Cloning and expression of the human vasoactive intestinal peptide receptor.
Vasoactive intestinal peptide (VIP) is a neuroendocrine mediator found in the central and peripheral nervous system. Distinct subsets of neural, respiratory, gastrointestinal, and immune cells bear specific high-affinity receptors for VIP, which are associated with a guanine nucleotide-binding (G) protein capable of activating adenylate cyclase. A cDNA clone (GPRN1) encoding the human VIP receptor was identified in libraries prepared from the Nalm 6 line of leukemic pre-B lymphoblasts and the HT-29 line of colon carcinoma cells. The deduced 362-amino acid polypeptide sequence encoded by GPRN1 shares a seven-transmembrane-segment hydropathicity profile with other G protein-coupled receptors. Northern blot analyses identified a 2.7-kilobase transcript of the VIP receptor in Nalm 6 and HT-29 cells as well as in tissues from rat brain, colon, heart, lung, kidney, spleen, and small intestine. COS-6 cells transfected with GPRN1 bound 125I-labeled VIP specifically with a dissociation constant (Kd) of 2.5 nM. VIP--and less effectively secretin, peptide histidine isoleucine (PHI), and glucagon competitively displaced bound 125I-VIP from transfected COS-6 cells, with potencies in the order VIP greater than secretin = PHI much greater than glucagon. VIP stimulated adenylate cyclase activity in stably transfected Chinese hamster ovary K1 cells, inducing a 3-fold increase in the intracellular level of cAMP. When the antisense orientation of the VIP receptor clone was introduced into HT-29 cells, there was a 50% suppression of the specific binding of 125I-VIP and of the VIP-induced increase in cAMP level, relative to untransfected cells. The VIP receptor cloned exhibits less than or equal to 24% homology with other receptors in the same superfamily and thus represents a subset of G protein-coupled receptors for peptide ligands.
Department of Medicine, University of California Medical Center, San Francisco 94143-0724.
S P
SP
Sreedharan
A
A
Robichon
K E
KE
Peterson
E J
EJ
Goetzl
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D003110:Colonic Neoplasms; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D007938:Leukemia; D008969:Molecular Sequence Data; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011964:Receptors, Gastrointestinal Hormone / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018005:Receptors, Vasoactive Intestinal Peptide; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
D012333:RNA, Messenger; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D011994:Recombinant Proteins; D011061:Poly A; D014660:Vasoactive Intestinal Peptide