Proc. Natl. Acad. Sci. USA Vol. 88, pp. 4986-4990, June 1991 Biochemistry
Cloning and expression of the human vasoactive intestinal peptide receptor (neuropeptides/G protein-coupled receptors)
SUNIL P. SREEDHARAN, ALAIN ROBICHON, KAREN E. PETERSON, AND EDWARD J. GOETZL Departments of Medicine and Microbiology/Immunology, University of California Medical Center, San Francisco, CA 94143-0724
Communicated by Y. W. Kan, March 8, 1991 (received for review December 17, 1990)
immune cells bear specific high-affinity receptors for VIP that consist of a single 46- to 73-kDa protein associated with a stimulatory guanine nucleotide-binding (G.) protein capable of activating adenylate cyclase (15). We previously identified specific high-affinity receptors for VIP on T and B lymphocytes (16). Here we report the cloning, sequencing, and expression of a cDNA that encodes the VIP receptors of Nalm 6 human leukemic pre-B cells and of HT-29 human colon adenocarcinoma cells, two cell lines that possess specific receptors for VIP (17, 18).*
Vasoactive intestinal peptide (VIP) is a neuroABSTRACT endocrine mediator found in the central and peripheral nervous systems. Distinct subsets of neural, respiratory, gastrointestinal, and immune cells bear specific high-affinity receptors for VIP, which are associated with a guaine 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-transmembranesegment hydropathicity proffle with other G protein-coupled receptors. Northern blot analyses identified a 2.7-kiobase 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 12I-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 'SI-VIP from transfected COS-6 cells, with potencies in the order VIP > secretin = PHI >> glucagon. VIP stimulated adenylate cyclase activity in stably transfected Chinese hamster ovary K1 cells, inducing a 3fold 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 doned exhibits
Cloning and expression of the human vasoactive intestinal peptide receptor (neuropeptides/G protein-coupled receptors)
SUNIL P. SREEDHARAN, ALAIN ROBICHON, KAREN E. PETERSON, AND EDWARD J. GOETZL Departments of Medicine and Microbiology/Immunology, University of California Medical Center, San Francisco, CA 94143-0724
Communicated by Y. W. Kan, March 8, 1991 (received for review December 17, 1990)
immune cells bear specific high-affinity receptors for VIP that consist of a single 46- to 73-kDa protein associated with a stimulatory guanine nucleotide-binding (G.) protein capable of activating adenylate cyclase (15). We previously identified specific high-affinity receptors for VIP on T and B lymphocytes (16). Here we report the cloning, sequencing, and expression of a cDNA that encodes the VIP receptors of Nalm 6 human leukemic pre-B cells and of HT-29 human colon adenocarcinoma cells, two cell lines that possess specific receptors for VIP (17, 18).*
Vasoactive intestinal peptide (VIP) is a neuroABSTRACT endocrine mediator found in the central and peripheral nervous systems. Distinct subsets of neural, respiratory, gastrointestinal, and immune cells bear specific high-affinity receptors for VIP, which are associated with a guaine 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-transmembranesegment hydropathicity proffle with other G protein-coupled receptors. Northern blot analyses identified a 2.7-kiobase 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 12I-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 'SI-VIP from transfected COS-6 cells, with potencies in the order VIP > secretin = PHI >> glucagon. VIP stimulated adenylate cyclase activity in stably transfected Chinese hamster ovary K1 cells, inducing a 3fold 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 doned exhibits
