Mutational analysis of the human vasoactive intestinal peptide receptor subtype VPAC(2): role of basic residues in the second transmembrane helix

1 We investigated the role of two conserved basic residues in the second tr ansmembrane helix arginine 172 (R172) and lysine 179 (K179) of the VPAC(2) receptor. 2 Vasoactive intestinal polypeptide (VIP) activated VPAC(2) receptors with an EC50 value of 7 nM, as compared to 150, 190 and 4000 nm at R172L, R172Q and K179Q-VPAC(2) receptors, respectively. It was inactive at K179I mutated VPAC(2) receptors. These results suggested that both basic residues were p robably implicated in receptor recognition and activation. 3 The VPAC(2)-sclective VIP analogue, [hexanoyl-His(1)]-VIP (C-6-VIP), had a higher affinity and efficacy as compared to VIP at the mutated receptors. 4 VIP, Asn(3)-VIP and Gln(3)-VIP activated adenylate cyclase through R172Q receptors with EC50 values of 190, 2 and 2 nm, respectively, and through R1 72L receptors with EC50 values of 150, 12 and 8 nM, respectively. Asn(3)-VI P and Gln(3)-VIP behaved as partial agonists at the wild type receptor, wit h E-max values (in per cent of VIP) of 75 and 52%, respectively. In contras t, they were more efficient than VIP (E-max values of 150 and 150% at the R 172Q VPAC(2) receptors, and of 400 and 360% at the R172L receptors, respect ively). These results suggested that the receptor's R172 and the ligand's a spartate 3 are brought in close proximity in the active ligand-receptor com plex. 5 The K179I and K179Q mutated receptors had a lower affinity than the wild- type receptors for all the agonists tested in this work: we were unable to identify the VIP amino acid(s) that interact with K179.