A DISULFIDE BOND BETWEEN CONSERVED CYSTEINES IN THE EXTRACELLULAR LOOPS OF THE HUMAN VIP RECEPTOR IS REQUIRED FOR BINDING AND ACTIVATION

The importance of two highly conserved cysteines in the human vasoacti ve intestinal peptide receptor 1 (hVIPR 1) was examined. By site-direc ted mutagenesis each Cys residue was converted into Ala or Ser. The mu tant and wild-type genes were transfected into HEK293 cells and tested for the ability to bind VIP and to activate cAMP production. Cys(215) -Ala/Ser and Cys(285)-Ala/Ser showed at least a 10-fold decrease in bi nding affinity and receptor potency when compared to the wild type. In contradiction to the wild-type receptor, bath mutations were insensit ive to dithiothreitol (DTT). The results indicate the existence of a d isulfide bond between Cys(215) and Cys(285), which is important for st abilising the receptor in the correct conformation for ligand binding and activation. (C) 1997 Federation of European Biochemical Societies.