary cells. The results in Table 1 indicate that no compound displays higher affinity for the human V₃R than the native hormone AVP itself, and none is selective for this receptor subtype. This underscores the need to develop new selective agonists for this receptor subtype. The availability of the CHO-V₃ cells should facilitate the screening and development of such selective ligands. As summarized above, little is known about the characteristics of endogenous human V₃Rs expressed in corticotroph cells or the level of expression of V₃Rs in human normal and tumorous corticotroph cells.

The pathway(s) linking activation of AVP receptors to regulation of specific effectors through G protein(s) is the object of our investigations. For instance, two pathways of activation of phosphoinositide hydrolysis mediated through G proteins can be distinguished by their sensitivity or insensitivity to PTX. Gß dimers have been proposed to mediate PTX-sensitive phosphoinositide hydrolysis, whereas members of the G_q/11 family participate in the PTX-insensitive activation of PLCß (36). Experiments by Exton and colleagues showed that the rat liver V₁ AVP receptor couples to two distinct G proteins (37), and our data with human platelets are consistent with the human V₁-vascular AVP receptor coupling to G_q/11 (38). In human myometrium, OT activates PLCß by interacting with at least two G proteins of the G_q and G_i families (39). The G protein(s) linked to the human V₃R is currently unknown. Our results suggest that the human V₃R is coupled to several G proteins of the G_q, G_i, and G_s families, depending on the level of expression of this receptor. Moreover, it appears that the coupling of V₃R to different effectors (PLC, PLA₂, MAPKs, and DNA synthesis) proceeds through different combinations of G proteins. PLC activation after AVP binding to the V₃R is only partially sensitive to PTX treatment, thus suggesting the involvement of proteins of the G_q/11 and G_i families. Moreover, the degree of stimulation of PLC seems to be positively related to the receptor expression level, without intervention of counterregulatory mechanisms. On the other hand, activation of arachidonic acid release after AVP binding to the V₃R was dependent on the level of receptor expression. Both low and medium densities of V₃Rs stimulated the release of arachidonic acid with a monophasic concentration-response pattern, whereas activation of CHO-V₃High resulted in a diminished response at relatively high concentrations of agonists compared with that in cells expressing medium receptor density. This result suggested the recruitment of additional regulatory mechanisms after activation of a high number of V₃Rs. The difference in PTX sensitivity of arachidonic acid release in CHO cells expressing different levels of V₃R could be interpreted as indicative of the recruitment of different forms of PLA₂ or different G proteins. Examination of these issues as well as identification of different forms of PLA₂ coupled to V₃R require further studies.

The uptake of [³H]thymidine triggered by the V₃R was clearly dependent on the level of receptor expression. The decreased stimulation of thymidine uptake by AVP observed in the CHO-V₃High cells appears to represent differential coupling of this receptor to adenylyl cyclase, as suggested by both the inhibition of this response driven by occupancy of the G_s-coupled V₂R and the significant increase in cAMP production that was only observed in agonist-treated CHO-V₃High. It is possible to hypothesize that after expression of high levels of V₃Rs, coupling of activated receptors to G proteins becomes less specific or relaxed, leading to the recruitment of additional transducers that are not engaged at lower receptor densities. This multiple signaling potential seems to represent a shared feature among G protein-coupled receptors, as shown by stimulation of both adenylate cyclase and PLC by four classical G_s-coupled receptors (LH, V₂, ß₁-, and ß₂-receptors) expressed at high levels in L cells (23). Nonetheless, the signaling pluripotential of G protein-coupled receptors is not necessarily caused by receptor overexpression, as exemplified by the endogenously expressed human TSH receptor that couples to all four G protein families, G_s, G_i, G_q/11, and G₁₂ (25), and by the dual coupling of endogenous ß-adrenergic receptors in COS-7 cells to adenylyl cyclase and MAPK activation (due to release of _s- and ß-subunits, respectively) (40).

