ASPARTATE-196 IN THE FIRST EXTRACELLULAR LOOP OF THE HUMAN VIP1 RECEPTOR IS ESSENTIAL FOR VIP BINDING AND VIP-STIMULATED CAMP PRODUCTION

VIP receptors belong to a subfamily of G protein-coupled receptors tha t includes secretin, glucagon, PTH and several other receptors. We hav e used site directed mutagenesis to investigate the requirement of som e highly conserved residues in the extracellular loops including aspar tate 196 (mutant D196A), leucine 199 (mutant L199A), tryptophane 286 ( mutant W286A) and tryptophane 294 (mutant W294A) for the ability of th e human VIP1 receptor to bind VIP and to mediate VIP-stimulated cAMP p roduction. After transfection of mutated cDNAs in Cos-7 cells, it appe ared that 1) mutants L199A, W286A and W294A bound VIP with the same di ssociation constant as the wild-type receptor whereas mutant D196A did not bind I-125-VIP; 2) mutants L199A, W286A and W294A mediate VIP-sti mulated cAMP production with the same EC(50) as the wildtype receptor whereas VIP displayed a 500-fold decrease of potency in promoting cAMP production through mutant D196A. Since all mutated receptor proteins were expressed and delivered at the plasma membrane (immunofluorescenc e studies), it is concluded that the first extracellular loop of the h uman VIP1 receptor contains a highly conserved aspartate residue which is essential for VIP binding and VIP-stimulated cAMP production. (C) 1997 Academic Press