CCK-B/gastrin receptor transmembrane domain mutations selectively alter synthetic agonist efficacy without affecting the activity of endogenous peptides
M. Blaker et al., CCK-B/gastrin receptor transmembrane domain mutations selectively alter synthetic agonist efficacy without affecting the activity of endogenous peptides, MOLEC PHARM, 58(2), 2000, pp. 399-406
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.