Spatial approximation between the C-terminus of VIP and the N-terminal ectodomain of the VPAC1 receptor.

Vasoactive intestinal peptide (VIP) exerts many biological functions through interaction with the VPAC1 receptor, a class II G protein-coupled receptor. Photoaffinity labeling studies associated with receptor mapping and three-dimensional molecular modeling demonstrated that the central part of VIP (6-24) interacts with the N-terminal ectodomain of VPAC1 receptor. However, the domain of the VPAC1 receptor interacting with the C-terminus of VIP is still unknown. A photoaffinity probe, Bpa28-VIP, was synthetized by substitution of amidated Asn28 of VIP by amidated photoreactive para-benzoyl-L-Phe (Bpa). Bpa28-VIP was shown to be a hVPAC1 receptor agonist in CHO cells expressing the recombinant VPAC1 receptor. After obtaining a covalent 125I-[Bpa28-VIP]/hVPAC1 complex, it was cleaved by CNBr, PNGase F, and endopeptidase Glu-C and the cleavage products were analyzed by electrophoresis. The data demonstrated that 125I-[Bpa28-VIP] was covalently bonded to the 121-133 fragment within the N-terminal ectodomain of the receptor. This fragment is adjacent to those covalently attached to the central part (6-24) of VIP.