CCK-B/gastrin receptor transmembrane domain mutations selectively alter synthetic agonist efficacy without affecting the activity of endogenous peptides

Recent efforts have focused on identifying small nonpeptide molecules that can mimic the activity of endogenous peptide hormones. Understanding the mo lecular basis of ligand-induced receptor activation by these divergent clas ses of ligands should expedite the process of drug development. Using the c holecystokinin-B/gastrin receptor (CCK-BR) as a model system, we have recen tly shown that both affinity and efficacy of nonpeptide ligands are markedl y affected by amino acid alterations within a putative transmembrane domain (TMD) ligand pocket. In this report, we examine whether residues projectin g into the TMD pocket determine the pharmacologic properties of structurall y 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 c ells and characterized. Binding affinities as well as ligand-induced inosit ol phosphate production at the mutant CCK-BRs were assessed for peptides (C CK-8 and CCK-4) and for peptoids (PD-135,158 and PD-136,450). Distinct as w ell as overlapping determinants of peptide and peptoid binding affinity wer e identified, supporting that both classes of ligands, at least in part, in teract 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 findin gs suggest that the mechanisms underlying activation of G-protein-coupled r eceptors by endogenous peptide hormones versus synthetic ligands may marked ly differ.