Abstract
The widespread 28-amino acid neuropeptide vasoactive intestinal peptide (VIP) exerts its many biological effects through interaction with serpentine class II G protein-coupled receptors named VPAC receptors. We previously provided evidence for a physical contact between the side chain at position 22 of VIP and the N-terminal ectodomain of the hVPAC1 receptor (Tan, Y. V., Couvineau, A., Van Rampelbergh, J., and Laburthe, M. (2003) J. Biol. Chem. 278, 36531-36536). We explored here the contact site between hVPAC1 receptor and the side chain at position 6 of VIP by photoaffinity labeling. The photoreactive para-benzoyl-l-Phe (Bpa) was substituted for Phe(6) in VIP resulting in [Bpa(6)]-VIP, which was shown to be a hVPAC1 receptor agonist in Chinese hamster ovary cells stably expressing the recombinant receptor. After obtaining the covalent (125)I-[Bpa(6)-VIP].hVPAC1 receptor complex, it was sequentially cleaved by cyanogen bromide, peptide N-glycosidase F, endopeptidase Glu-C, and trypsin, and the cleavage products were analyzed by electrophoresis. The data demonstrated that (125)I-[Bpa(6)-VIP] were covalently attached to the short 104-108 fragment within the N-terminal ectodomain of the receptor. The data were confirmed by creation of a receptor mutant with new CNBr cleavage site. In a three-dimensional model of the receptor N-terminal ectodomain, this fragment was located on one edge of the putative VIP-binding groove and was adjacent to the fragment covalently attached to the side chain at position 22 of VIP. Altogether these data showed that the central part of VIP, at least between Phe(6) and Tyr(22), interacts with the N-terminal ectodomain of the hVPAC1 receptor.
Highlights
From the INSERM U410, Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santeet de la Recherche Medicale, Facultede Medecine Xavier Bichat, Paris F-75018, France
After obtaining the covalent 125I-[Bpa6VIP]1⁄7hVPAC1 receptor complex, it was sequentially cleaved by cyanogen bromide, peptide N-glycosidase F, endopeptidase Glu-C, and trypsin, and the cleavage products were analyzed by electrophoresis
This site of incorporation was selected for several reasons: (i) Phe6 is important for the biological activity of vasoactive intestinal peptide (VIP) [9] and is strictly conserved in all natural hormones structurally related to VIP [1]. (ii) The substitution of Bpa for phenylalanine keeps an aromatic residue, and we expected that, even though Phe6 is important for biological activity, the [Bpa6]-VIP probe should keep reasonable affinity for the receptor. (iii) Position 6 and the previously explored position 22 [8] are at the two ends of the central ␣-helical domain of VIP [9, 10]
Summary
Peptide (VIP) and Further Insights into the Interaction of VIP with the N-terminal Ectodomain of Human VPAC1 Receptor by Photoaffinity Labeling with [Bpa6]-VIP*. The VPAC1 receptor is prototypic of class II G protein-coupled receptors and has been extensively studied by molecular biology techniques including site-directed mutagenesis and molecular chimerism (for review see Ref. 3) These studies made it possible to delineate the receptor domains involved in high affinity VIP binding [3], selectivity toward some natural peptide agonists [4, 5] and activation of adenylyl cyclase [6]. It is well known that VIP has diffuse pharmacophoric domains, with the amino acid residues important for biological activity being distributed along the whole 28-amino acid peptide chain [9] In this context, we further explored the contact sites between VIP and the hVPAC1 receptor by incorporating a photoactivable benzophenone group on the side chain at position 6 of VIP.
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