The mechanisms underlying dual activation of G proteins are not completely understood. As far as AVP receptors are concerned, a recent publication by Liu and Wess provides useful information about the receptor domains involved in functional coupling (41). Construction of rat V₁/human V₂ AVP receptor hybrids showed that the second intracellular loop of the V₁ receptor activates PLC, whereas receptors containing the third intracellular loop of the V₂ receptor stimulate cAMP production. This bifunctionality of the hybrid AVP receptors was not due to a totally relaxed interaction with G proteins, as no coupling of G_i to this receptor was observed. Conversely, Wong and Ross (42) reported that chimeric muscarinic cholinergic:ß-adrenergic receptors (third intracellular loop of one receptor was replaced by that of the other one) were promiscuous in their coupling to G proteins. Obviously, additional studies are required to decipher the molecular domains responsible for multiple activation of G proteins after occupancy of different levels of cognate receptors by agonists.

Receptor tyrosine kinases are involved in the regulation of cell proliferation and differentiation. Several kinases participate in this process, including the phosphoinositide 3-kinase, S6 kinase, MAPK, and Jak/STAT pathways. The role of each pathway in cell signaling varies with the cell type and agonist studied. The MAPKs are a point of convergence for mitogenic signals triggered by several classes of cell surface receptors, including the GPCRs. G_i- and G_q-coupled receptors stimulate MAPK activation via distinct signaling pathways (43). In transfected COS-7 cells, MAPKs can be stimulated by G_s, G_i, and G_q through participation of both - and ß-subunits (44). AVP stimulation of V₁-vascular receptors transiently activates MAPKs through a PTX-independent pathway (45). At least two major pathways link V₁-vascular receptors to MAPK (46): one involves TPA-sensitive PKC, and the other involves wortmannin-sensitive molecules such as phosphoinositide 3-kinase. Among other members of the AVP/OT family of receptors, OT receptors of human uterine myometrium stimulate MAPK phosphorylation via a PTX-sensitive G protein (47). In the studies here described, we observed that activation of V₃Rs leads to stimulation of the MAPK pathway. This action is specifically blocked by a V₁/V₃ antagonist, whereas both V₁- and V₂-selective antagonists are ineffective. This activation of MAPK after occupancy of V₃R is physiologically relevant, as the corresponding EC₅₀ values are in agreement with the K_d measured in the binding experiments. The lack of effect of PTX on AVP-induced MAPK phosphorylation suggests participation of a PTX-insensitive G protein/s, presumably of the G_q/11 family. This toxin insensitivity is unlikely to reflect a partial uncoupling of the V₃R to G_i due to the fact that this rather extensive treatment (0.5 µg/ml PTX, 18–24 h) results in almost complete inhibition of signaling driven by ATP receptors expressed in the same CHO cell line (48) and in approximately 50% inhibition of AVP-dependent arachidonic acid release. MAPK activation is PLC and PKC dependent, as suggested by the experiments performed in the presence of neomycin or staurosporine. This profile is different from that of angiotensin II, which activates MAPK in a PKC-independent manner in vascular smooth muscle cells (49). The down-regulation of PKC activity in CHO cells by chronic exposure to phorbol ester completely blocks G_q/11-mediated mitogenic signals, whereas G_i-mediated signals are unaffected (43). These findings are in agreement with our own data.

Cellular responses are determined by the duration of MAPK activation. The ability of a receptor to produce longer or more sustained activation of MAPK has been shown to determine the ability of a cell to differentiate or proliferate, depending on the cell type. For instance in PC12 cells, sustained MAPK activation is associated with translocation of MAPKs to the nucleus, whereas transient activation does not lead to nuclear activation (50). Along the same line, differences in V₃R receptor number markedly affected the duration of MAPK activation. Indeed, MAPK stimulation induced by AVP in CHO cells expressing high densities of V₃Rs was more intense and long lasting than in the other cell types.

These findings raise the issue of the role of V₃R in the development of ACTH-secreting tumors, which have been shown to express high levels of V₃Rs (8, 9). Two of our observations appear linked to the characteristics of ACTH-secreting corticotroph tumors. First, initial up-regulation of V₃Rs in these tumors and the augmented uptake of [³H]thymidine driven by medium range densities of this receptor, suggest that AVP may play at least a contributing role in uncontrolled corticotroph proliferation. Second, and considering the presence of cAMP-responsive elements in the POMC gene (51) and the recognized effect of cAMP-elevating agents in ACTH secretion, the increase in levels of this second messenger after activation of high densities of V₃R may imply the participation of AVP in the augmented secretion of ACTH in these tumors.

