title: A disulfide bond between conserved cysteines in the extracellular loops of the human VIP receptor is required for binding and activation.
abstract: The importance of two highly conserved cysteines in the human vasoactive intestinal peptide receptor I (hVIPR 1) was examined. By site-directed mutagenesis each Cys residue was converted into Ala or Ser. The mutant and wild-type genes were transfected into HEK293 cells and tested for the ability to bind VIP and to activate cAMP production. Cys215-Ala/Ser and Cys285-Ala/Ser showed at least a 10-fold decrease in binding affinity and receptor potency when compared to the wild type. In contradiction to the wild-type 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 stabilising the receptor in the correct conformation for ligand binding and activation.
authors.affiliation: Department of Clinical Biochemistry, Bispebjerg Hospital University of Copenhagen, Copenhagen NV, Denmark. smk@biobase.dk
authors.forename: S M
authors.initials: SM
authors.lastname: Knudsen
authors.forename: J W
authors.initials: JW
authors.lastname: Tams
authors.forename: B S
authors.initials: BS
authors.lastname: Wulff
authors.forename: J
authors.initials: J
authors.lastname: Fahrenkrug
mesh_terms: D001667:Binding, Competitive; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D018005:Receptors, Vasoactive Intestinal Peptide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
chemical_list: D004220:Disulfides; D018005:Receptors, Vasoactive Intestinal Peptide; D014660:Vasoactive Intestinal Peptide; D000242:Cyclic AMP; D003545:Cysteine; D004229:Dithiothreitol