MOLECULAR-CLONING, SEQUENCING, AND FUNCTIONAL EXPRESSION OF A CDNA-ENCODING THE HUMAN-V(1A) VASOPRESSIN RECEPTOR

Vasopressin (AVP), the antidiuretic hormone, is a cyclic nonapeptide t hat acts through binding to G protein-coupled specific membrane recept ors pharmacologically divided into three subtypes (V1a, V1b, and V2) l inked to distinct second messengers. Within the family of human AVP re ceptors, the V2 AVP receptor has been cloned, but the structure of the human V1a and V1b AVP receptors remains unknown. We report here the s tructure and functional expression of a human V1a AVP receptor complem entary DNA isolated from human liver cDNA libraries. Cloning and seque ncing of a full-length clone isolated a 1472-nucleotide sequence encod ing a 418-amino acid polypeptide with seven putative transmembrane dom ains typical of G protein-coupled receptors. Amino acid sequence ident ity with the rat liver V1a AVP receptor, the human and rat V2 AVP rece ptors, and the human oxytocin receptor was 72, 36, 37, and 45%, respec tively. Functional characterization of the cloned receptor was done by transient expression in COS-7 cells and stable expression in Chinese hamster ovary cells. Localization of the expressed receptor at the cel lular surface was illustrated by using the fluorescent linear analog p henylacetyl-D-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2 coupled to fluoresce in-avidin by dodecabiotin. Competition binding experiments with cetyl- D-Tyr(Et)-Phe-Val-Asn-Lys-Pro-[I-125]Tyr-NH2 and AVP analogs revealed high affinity specific binding sites of the V1a subtype. Saturation bi nding experiments with [H-3]AVP confirmed the presence of a single cla ss of high affinity binding sites. Measurement of AVP-induced inositol phosphate production and calcium mobilization confirmed that the expr essed V1a AVP receptor is coupled to phospholipase C via a pertussis t oxin-insensitive pathway. Thus, the human V1a AVP receptor belongs to' 'the superfamily of seven-transmembrane segment receptors with a signi ficant sequence identity with the other members of the AVP-oxytocin fa mily of receptors.