VASOACTIVE-INTESTINAL-PEPTIDE (VIP)(1) RECEPTOR - 3 NONADJACENT AMINO-ACIDS ARE RESPONSIBLE FOR SPECIES SELECTIVITY WITH RESPECT TO RECOGNITION OF PEPTIDE HISTIDINE ISOLEUCINEAMIDE

Vasoactive intestinal peptide (VIP)(1) receptors in rats and humans re cognize peptide histidine isoleucineamide (PHI) with high and low affi nity, respectively. We took advantage of this phenotypic difference to identify the domain responsible for the selective recognition of PHI by rat and human receptors which display >80% sequence identity. After transfection of human and rat receptors in COS cells, the ratio of IC 50 for PHI/IC50 for VIP (referred to as P/V) in inhibiting I-125-VIP b inding was shown to be >1,000 and <40, respectively. Construction of e ight rat/human receptor chimerae by overlap polymerase chain reaction and determination of their P/V ratios demonstrated that the critical d omain for PHI recognition is present within a sequence comprising part of the first extracellular loop and third transmembrane domain. This domain contains three different amino acids numbered according to huma n and rat sequences, respectively, e.g. Gln(207) (human) versus His(20 8) (rat), Gly(211) versus Ala(212) and Met(219) versus Val(220). Site- directed mutagenesis introducing individual, double, or triple mutatio ns in a chimeric construct revealed that all three amino acids were in volved in the recognition of PHI. Triple mutations were then introduce d in the wild type receptors i.e. Q207H, G211A, M219V human VIP1 recep tor and H208Q, A212G, V220M rat VIP1 receptor, resulting in a complete change in their phenotype from human to rat and from rat to human, re spectively. The results demonstrate that three nonadjacent amino acids are responsible for the selective recognition of PHI by human and rat VIP1 receptors.