Also, the question remains as to whether other kinases contribute to the mitogenic response triggered by V₃Rs and the role of activation of these kinases by V₃R in normal and pathological corticotroph cell growth. A counterintuitive observation was that although activation of both medium and high densities of V₃Rs triggered a significant activation of p44/p42 MAPKs, only the former resulted in increased [³H]thymidine uptake. Incubation of cells with a MAP kinase kinase inhibitor (10 µM PD98059) completely prevented AVP-induced activation of p42/p44 on CHO-V₃Medium cells, but resulted in only a 30–50% inhibition of agonist-induced [³H]thymidine uptake (data not shown). This indicates that the effect of AVP on cell proliferation implies the recruitment of both p42/p44 MAPK-dependent and -independent pathways. This opens the possibility that occupancy of the V₃R by agonist may trigger simultaneous activation of other MAPK cascades, such as p38 or Jun-N-terminal kinase/stress-activated protein kinase, which are more critical for the proliferative response. In this context, the observation that arachidonic acid mediates activation of Jun-N-terminal kinase/stress-activated protein kinase (52) may explain the partial proliferative response triggered by AVP in CHO-V₃High compared with CHO-V₃Medium cells. It could be argued that the reduced proliferative effects triggered by high concentrations of AVP in CHO-V₃High cells result from an increase in cAMP levels, leading to activation of PKA and raf inhibition (53). Indeed, there is a cross-talk between the MAPK pathway and the cAMP signaling pathway, leading to a dual effect: inhibition by PKA and stimulation by G-protein ß dimers (54, 55). As expected, we have observed a partial inhibition of AVP-dependent p42/p44 activation after treatment of CHO-V₃Medium with 10 µM forskolin (results not shown). These observations indicate that although increased levels of cAMP and PKA activity inhibit agonist-dependent MAPK activation, this cannot explain the reduction in cell proliferation by AVP in CHO-V₃High cells due to the combined evidence that these cells exhibit the highest stimulation of p42/p44 and that complete inhibition of AVP-induced MAPK activation (using PD98059 results only in partial inhibition of cell proliferation. This suggests that 1) AVP recruits both MAPK-dependent and -independent pathways leading to cell proliferation; 2) elevation of cAMP levels blocks both pathways; and 3) the MAPK-independent pathway appears quantitatively more important (based on both the partial effect of the MAP kinase kinase inhibitor on thymidine uptake in CHO-V₃Medium cells and the robust activation of p42/p44 by AVP in CHO-V₃High cells. Although these studies in transfected CHO cells represent an important step in defining the signaling pathways coupled to activation of different densities of V₃Rs, it is important to mention that the relevant downstream effectors may vary in each host cell, reflecting conditions such as relative expression of G proteins and/or effector subtypes. Clearly, the host cell specificity is an important consideration in extrapolating the conclusions from this study to the phenotype of corticotroph tumors. This phenotypic modulation was recently illustrated by Calleja et al., who showed that the effect of cAMP on cell proliferation depended on both the type of tyrosine kinase receptor present and the cell type involved (56). Comparative studies examining AVP-dependent activation of multiple MAPK pathways after stimulation of corticotroph cell lines (such as AtT20) expressing different densities of V₃Rs will define this aspect in more detail.

From these experiments using CHO cells expressing various densities of V₃Rs, several conclusions can be drawn. More than one G protein seems to participate in signal transduction pathways linked to V₃Rs. The pattern of activation of a given signal is dependent on both the level of V₃R expression and the concentration of agonist. For some cellular responses, including activation of PLC and MAPKs, the degree of stimulation by AVP is directly proportional to both the concentration of agonist and the level of expression of V₃Rs. Moreover, only G protein(s) of the G_q/11 class seem to be involved in these signals. For other cellular responses, including activation of PLA₂, cAMP production, and thymidine uptake, high levels of V₃R expression seem to couple simultaneously to both stimulatory and inhibitory components in the presence of high concentrations of AVP. Also, several G proteins, including G_s, G_i and G_q/11 classes, mediate recruitment of these pathways. The increased synthesis of DNA and cAMP levels observed in cells expressing medium and high levels of V₃Rs, respectively, may represent important events in the induction and phenotype maintenance of ACTH-secreting tumors. The different intracellular pathways linked to the human V₃R are summarized in Fig. 10.

