Expression cloning of the human V2 vasopressin receptor

Regulatory Peptides, 45 (1993) 61-66 © 1993 Elsevier Science Publishers B.V. All fights reserved 0167-0115/93/$06.00

61

REGPEP 01344

Expression cloning of the human V 2 vasopressin receptor C. Barberis b, A. Seibold a, M. Ishido c, W. Rosenthal a and M. Birnbaumer a aDepartment of Cell Biology, Baylor College of Medicine, Houston, TX (USA), b Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Mon~ellier (France) and Cinstitute of Applied Biochemistry, University of Tsukuba, Ibaraki-ken (Japan)

Key words: Vasopressin receptor; Human kidney; Amino acid sequence

Introduction Vasopressin and oxytocin are the neurohypophysial hormones in mammalian species. The nonapeptide structure of these hormones appears wellconserved in evolution. In vertebrates, ten peptides which are largely homologous, have been characterized [1]. These peptides control a broad range of physiological effects (among those, the antidiuretic, pressor, uterotonic and milk-ejecting activities are the best known). These effects are obtained through the activation of a family of G-protein coupled receptors. This family of receptors display a remarkable diversity in its pharmacological, functional and biological properties. Four subtypes of receptors have been distinguished on a functional and/or pharmacological basis. Renal vasopressin receptors (V2-type) that mediate the antidiuretic response were among the first membrane receptors for which a functional coupling with adenylate cyclase was demonstrated [2]. Vasopressin receptors of the liver (Via-type) and adenohypophysis (Vlb-type) and oxytocin receptors of

Correspondence to: Correspondance to: C. Barberis, Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Rue de la Cardonille, 34094 Montpellier, Cedex 5 France.

the uterus and mammary gland act through increased inositol turn over to mobilize intracellular Ca 2+ [3 ]. A tremendous amount of analogues of neurohypophysial hormones have been synthesized. Some of them are potent and selective agonists and antagonists of the antidiuretic and vasopressor responses to vasopressin and of the uterotonic response to oxytocin [4]. These peptides were extensively used as pharmacological probes for the characterization of vasopressin and oxytocin receptors. They have allowed the detection of differences in the ligand selectivity ofvasopressin and oxytocin receptors among mammalian species. Examples of these differences are the characterization of the Via and Vlb type of vasopressin receptors, as well as the uncovering of ligand selectivity of renal V2 vasopressin receptors from the three mammalian species which have been the more extensively studied: human, rat and pig. Worth mentioning is the analogue 1-deamino penicillamine-[4-valine,8D-arginine]vasopres sin, which is an antagonist of vasopressin-induced adenylate cyclase activation in the pig kidney, and a potent activator of rat kidney adenylate cyclase. It seems that the extraordinary complexity of this family of receptors makes the molecular identification and characterization of the different members of the family very valuable to further explore their properties, i.e., the functional domains such as the ligand

62 interaction, G-protein interaction, desensitization and down regulation. Here, we report the cloning of a complementary DNA encoding the V2 vasopressin receptor from the human kidney. This receptor, when activated by vasopressin, promotes water permeabilization of the collecting duct [5,6] and stimulation of sodium, calcium, magnesium and chloride reabsorption by the ascending limb [5,7] through an increase in adenylyl cyclase activity.

ance of a new property: the ability to respond to AVP. These cells were subcultured and one of the

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The strategy used for cloning the V2 vasopressin receptor is based on the transformation of the mouse L t k - cell from V2R- to V2R ÷ by DNA mediated gene transfer [8]. Except for prostaglandins, the adenylyl cyclase of these cells is insensitive to any of the following hormones: LH, FSH, TSH, ACTH, calcitonin, PTH, CRF, GRF, glucagon, secretin, VIP, vasopressin (AVP), and noradrenaline. The cells respond however to forskolin and manipulations that activate Gs, such as addition of NaF and GTP analogs. These results indicated that L t k - cells have a normal adenylyl cyclase system susceptible to stimulation by Gs-coupled stimulatory receptor(s). Mouse L t k - cells were co-transfected with human genomic DNA and the selectable marker gene thymidine kinase (see Fig. 1 and Ref. 8). They were then plated in 96-well microtitration plates at a density such that after HAT (100 # M hypoxanthine, 1 # M aminopterine and 10 # M thymidine) selection, each well contained, on the average, two to three Ltk ÷ cell clones. Then, they were screened for hormonestimulated adenylyl cyclase activity in each individual well, using a mixture of the 12 stimulatory peptide hormones listed above. Applying this methodology, a primary transformant which expresses a human peptide receptor was obtained after analyzing 36 96-well plates of transfected and HAT-selected cells. Those positive cells were designated HTB cells. It was shown that the elevated adenylyl cyclase activity of HTB cells was due to the appear-

