DIFFERENT VASOACTIVE INTESTINAL POLYPEPTIDE RECEPTOR DOMAINS ARE INVOLVED IN THE SELECTIVE RECOGNITION OF TWO VPAC2-SELECTIVE LIGANDS

A vasoactive intestinal polypeptide (VIP) analog, acylated on the amino-terminal histidine by hexanoic acid (C 6 -VIP), behaved as a VPAC 2 preferring agonist in binding and functional studies on human VIP receptors, and radioiodinated C 6 -VIP was a suitable ligand for binding studies on wild-type and chimeric receptors. We evaluated the properties of C 6 -VIP, its analog AcHis 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 efficacy on the chimeras starting with the amino-terminal VPAC 2 receptor sequence. The binding and functional profile of these chimeric receptors suggested that the high affinity of Ro 25-1553 for VPAC 2 receptors is supported 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)-EC 3 receptor domain. These results further support the hypothesis that the central and carboxyl-terminal regions of the peptide (modified in RO 25-1553) recognize the extracellular amino-terminal region domain, whereas the amino-terminal VIP amino acids bind to the TM receptor core. VIP had a reduced affinity and efficacy on the N-VPAC 1 /VPAC 2 and on the N→EC 2 -VPAC 1 /VPAC 2 chimeric receptors. C 6 -VIP behaved as a high-affinity agonist on these 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-type receptors and for the two latter chimeras, supporting the hypothesis that these chimeras were properly folded but unable to reach the high-agonist-affinity, active receptor conformation in response to VIP binding.