Role of second extracellular loop in the function of human vasoactive intestinal polypeptide/pituitary adenylate cyclase activating polypeptide receptor 1 (hVPAC(1)R)

To elucidate the functional role of the second extracellular loop of human vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating po lypeptide (VIP/PACAP) receptor (hVPAC(1)R), surface expression, ligand bind ing, and receptor activation were analyzed. Amino acids in the entire secon d extracellular loop were individually substituted by alanine by site-direc ted mutagenesis. The mutant and wild-type receptors were transiently expres sed in HEK293 cells and purified cell membranes were tested for the ability to bind VIP, while the receptor activity was measured as potency of cAMP p roduction analysed on intact cells. Surface expression of the substituted c onserved residues, W286A, I289A, W294A, and W295A, was evidently decreased to 20-30% compared to the wild-type expression; W286A also showed an signif icantly reduced potency of cAMP production. Substituted residues as F280A, E281A, and G284A showed a significant reduction in the potency of stimulate d cAMP production amounting to 8-46-fold, compared to the wild-type with un affected surface expression and VIP binding. These results indicate that so me residues in the second extracellular loop of the human VPAC(1)R particip ate in the active mechanism of a ligand-mediated response without being dir ectly involved in the binding of VIP.