Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6783-6787, July 1995 Neurobiology Extrapituitary expression of the rat Vlb vasopressin receptor gene (G protein-coupled receptor/neurohypophysial hormones/in situ hybridization/rat brain) STEPHEN J. LOLAIT*t, ANNE-MARIE O'CARROLL*, LAWRENCE C. MAHANt, CHRISTIAN C. FELDER*, DONALD C. BUTrON*, W. SCOrr YOUNG III*, EVA MEZEY§, AND MICHAEL J. BROWNSTEIN* *Laboratory of Cell Biology, Building 36, Room 3A-17, National Institute of Mental Health, and *Laboratory of Neurochemistry, and §The Clinical Neuroscience Branch, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, MD 20892 Communicated by Julius Axelrod, National Institute of Mental Health, Bethesda, MD, December 19, 1995 ABSTRACT [Arg8]vasopressin (AVP) stimulates adreno- corticotropic hormone release from the anterior pituitary by acting on the Vlb AVP receptor. This receptor can be distin- guished from the vascular/hepatic Vla and renal V2 AVP receptors by its differential binding affinities for structural analogous of AVP. Recent studies have shown that the cloned Vla and V2 receptors are structurally related. We have isolated a clone encoding the Vlb receptor from a rat pituitary cDNA library using polymerase chain reaction (PCR)-based methodology. The rat Vlb receptor is a protein of 421 amino acids that has 37-50% identity with the Vla and V2 receptors. Homology is particularly high in the seven putative mem- brane-spanning domains of these guanine nucleotide-binding protein-coupled receptors. Expression of the recombinant receptor in mammalian cells shows the same binding speci- ficity for AVP agonists and antagonists as the rat pituitary Vlb receptor. AVP-stimulated phosphotidylinositol hydrolysis and intracellular Ca2+ mobilization in Chinese hamster ovary or COS-7 cells expressing the cloned receptor suggest second messenger signaling through phospholipase C. RNA blot analysis, reverse transcription PCR, and in situ hybridization studies reveal that Vlb receptor mRNA is expressed in the majority of pituitary corticotropes as well as in multiple brain regions and a number of peripheral tissues, including kidney, thymus, heart, lung, spleen, uterus, and breast. Thus, the Vlb receptor must mediate some of the diverse biological effects of AVP in the pituitary as well as other organs. The neurohypophysial hormone [Arg8]vasopressin (AVP), a nonapeptide synthesized primarily in the hypothalamus, has diverse hormonal actions in peripheral tissues, including the inhibition of diuresis, contraction of vascular smooth muscle, stimulation of hepatic glycogenolysis, and release of adreno- corticotropic hormone (ACTH) (1, 2). In the brain, AVP may act as a neurotransmitter or neuromodulator in various phys- iological responses such as thermoregulation, cardiovascular homeostasis, display of social behavior, as well as modulation of learning and memory (3). AVP exerts its actions by binding to specific guanine nucleotide-binding protein (G protein)- coupled receptors that have been defined on the basis of their tissue distribution and pharmacology. At least three receptors for AVP (Vla, Vlb, and V2) and one receptor for the related hormone, oxytocin (OT), have been described (1). The Vla receptor (R) is found in many tissues, including brain, pitu- itary, liver, blood vessels, and kidney, while the V1bR, distin- guished from the Vla subtype by different agonist and antag- onist affinities (4-6), is found in the anterior pituitary where it modulates ACTH secretion from corticotropes (1, 2). Both VlRs (and the OTR) act via phosphotidylinositol hydrolysis and mobilization of intracellular Ca2 . On the other hand, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. V2Rs positively couple to adenylate cyclase and are predom- inantly expressed in the kidney where they mediate the anti- diuretic effect of AVP (1). Recently, the structures of the rat VlaR and human OTR cDNAs have been elucidated (7, 8). Subsequently, the V2Rs (9-11), a teleost fish arginine vasotocin (AVT) receptor (12), and species homologues of the OTR and VlaR (11, 13, 14) have been cloned. These receptors form a distinct subfamily of G protein-coupled receptors with 35-60% amino acid identity; most homology resides in their seven putative transmembrane (TM) domains. Regulation of ACTH secretion is a critical component in the mammalian response to stress. Although in most species corticotropin-releasing factor (CRF) plays the dominant role in stimulating transcription of the gene encoding the ACTH precursor, proopiomelanocortin (POMC), AVP powerfully synergizes with CRF in releasing ACTH (2). An understanding of the precise molecular basis for AVP effects at the pituitary VlbR has been hampered by the lack of VlbR antagonists, radioligands, and cloned receptor cDNAs. In the present study, we have utilized the polymerase chain reaction (PCR) and AVP/OTR sequence homology to clone the rat pituitary V1bR.V Expression of the cDNA confers Vlb-type pharma- cology on transfected mammalian cells. In addition to the pituitary, the VlbR gene is widely expressed in brain and peripheral tissues, suggesting that AVP may have novel actions on extrapituitary VlbRs. MATERIALS AND METHODS Materials. AVP, OT, AVT, or 1-desamino-3,4-cyclopenta- methylene propionic acid (O-methyltyrosine)AVP {mVP or [d(CH2)s,Tyr(Me)2]AVP}, 1-desamino[penicillamine,O- methyltyrosine]AVP {dPVP or [dPen,Tyr(Me)2]AVP}, 1-des- amino[8-D-arginine]vasopressin (dDAVP), d(CH2)5,[Tyr(Me)2, Thr4,Orn8,Tyr-NH29]vasotocin (OTA), and [d(CH2)5-D-Ile2, Ile4,Arg8]vasopressin ([dIle2,Ile4]AVP) were purchased from Peninsula Laboratories. Desamino[D-3-(pyridyl)Ala2,Arg8]- vasopressin (d-3PAL) was from Bachem. [3H]AVP (specific activity, 64.8 Ci/mmol; 1 Ci = 37 GBq) was obtained from DuPont/NEN. Abbreviations: AVP, [Arg8]vasopressin; OT, oxytocin; G protein, guanine nucleotide-binding protein; ACTH, adrenocorticotropic hor- mone; POMC, proopiomelanocortin; TM, transmembrane; R, recep- tor; CRF, corticotropin-releasing factor; IP, inositol phosphate; IP3, inositol 1,4,5-trisphosphate; CHO, Chinese hamster ovary; RT-PCR, reverse transcription polymerase chain reaction; ISH, in situ hybrid- ization; AVT, arginine vasotocin; mVP, 1-desamino-34,3-cyclopenta- methylene propionic acid (O-methyltyrosine)AVP; dPVP, 1-desamino- [penicillamine,O-methyltyrosine]AVP; dDAVP, 1-desamino[8-D- arginine]vasopressin; OTA, d(CH2)s,[Tyr(Me)2,Thr4,Orn8,Tyr- NH29]vasotocin; [dIle2,Ile4]AVP, [d(CH2)5-D-Ile2,Ile4,Arg8]- vasopressin; d-3PAL, desamino[D-3-(pyridyl)Ala2,Arg8]vasopressin. tTo whom reprint requests should be addressed. 5The sequence reported in this paper has been deposited in the GenBank data base (accession no. U27322). 6783