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63 subcultured cells which continued to exhibit higher adenylyl cyclase activity than the others on stimulation by AVP was designated HTB-1 cells. They were estimated to contain 12.106 base pairs of human DNA. Genomic D N A was extracted from the cloned HTB-1 cell line and secondary transfections were carried out, utilizing this genomic D N A as the source of human D N A containing the V 2 vasopressin receptor gene. A new cell line, called HTB-2, which contains around 500.103 base pairs of human D N A and also expressed the human V 2 vasopressin receptor was obtained. Genomic D N A was extracted from the cloned HTB-2 cell line and used to obtain a tertiary transformant, called HTB-3, which contained about 50.103 base pairs of human DNA, and which also expressed the human V2 vasopressin receptor [9]. Genomic D N A was extracted from the cloned HTB-3 cell line and used to construct a genomic library in the phage 2EMBL-3. This genomic library was screened for clones positive for human repetitive DNA sequence (Alu), using labelled total genomic DNA as a probe [10]. Seven clones positive for human D N A were obtained. They were used to transfect mouse L t k - cells. One of them ( 2 E M B L 3 14) was able to confer arginine vasopressin responsiveness to the new cell transformants (called LV 2 cells). Several of these LV 2 cells, the LV>G9, LV2.D4 and LV2.E7 expressed the human V 2 vasopressin receptor at a very high level (200,000 receptor sites per cell). A specific oligo(dT) primed complementary D N A library was constructed in 2gtl0 using poly(A) + RNA from the LV2.G9 cells as template. This library was screened for recombinant phages with a 2.2 kb Barn HI D N A fragment, isolated from the phage 2EMBL3-14, which was previously shown to be able to confer arginine vasopressin responsiveness to L cell adenylyl cyclase when tested in stable transfection assays [9]. One positive clone was isolated (V:R-G9.3). It was 1663 base pairs long. It was sequenced after subcloning into Bluescript KS ( + ) .

Fig. 2 shows the deduced amino-acid sequence of the cDNA. This c D N A predicts a protein of 371 aminoacids with a calculated M r of 40,225 in the absence of post-translational modifications. It has one potential glycosylation site in the amino terminus, two potential phosphorylation sites for protein kinase C and one potential phosphorylation site for casein kinase. Hydropathicity analysis of the translated protein sequence suggests the presence of seven transmembrahe domains that are characteristic of G proteincoupled receptors. To ascertain the identity of the protein coded by V2R-G9.3, we first examined the tissue distribution of its transcript by Northem blot analysis, using the 1,663 base pairs human kidney c D N A as a probe. An mRNA of about 1.9-2.0 kilobases was found in kidney RNA from two species: rat and rabbit. A smaller transcript was found in the porcine kidney cell L L C P K 1, known to express a V 2 lysine vasopressin receptor. A new cDNA (V2R-hk.5) was cloned from a human kidney c D N A library using two oligonucleotides based on the sequence of the V2RG9.3 clone as screening probes. The nucleotide composition of V2R-hk.5 c D N A differed from that derived from the V2R-G9.3 cells only in that it was four nucleotide longer at its 5' end. The pharmacological characteristics of the receptor encoded by V2R-G9.3 were studied by expressing the c D N A in eukaryotic cells. Arginine vasopressin increased the adenylyl cyclase activity of COS.M6 cells transiently transfected with the receptor c D N A subcloned into the expression plasmid p K N H [ 11 ].

Discussion Amino acid identities to other members of the superfamily are concentrated within the transmembrane domains. The human V 2 vasopressin receptor is not particularly closely related to the other G protein-related receptors in terms of sequence similarity (25-35~o identity in the core covering trans-

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membrane segments I-VII). However, the neurohypophysial hormone receptors are strikingly similar to each other in both size and amino acid sequences. The human V2 vasopressin receptor has 50~o similarity with the rat V xvasopressin receptor and human oxytocin receptor (Fig. 2). This similarity is comparable to those observed between human fiX and r2 adrenergic receptor subtypes (54 ~o) and porcine M l and M 2 muscarinic receptor subtypes (43 ~o) [ 12,13 ]. The sequence similarity is remarkable not only in the putative transmembrane domains and their flanking sequences, but also in their first and second extracellular loop. These conserved regions are plausible candidates to form part of the peptide recognition site. It should then be possible to highlight the structural domains confering the highest selectivity for the different subtypes of vasopressin and oxytocin receptors and to pin-point the more prominent species differences in the structure responsible for the ligand selectivity of vasopressin and oxytocin analogues. And it would not be surprising to find several subtypes of V 1, V2 and OT receptors not yet distinguished on a pharmacological basis. The third cytoplasmic loop, the domaine thought to be important in the coupling to G proteins is the segment with the highest diversity between the V2 vasopressin receptor (which is coupled to cAMP production) on one hand and the V 1 vasopressinand oxytocin- receptors (which are coupled to IP 3 production and Ca 2+ mobilization) on the other hand. Finally the amino-terminal and carboxyterminal regions also differ between the three receptors. The definition of the different functional domains of these receptors can now be attempted. Regulations such as the down regulation of the receptors and the modulation of the generation of

cAMP by the process of desensitization can be explored. Even more important, it should be possible to establish the biochemical basis underlying the multiple biological effects of vasopressin and oxytocin in mammals.

