Cloning and expression of the human vasoactive intestinal peptide receptor.

Abstract

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.

• Authors: S P Sreedharan, A Robichon, K E Peterson, E J Goetzl
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 88 Issue 11 Pg. 4986-90 (Jun 01 1991) ISSN: 0027-8424 [Print] United States
• PMID: 1675791 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• RNA, Messenger
• Receptors, Gastrointestinal Hormone
• Receptors, Vasoactive Intestinal Peptide
• Recombinant Proteins
• Poly A
• Vasoactive Intestinal Peptide

Topics

• Amino Acid Sequence
• Animals
• Blotting, Northern
• Cell Line
• Cloning, Molecular (methods)
• Colonic Neoplasms
• Gene Library
• Humans
• Kinetics
• Leukemia
• Molecular Sequence Data
• Poly A (genetics, isolation & purification)
• Protein Conformation
• RNA, Messenger (genetics, isolation & purification)
• Rats
• Receptors, Gastrointestinal Hormone (genetics, isolation & purification, metabolism)
• Receptors, Vasoactive Intestinal Peptide
• Recombinant Proteins (isolation & purification, metabolism)
• Sequence Homology, Nucleic Acid
• Transcription, Genetic
• Transfection
• Vasoactive Intestinal Peptide (metabolism)