MUTATIONAL ANALYSIS OF CYSTEINE RESIDUES WITHIN THE EXTRACELLULAR DOMAINS OF THE HUMAN VASOACTIVE-INTESTINAL-PEPTIDE (VIP) 1-RECEPTOR IDENTIFIES 7 MUTANTS THAT ARE DEFECTIVE IN VIP BINDING

We have used site-directed mutagenesis to investigate the requirement of cysteine residues in the extracellular domains of the human VIP 1 receptor for binding VIP. Cys(37), Cys(50), Cys(63), Cys(72), Cys(86), Cys(105) and Cys(122) (N-terminal extracellular domain), Cys(208) and Cys(215) (second extracellular loop) and Cys(288) (third extracellular loop) were mutated into glycine, and mutated cDNAs tranfected into Cos-7 cells. It appeared that mutants C50G, C63G, C72G, C86G, C105G, C122G, and C288G did not bind VIP whereas mutants C37G, C208G and C215G bound VIP with the same dissociation constant (# 0.5 nM) as the wildtype receptor. Ale mutated receptor proteins were synthesized by Cos-7 cells (Western blot) and delivered at the plasma membrane level (confocal microscopy). The fact that C208G and C215G mutants retained a complete binding activity while the C288G mutant was inactive does not suggest the presence of a functionally relevant disulfide bond between the second and third extracellular loop of the human VIP receptor contrary to what has been shown in several other heptahelical receptors. It is also conducted that the six crucial cysteine residues, e.g., Cys(50), Cys(63), Cys(72), Cys(861) Cys(105) and Cys(122) in the N-terminal extracellular domain, may be functionally important by forming intramolecular disulfide bonds which help to maintain the topology for ligand binding in human VIP I receptors. (C) 1995 Academic Press, Inc.