Different vasoactive intestinal polypeptide receptor domains are involved in the selective recognition of two VPAC(2)-selective ligands

A vasoactive intestinal polypeptide (VIP) analog, acylated on the amino-ter minal histidine by hexanoic acid (C-6-VIP), behaved as a VPAC(2) preferring agonist in binding and functional studies on human VIP receptors, and radi oiodinated C-6-VIP was a suitable ligand for binding studies on wild-type a nd chimeric receptors. We evaluated the properties of C-6-VIP, its analog A cHis(1)-VIP, and the VPAC(2)-selective agonist Ro 25-1553 on the wild-type VPAC(1) and VPAC(2) receptors and on the chimeric receptors exchanging the different domains between both receptors. VIP had a normal affinity and eff icacy on the chimeras starting with the amino-terminal VPAC(2) receptor seq uence. The binding and functional profile of these chimeric receptors sugge sted that the high affinity of Ro 25-1553 for VPAC(2) receptors is supporte d by the amino-terminal extracellular domain, whereas the ability to prefer C-6-VIP over VIP is supported by the VPAC(2) fifth transmembrane (TM5)-EC3 receptor domain. These results further support the hypothesis that the cen tral and carboxyl-terminal regions of the peptide (modified in RO 25-1553) recognize the extracellular aminoterminal region domain, whereas the amino- terminal VIP amino acids bind to the TM receptor core. VIP had a reduced af finity and efficacy on the N-VPAC(1)/VPAC(2) and on the N-->EC2-VPAC(1)/VPA C(2) chimeric receptors. C-6-VIP behaved as a high-affinity agonist on thes e constructions. The antagonists [AcHis(1),D-Phe(2),Lys(15),Arg(16),Leu(27) ]VIP(3-7)/GRF(8-27) and VIP(5-27) had comparable affinities for the wild-ty pe receptors and for the two latter chimeras, supporting the hypothesis tha t these chimeras were properly folded but unable to reach the high-agonist- affinity, active receptor conformation in response to VIP binding.