Molecular cloning, sequencing, and functional expression of a cDNA encoding the human V1a vasopressin receptor.
Vasopressin (AVP), the antidiuretic hormone, is a cyclic nonapeptide that acts through binding to G protein-coupled specific membrane receptors pharmacologically divided into three subtypes (V1a, V1b, and V2) linked to distinct second messengers. Within the family of human AVP receptors, the V2 AVP receptor has been cloned, but the structure of the human V1a and V1b AVP receptors remains unknown. We report here the structure and functional expression of a human V1a AVP receptor complementary DNA isolated from human liver cDNA libraries. Cloning and sequencing of a full-length clone isolated a 1472-nucleotide sequence encoding a 418-amino acid polypeptide with seven putative transmembrane domains typical of G protein-coupled receptors. Amino acid sequence identity with the rat liver V1a AVP receptor, the human and rat V2 AVP receptors, and the human oxytocin receptor was 72, 36, 37, and 45%, respectively. 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 cellular surface was illustrated by using the fluorescent linear analog phenylacetyl-D-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2 coupled to fluorescein-avidin by dodecabiotin. Competition binding experiments with phenylacetyl-D-Tyr(Et)-Phe-Val-Asn-Lys-Pro-[125I]Tyr-NH2 and AVP analogs revealed high affinity specific binding sites of the V1a subtype. Saturation binding experiments with [3H]AVP confirmed the presence of a single class of high affinity binding sites. Measurement of AVP-induced inositol phosphate production and calcium mobilization confirmed that the expressed V1a AVP receptor is coupled to phospholipase C via a pertussis toxin-insensitive pathway. Thus, the human V1a AVP receptor belongs to the superfamily of seven-transmembrane segment receptors with a significant sequence identity with the other members of the AVP-oxytocin family of receptors.
Thibonnier
M
M
Department of Medicine, University Hospitals of Cleveland, Ohio.
Auzan
C
C
Madhun
Z
Z
Wilkins
P
P
Berti-Mattera
L
L
Clauser
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D001483:Base Sequence; D001667:Binding, Competitive; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D006224:Cricetinae; D015342:DNA Probes; D018076:DNA, Complementary / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D037342:Pertussis Toxin; D051381:Rats; D018045:Receptors, Oxytocin / Q000235:genetics; D017483:Receptors, Vasopressin / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D015342:DNA Probes; D018076:DNA, Complementary; D007295:Inositol Phosphates; D009838:Oligodeoxyribonucleotides; D018045:Receptors, Oxytocin; D017483:Receptors, Vasopressin; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D001127:Arginine Vasopressin; D037342:Pertussis Toxin