View larger version (49K): [in this window] [in a new window]   Figure 10. Intracellular pathways coupled to the activation of different densities of human AVP V3 pituitary receptors. The different signals triggered by activation of different densities of V3 pituitary receptors are linked by solid arrows for stimulatory events and by broken arrows for inhibitory events.

	Acknowledgments

 
We thank Mark Wagner for skilled technical assistance. The MAPK immunoblotting experiments shown in Figs. 6–9 were performed by N.D. in Dr. J. Douglas’s laboratory.

	Footnotes

 
¹ This work was supported in part by NIH Grants RO1-HL-39757 and PO1-HL-41618 (Program Project Grant Director Dr. J. Douglas; to M.T.), R29-DK-44097 (to L.N.B.-M.), and ACS Grant RPG-97–008-01-BE (to R.M.). Salary support for N.D. and J.P. was provided by Grant PO1-HL-41618.

Received February 10, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Thibonnier M 1993 Cytoplasmic and nuclear signalling pathways of V₁-vascular vasopressin receptors. Regul Pept 45:79–84[CrossRef][Medline]
2. Michell RH, Kirk JC, MMB 1979 Hormonal stimulation of phosphatidylinositol breakdown with particular reference to the hepatic effects of vasopressin. Biochem Soc Trans 7:861–865[Medline]
3. Antoni F 1984 Novel ligand specificity of pituitary vasopressin receptors in the rat. Neuroendocrinology 39:186–188[Medline]
4. Baertschi AJ, Friedli M 1985 A novel type of vasopressin receptor on anterior pituitary corticotrophs? Endocrinology 116:499–502[Abstract]
5. Arsenijevic Y, Dubois-Dauphin M, Tribollet E, Manning M, Sawyer WH, Dreifuss JJ 1994 Vasopressin-binding sites in the pig pituitary gland: competition by novel vasopressin antagonists suggests the existence of an unusual receptor subtype in the anterior lobe. J Endocrinol 141:383–391[Abstract/Free Full Text]
6. Jard S, Gaillard RC, Guillon G, Marie J, Schoenenberg P, Muller AF, Manning M, Sawyer WH 1986 Vasopressin antagonists allow demonstration of a novel type of vasopressin receptor in the rat adenohypophysis. Mol Pharmacol 30:171–177[Abstract]
7. De Keyser Y, Auzan C, Lenne F, Beldjord C, Thibonnier M, Bertagna X, Clauser E 1994 Molecular cloning, sequencing, and functional expression of a cDNA encoding the human V₃-pituitary vasopressin receptor. FEBS Lett 356:215–220[CrossRef][Medline]
8. Dahia PLM, Ahmed-Shuaib A, Jacobs RA, Chew SL, Honegger J, Fahlbusch R, Besser GM, Grossman AB 1996 Vasopressin receptor expression and mutation analysis in corticotropin-secreting tumors. J Clin Endocrinol Metab 81:1768–1771[Abstract]
9. De Keyser Y, Lenne F, Auzan C, Jégou S, René P, Vaudry H, Kuhn JM, Luton JP, Clauser E, Bertagna X 1996 The pituitary V3 vasopressin receptor and the corticotroph phenotype in ectopic ACTH syndrome. J Clin Invest 97:1311–1318[Medline]
10. Aguilera G 1994 Regulation of pituitary ACTH secretion during chronic stress. Front Neuroendocrinol 15:321–350[CrossRef][Medline]
11. Giguere V, Labrie F 1982 Vasopressin potentiates cyclic AMP accumulation and ACTH release induced by corticotropin-release factor in rat anterior pituitary cells in culture. Endocrinology 111:1752–1754[Medline]
12. Knepel W, Homolka L, Vlakovska M, Nutto D 1984 In vitro adrenocorticotropin/ß-endorphin-releasing activity of vasopressin analogs is related neither to pressor nor to antidiuretic activity. Neuroendocrinology 38:344–350[Medline]
