Importance of conserved cysteines in the extracellular loops of human PACAP/VIP1 receptor for ligand binding and stimulation of cAMP production

The importance of two highly conserved cysteines in the human pituitary adenylate cyclase activating polypeptide (PACAP)/vasoactive intestinal peptide 1 (VIP1) receptor was examined. Using site-directed mutagenesis, each Cys residue was converted into Ala or Ser. The mutant and wildtype genes were transfected into HEK293 cells and tested for the ability to bind VIP and to activate cAMP production. Cys215Ala/Ser and Cys285Ala/Ser showed at least a tenfold decrease in binding affinity and receptor potency when compared to the wildtype. In contradiction to the wildtype receptor, both mutations were insensitive to dithiothreitol (DTT), The results indicate the existence of a disulfide bond between Cys215 and Cys285, which is important for stabilizing the receptor in the correct conformation for ligand binding and activation.