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.
Footnotes
- Received December 7, 1999.
- Accepted May 9, 2000.
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Send reprint requests to: Alan S. Kopin, M.D., Department of Medicine and GRASP Digestive Disease Center, New England Medical Center, Box 239, 750 Washington St., Boston, MA 02111. E-mail:akopin{at}lifespan.org
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↵1 Equal contributions were made by both authors.
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↵2 Present address: Medizinische Kernklinik und Poliklinik, Universitäts-Krankenhaus Eppendorf, 20246 Hamburg, Germany.
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This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK46767 and an American Gastroenterological Association Industry Research Scholar Award (to M. Beinborn). A.K. is a New England Medical Center Molecular Cardiology Research Institute investigator. M. Bläker is supported by the Deutsche Forschungsgemeinschaft.
- The American Society for Pharmacology and Experimental Therapeutics
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