We developed previously VPAC1 [vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (PACAP) receptor]>VPAC2 receptor selective ligands. Replacement of the VIP-Thr11 by an Arg11 in these ligands contributed to their selectivity: Arg11-VIP had a 200-fold lower affinity when compared with VIP at VPAC2 receptors as opposed to 3- to 5-fold higher affinity at VPAC1 receptors. Comparison of the binding and functional properties of related VIP analogues suggested that the VPAC1 selectivity of Arg11-VIP was due to the loss of a hydrogen bond between the hydroxy group of Thr residue and the VPAC2 receptor, steric hindrance between the Arg side chain and the VPAC2 receptor and charge attraction by the VPAC1 receptor. Comparison of the ability of VIP analogues to activate adenylate cyclase through chimaeric VPAC1/VPAC2 and VPAC2/VPAC1 receptors indicated that the first extracellular receptor loop carried most of the VPAC2 receptors’ ability to discriminate VIP from Arg11-VIP. Based on results obtained for a truncated VPAC2 receptor and the closely related PACAP-preferring receptor (PAC1) and secretin receptors, we hypothesized that Thr11 interacted with the VPAC2 receptor Tyr184 (similar to the VPAC1 receptor Phe200 residue). The Y184F (Tyr184→Phe) VPAC2 mutant lost the ability to discriminate VIP from Val11-VIP, and the F200Y VPAC1 mutant acquired the ability to discriminate the natural peptide from Val11-VIP. These results support the hypothesis that the hydroxy group of the native VIP-Thr11 side chain can indeed form a hydrogen bond with the Tyr side chain in the VPAC2 receptor.

Abbreviations used: ECL, extracellular loop; GRF, growth hormone-releasing factor; PACAP, pituitary adenylate cyclase-activating peptide; PAC, PACAP-preferring receptor; TM, transmembrane helix; VIP, vasoactive intestinal peptide; VPAC, VIP/PACAP receptor; Y184F, Tyr184→Phe.

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