Photoaffinity labeling demonstrates physical contact between vasoactive intestinal peptide and the N-terminal ectodomain of the human VPAC1 receptor.
Vasoactive intestinal peptide (VIP) is a prominent neuropeptide whose actions are mediated by VPAC receptors belonging to class II G protein-coupled receptors. To identify contact sites between VIP and its VPAC1 receptor, an analog of VIP substituted with a photoreactive para-benzoyl-l-Phe (Bpa) at position 22 has been synthesized and evaluated in Chinese hamster ovary cells stably expressing the recombinant human receptor. Bpa22-VIP and native VIP are equipotent in stimulating adenylyl cyclase activity in cell membranes. Cyanogen bromide cleavage of the covalent 125I-[Bpa22-VIP]-hVPAC1R complex yielded a single labeled fragment of 30 kDa that shifted to 11 after deglycosylation, most consistent with the 67-137 fragment of the receptor N-terminal ectodomain. Further cleavage of this fragment with V8 endoproteinase and creation of receptor mutants with new CNBr cleavage sites (XàMet), demonstrated that 125I-[Bpa22-VIP] was covalently attached to the short receptor 109-120 fragment (GWTHLEPGPYPI). In a three-dimensional model of the receptor N-terminal ectodomain, this fragment is located on one edge of the putative VIP binding groove and encompasses several amino acids previously shown to be crucial for VIP binding (reviewed in Laburthe, M., Couvineau, A., and Marie, J. C. (2002) Receptors Channels 8, 137-153). Our data provide the first direct evidence for a physical contact between VIP and the N-terminal ectodomain of the hVPAC1 receptor.
Tan
Yossan-Var
YV
INSERM U410, Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine Xavier Bichat, Paris F-75018, France.
Couvineau
Alain
A
Van Rampelbergh
Jean
J
Laburthe
Marc
M
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D003488:Cyanogen Bromide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008027:Light; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D019920:Photoaffinity Labels / Q000494:pharmacology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018005:Receptors, Vasoactive Intestinal Peptide / Q000737:chemistry*; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014660:Vasoactive Intestinal Peptide / Q000737:chemistry*
D008024:Ligands; D010455:Peptides; D019920:Photoaffinity Labels; D018005:Receptors, Vasoactive Intestinal Peptide; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D011993:Recombinant Fusion Proteins; D014660:Vasoactive Intestinal Peptide; D003488:Cyanogen Bromide