Abstract
The human receptor subtype for VIP and PACAP, referred to as VPAC1 receptor, has a large N-terminal extracellular domain which is critical for VIP binding. We further investigated this domain by mutating 12 amino acid residues which could participate in the formation of a tight bend (W67) or a coiled coil motif. They were changed to alanine (A) and the cDNAs were transiently transfected into Cos cells. All mutants but W67A exhibited Kd values similar to that of the wild-type receptor. For the W67A mutant, no specific 125I-VIP binding could be observed. Mutants at the W67 site were further characterized after stable transfection of epitope-tagged VPAC1 receptor–GFP fusion proteins into CHO cells. W67A, W67E, W67H, and W67K mutants neither bound VIP nor mediated adenylyl cyclase activation by VIP. The W67F mutant mediated stimulation of adenylyl cyclase only at high VIP concentrations. Microscopic analysis and antibody binding experiments showed that all mutants were similarly expressed at the cell surface of CHO cells. Therefore tryptophan 67 in the human VPAC1 receptor plays a crucial role in VIP binding due, in part, to its aromatic moiety.
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