HIGHLY CONSERVED ASPARTATE-68, TRYPTOPHANE-73 AND GLYCINE-109 IN THE N-TERMINAL EXTRACELLULAR DOMAIN OF THE HUMAN VIP RECEPTOR ARE ESSENTIAL FOR ITS ABILITY TO BIND VIP

The human VIP receptor belongs to a subfamily of G protein-coupled rec eptors that includes secretin, glucagon and several other receptors. W e have used site-directed mutagenesis to investigate the requirement o f highly conserved aspartate 68 (mutant D68G), tryptophane 73 (mutant W73G), proline 87 (mutant P87G), glycine 109 (mutant Delta 109) and tr yptophane 110 (mutant W110G) for the ability of the human VIP receptor to bind VIP. After transfection of mutated cDNAs in Cos-7 cells, it a ppeared that mutants G87P and W110G bound VIP with the same dissociati on constant as the wild type receptor whereas mutants W73G, P87G and D elta 109 did not bind VIP. Since all mutated receptor proteins were sy nthesized by Cos-7 cells (Western blot) and expressed at the plasma me mbrane level (immunofluorescence studies), it is concluded that the N- terminal extracellular domain of the human VIP receptor contains highl y conserved amino acid residues which are essential for its intrinsic binding activity. (C) 1995 Academic Press, Inc.