title: CCK-B/Gastrin receptor transmembrane domain mutations selectively alter synthetic agonist efficacy without affecting the activity of endogenous peptides.
abstract: Recent efforts have focused on identifying small nonpeptide molecules that can mimic the activity of endogenous peptide hormones. Understanding the molecular basis of ligand-induced receptor activation by these divergent classes of ligands should expedite the process of drug development. Using the cholecystokinin-B/gastrin receptor (CCK-BR) as a model system, we have recently shown that both affinity and efficacy of nonpeptide ligands are markedly affected by amino acid alterations within a putative transmembrane domain (TMD) ligand pocket. In this report, we examine whether residues projecting into the TMD pocket determine the pharmacologic properties of structurally diverse CCK-BR ligands, including peptides and synthetic peptide-derived partial agonists (peptoids). Nineteen mutant human CCK-BRs, each including a single TMD amino acid substitution, were transiently expressed in COS-7 cells and characterized. Binding affinities as well as ligand-induced inositol phosphate production at the mutant CCK-BRs were assessed for peptides (CCK-8 and CCK-4) and for peptoids (PD-135,158 and PD-136, 450). Distinct as well as overlapping determinants of peptide and peptoid binding affinity were identified, supporting that both classes of ligands, at least in part, interact with the CCK-BR TMD ligand pocket. Eight point mutations resulted in marked increases or decreases in the functional activity of the synthetic peptoid ligands. In contrast, the functional activity of both peptides, CCK-8 and CCK-4, was not affected by any of the CCK-BR mutations. These findings suggest that the mechanisms underlying activation of G-protein-coupled receptors by endogenous peptide hormones versus synthetic ligands may markedly differ.
authors.affiliation: Department of Medicine and the GRASP Digestive Disease Center, Tupper Research Institute, New England Medical Center, Boston, Massachusetts 02111, USA.
authors.forename: M
authors.initials: M
authors.lastname: Bläker
authors.forename: Y
authors.initials: Y
authors.lastname: Ren
authors.forename: L
authors.initials: L
authors.lastname: Seshadri
authors.forename: E W
authors.initials: EW
authors.lastname: McBride
authors.forename: M
authors.initials: M
authors.lastname: Beinborn
authors.forename: A S
authors.initials: AS
authors.lastname: Kopin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008536:Meglumine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D034444:Peptoids; D011869:Radioligand Assay; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D007211:Indoles; D008565:Membrane Proteins; D010455:Peptides; D034444:Peptoids; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin; C065600:PD 135158; D008536:Meglumine