title: Mutations within the cholecystokinin-B/gastrin receptor ligand 'pocket' interconvert the functions of nonpeptide agonists and antagonists.
abstract: We have reported previously that the transmembrane domains of the cholecystokinin-B/gastrin receptor (CCK-BR) comprise a putative ligand binding pocket. In the present study, we examined whether amino acid substitutions within the CCK-BR pocket altered the affinities and/or functional activities of L-365,260 (the prototypical nonpeptide CCK-BR antagonist) and two structural derivatives, YM022 (a higher affinity antagonist) and L-740,093S (a partial agonist). Eight amino acids that project into the CCK-BR pocket were individually replaced by alanine, using site-directed mutagenesis. Affinities for the nonpeptide molecules, as well as ligand-induced inositol phosphate production, were assessed with the wild-type and mutant receptors. For each of the nonpeptide ligands examined, a distinct series of mutations altered the affinity, suggesting that each ligand possessed a characteristic pattern of interactions within the CCK-BR pocket. Basal signaling levels and inositol phosphate formation induced by the full agonist CCK octapeptide were comparable for the wild-type receptor and all of the mutant CCK-BR forms. In contrast to the peptide agonist CCK octapeptide, the functional activities of the nonpeptide molecules were selectively altered by single point mutations within the CCK-BR pocket, resulting in interconversion of agonists and antagonists. These findings suggest that interactions between nonpeptide molecules and transmembrane domain amino acids of the CCK-BR can determine the functional activity and affinity of the ligands.
authors.affiliation: Division of Gastroenterology and GRASP Digestive Disease Center, Tupper Research Institute, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
authors.forename: M
authors.initials: M
authors.lastname: Bläker
authors.forename: Y
authors.initials: Y
authors.lastname: Ren
authors.forename: M C
authors.initials: MC
authors.lastname: Gordon
authors.forename: J E
authors.initials: JE
authors.lastname: Hsu
authors.forename: M
authors.initials: M
authors.lastname: Beinborn
authors.forename: A S
authors.initials: AS
authors.lastname: Kopin
mesh_terms: D000818:Animals; D001569:Benzodiazepines / Q000378:metabolism / Q000494:pharmacology; D001570:Benzodiazepinones / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D019556:COS Cells / Q000378:metabolism / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D006727:Hormone Antagonists / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D007457:Iodine Radioisotopes; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010671:Phenylurea Compounds / Q000378:metabolism / Q000494:pharmacology; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D012844:Sincalide / Q000378:metabolism
chemical_list: D001570:Benzodiazepinones; D018076:DNA, Complementary; D006727:Hormone Antagonists; D007295:Inositol Phosphates; D007457:Iodine Radioisotopes; D008024:Ligands; D010671:Phenylurea Compounds; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin; D001569:Benzodiazepines; C087260:YM 022; C090649:L 740093; C058121:L 365260; D012844:Sincalide