13. Levy A, Lightman SL, Hoyland J, Mason WT 1990 Inositol phospholipid turnover and intracellular Ca⁺⁺ responses to thyrotropin-releasing hormone, gonadotropin-releasing hormone and arginine vasopressin in pituitary corticotroph and somatotroph adenomas. Clin Endocrinol (Oxf) 33:73–79[Medline]
14. Liu JP 1994 Studies of the mechanisms of action of corticotropin-releasing factor (CRF) and vasopressin (AVP) in the ovine anterior pituitary: evidence that CRF and AVP stimulate protein phosphorylation and dephosphorylation. Mol Cell Endocrinol 106:57–66[CrossRef][Medline]
15. Liu JP, Engler D, Funder JW, Robinson PJ 1994 Arginine vasopressin (AVP) causes the reversible phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein in the ovine anterior pituitary: evidence that MARCKS phosphorylation is associated with adrenocorticotropin (ACTH) secretion. Mol Cell Endocrinol 105:217–226[CrossRef][Medline]
16. Holmes MC, Antoni FA, Szentendrei T 1984 Pituitary receptors for corticotropin-releasing factor: no effect of vasopressin on binding or activation of adenylate cyclase. Neuroendocrinology 39:162–169[Medline]
17. Thibonnier M, Auzan C, Wilkins P, Berti-Mattera L, Madhun Z, Clauser E 1994 Cloning, sequencing, and functional expression of the cDNA coding for the human V_1a vasopressin receptor. J Biol Chem 269:3304–3310[Abstract/Free Full Text]
18. Birnbaumer M, Seibold A, Gilbert S, Ishido M, Barberis C, Antaramian A, Brabet P, Rosenthal W 1992 Molecular cloning of the receptor for human antidiuretic hormone. Nature 357:333–335[CrossRef][Medline]
19. Kimura T, Tanizawa O, Mori K, Brownstein MJ, Okayama H 1992 Structure and expression of a human oxytocin receptor. Nature 356:526–529[CrossRef][Medline]
20. Morel A, O’Carroll AM, Brownstein MJ, Lolait SJ 1992 Molecular cloning and expression of a rat V_1a arginine vasopressin receptor. Nature 356:523–526[CrossRef][Medline]
21. Lolait SJ, O’Carroll AM, McBride OW, Konig M, Morel A, Brownstein MJ 1992 Cloning and characterization of a vasopressin V₂ receptor and possible link to nephrogenic diabetes insipidus. Nature 357:336–339[CrossRef][Medline]
22. George ST, Berrios M, Hadcock JR, Wang HY, Malbon CC 1988 Receptor density and cAMP accumulation: analysis in CHO cells exhibiting stable expression of a cDNA that encodes the beta₂-adrenergic receptor. Biochem Biophys Res Commun 150:665–672[CrossRef][Medline]
23. Zhu X, Gilbert S, Birnbaumer M, Birnbaumer L 1994 Dual signaling potential is common among G_s-coupled receptors and dependent on receptor density. Mol Pharmacol 46:460–469[Abstract]
24. Birnbaumer M 1995 Mutations and diseases of G protein coupled receptors. J Recept Signal Transduction Res 15:131–160[Medline]
25. Laugwitz KL, Allgeier A, Offermanns S, Spicher K, Van Sande J, Dumont JE, Schultz G 1996 The human thyrotropin receptor: a heptahelical receptor capable of stimulating members of all four G protein families. Proc Natl Acad Sci USA 93:116–120[Abstract/Free Full Text]
26. Malerbi DA, Mendonça BB, Liberman B 1993 The desmopressin stimulation test in the differential diagnosis of Cushing’s disease. Clin Endocrinol (Oxf) 38:463–472[Medline]
27. Thibonnier M, Bayer AL, Madhun Z, Linear V 1993 1-Vascular vasopressin antagonists suitable for radioiodination, biotinylation, and fluorescent labeling. Am J Physiol 265:E906–E913