Tryptophan 67 in the human VPAC(1) receptor: Crucial role for VIP binding

The human receptor subtype for VIP and PACAP, referred to as VPAC(1) recept or, has a large N-terminal extracellular domain which is critical for VIP b inding. We further investigated this domain by mutating 12 amino acid resid ues which could participate in the formation of a tight bend (W67) or a coi led coil motif. They were changed to alanine (A) and the cDNAs were transie ntly transfected into Cos cells. All mutants but W67A exhibited K-d values similar to that of the wildtype receptor. For the W67A mutant, no specific I-125-VIP binding could be observed. Mutants at the W67 site were further c haracterized after stable transfection of epitope-tagged VPAC(1) receptor-G FP fusion proteins into CHO cells. W67A, W67E, W67H, and W67H mutants neith er bound VIP nor mediated adenylyl cyclase activation by VIP. The W67F muta nt mediated stimulation of adenylyl cyclase only at high VIP concentrations . Microscopic analysis and antibody binding experiments showed that all mut ants were similarly expressed at the cell surface of CHO cells. Therefore t ryptophan 67 in the human VPAC(1) receptor plays a crucial role in VIP bind ing due, in part, to its aromatic moiety. (C) 2000 Academic Press.