Characterization of the new photoaffinity probe (Bz2-K24)-VIP

Site-directed mutagenesis and molecular modeling demonstrated that the N-terminal ectodomain of the VPAC1 receptor is a major site of vasoactive intestinal peptide (VIP) binding. Previous studies with the [Bpa6]-VIP and [Bpa22]-VIP probes (substitution with the photoactivable Bpa for the residues 6 and 22 in VIP) showed spatial approximation between the amino acids 6 and 22 of VIP and the 104-108 and 109-119 sequences within the N-terminal ectodomain of the receptor, respectively. Here, we characterize the new probe (Bz2-K24)-VIP (substitution with the photoreactive Bz2-K for the residue 24 in VIP). After photolabeling and sequential digestions of the receptor, the 121-133 sequence of the N-terminal ectodomain was identified as the site of interaction. The N-terminal ectodomain of the VPAC1 receptor is therefore an affinity trap for the central part of VIP, at least between residues 6 and 24.