VASOACTIVE INTESTINAL POLYPEPTIDE AND PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE RECEPTOR CHIMERAS REVEAL DOMAINS THAT DETERMINE SPECIFICITY OF VASOACTIVE INTESTINAL POLYPEPTIDE BINDING AND ACTIVATION

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclas e-activating polypeptide (PACAP) receptors are closely related G prote in-coupled receptors with seven-transmembrane domains. The VIP recepto r can bind both VIP and PACAP with high affinity, whereas the PACAP re ceptor binds only PACAP with high affinity. To elucidate the structura l domains involved in a selectivity for VIP binding and the subsequent receptor activation, a series of chimeric receptors between the VIP a nd PACAP receptors was constructed, expressed in COS-7 cells, and anal yzed for ligand binding and cAMP generation. All chimeric constructs b ound PACAP with high affinity and subsequently activated cAMP generati on similarly to the wild-type receptors. In contrast, profound differe nces were observed in the potencies of VIP for competition of I-125-la beled PACAP binding to both wild-type receptors and the chimeric recep tors. The cAMP responses of these receptor generally correlated with t he ability of VIP to compete for PACAP radioligand binding with the ex ceptions for some particular chimeras. In this report we demonstrate t hat several domains, including the amino-terminal extracellular domain , the transmembrane domains I and II, and the first extracellular loop of the VIP receptor, are important for the selectivity for VIP bindin g and responsiveness to VIP. We further show that the third extracellu lar loop and its proximal domains of the VIP receptor appear to be inv olved in the VIP recognition, especially the receptor activation proce ss. On the other hand, the direct binding experiments of the VIP radio ligand demonstrated that both wild-type receptors and all chimeric rec eptors have a high affinity binding site for VIP, although this high a ffinity VIP binding resulted in a biological response only in the VIP receptor or VIP receptor-like chimeras. This suggests that there is a non-biologically relevant high affinity VIP-binding site within the ra t PACAP receptor.