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.