The N-terminal parts of VIP and antagonist PG97-269 physically interact with different regions of the human VPAC1 receptor.

Vasoactive intestinal peptide (VIP) is a widespread neuropeptide, which exerts many biological functions through interaction with the VPAC1 receptor, a class II G protein-coupled receptor. Photoaffinity labeling studies combined with 3D molecular modeling demonstrated that the central and C-terminal parts of VIP (segment 6-28) have physical contacts with the N-terminal ectodomain (N-Ted) of VPAC1 receptor. However, the domain of the hVPAC1 receptor interacting with the N-terminus of VIP (1-5) is still unknown. We have synthesized a photoreactive probe Bpa0-VIP. After photolabeling and receptor cleavage, Nu-PAGE analysis revealed a 5-kDa labeled fragment corresponding to the 130-137 sequence of hVPAC1 receptor, indicating that the N-terminus of VIP also interacts with the N-ted. A photoreactive probe, Bpa0-PG97-269, was also synthesized with the specific peptide antagonist PG97-269. After photoaffinity labeling, a glycosylated 15-kDa fragment is identified by cyanogen bromide (CNBr) cleavage and corresponds to the 43-66 sequence of the hVPAC1 receptor N-ted. These results indicate that: (1) the N-terminal part of VIP physically interacts with the N-ted in the continuity of 6-28 VIP sequence; (2) the N-terminal part of VIP and the selective peptide antagonist (PG97-269) have different sites of interaction with the VPAC1 receptor N-ted.