References 1 Acher, R. and Chauvet, J., Structure, processing and evolution of the neurohypophysial hormone-neurophysin precursors, Biochimie, 70 (1988) 1197-1207. 2 Bockaert, J., Roy, C., Rajerison, R. and Jard, S., Specific binding of [3H ]-lysine vasopressin to pig kidney plasma membranes. Relationship of receptor occupancy to adenylate cyclase activation, J. Biol. Chem., 248 (1973) 5922-5931. 3 Jard, S., Elands, J., Schmidt, A. and Barberis, C., Vasopressin and oxytocin receptors: an overview. In H. Imura and K. Shizume (Eds.), Progress in Endocrinology, Elsevier, Amsterdam, 1988, pp. 1183-1188. 4 Manning, M. and Sawyer, W.H., Antagonists of vasopressin and oxytocin: current status and future perspectives. In S. Jard and R. Jamison (Eds.), Vasopressin, John Libbey, Paris, 1991, pp. 297-309. 5 Elalouf, J.M., Di Stefano, A. and De Rouffignac, C., Sensitivities of rat kidney thick ascending limbs and collecting ducts to vasopressin in vivo, Proc. Natl. Acad. Sci. USA, 83 (1986) 2276-2280. 6 Handles, J.S. and Orloff, J.,Antidiuretic hormone, Annu. Rev. Physiol., 43 (1981) 611-624. 7 Wittner, M., Di Stefano, A., Wangemann, P., Nitschke, R., Greger, R., Bailly, C., Amid, C. and De Rouffignac, C., Differential effects of ADH on sodium, chloride, potassium, calcium and magnesium transport in cortical and medullary thick ascending limbs of mouse nephron, PflOegers Arch., 412 (1988) 516-523. 8 Birnbaumer, M., Hinrichs, V. and Themen, P.N., Development and characterization of a mouse cell line expressing the human V2 vasopressin receptor gene, Mol. Endocrinol., 4 (1990) 245-254. 9 Birnbaumer, M., Seibold, A., Gilbert, S., Ishido, M., Barberis,

Fig. 2. Primary sequence comparison between the neurohypophysial peptide receptors. Gaps were introduced to maximize sequence identities. Asterisks denote predicted transmembrane regions I through VII. h V2, amino acid sequence of the human V2 vasopressin receptor [9], r V2, amino acid sequence of the rat V2 vasopressin receptor [ 14], r V1, amino acid sequence of the rat Via vasopressin receptor [15], h OT, amino acid sequence of the human oxytocin receptor [16]. The similarities between h V2 and r V2, r V1, h OT are 87~o, 48~o, 49~/o, respectively.

66 C., Antaramian, A., Brabet, P. and Rosenthal, W., Molecular cloning of the receptor for human antidiuretic hormone, Nature, 357 (1992) 333-335. 10 Ktlhn, L.C., McClelland, A. and Ruddle F.H., Gene transfer, expression and molecular cloning of the human transferrin receptor gene, Cell, 37 (1984) 95-103. 11 Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kangawa, K., Minamino, N., Mataro, H., Ueda, M., Hanaoka, M., Horose, T. and Numa, S., Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor, Nature, 339 (1989) 439-445. 12 Lefkowitz, R.J. and Caron, M.G., Adrenergic receptors. Models for the study of receptors coupled to guanine nucleotide regulatory proteins, J. Biol. Chem., 263 (1988) 4993-4996.

13 Liao, C.F., Themmen, A.P.N., Joho, R., Barberis, C., Birnbaumer, M. and Birnbaumer, L., Molecular cloning and expression of a fifth muscarinic acetylcholine receptor, J. Biol. Chem., 264 (1989) 7328-7337. 14 Lolait, S.J., O'Carrol, A.M., McBride, O.W., Konig, M., Morel, A. and Brownstein, M.J., Cloning and characterization of a vasopressin V2 receptor and possible link to nephrogenic diabetes insipidus, Nature, 357 (1992) 336-339. 15 Morel, A., O'Caroll, A.M., Brownstein M.J. and Lolait S.J., Molecular cloning and expression of a rat Via arginine vasopressin receptor, Nature, 356 (1992) 523-526. 16 Kimura, T., Tanizawa, O., Mori, K., Brownstein, M.J. and Okayama, H., Structure and expression of a human oxytocin receptor, Nature, 356 (1992) 526-529.