Molecular Mechanism Underlying Partial and Full Agonism Mediated by the Human Cholecystokinin-1 Receptor

Abstract

The cholecystokinin-1 receptor (CCK1R) is a G protein-coupled receptor (GPCR) that regulates important physiological functions. As for other GPCRs, the molecular basis of full and partial agonism is still far from clearly understood. In the present report, using both laboratory experiments and molecular modeling approaches, we have investigated the partial agonism mechanism of JMV 180, on the human CCK1R. We first showed that efficacy of the CCK1R to activate phospholipase C is dependent on the correct orientation of the C-terminal end of peptidic ligands toward residue Phe 330 of helix VI. We have previously reported that a single mutation of Met 121 (helix III) markedly reduced the receptor-mediated inositol phosphate production upon stimulation by CCK. Computational simulations predicted that residue 121 affected orientation of the C-terminal end of CCK, thus suggesting that the molecular complex with a reduced inositol phosphate production observed with the mutated CCK1R resembles that resulting from binding of JMV 180 to the WT-CCK1R. Pharmacological, biochemical, and functional characterizations of the two receptor⅐ligand complexes with decreased abilities to signal were carried out in different cell types. We found that they presented the same features, such as total dependence of inositol phosphate production to G␣ q expression, single affinity of binding sites, insensitivity of binding to non-hydrolyzable GTP, absence of GTP␥ [S 35 ] binding following agonist stimulation, similarity of dose-response curves for amylase secretion, and incapacity to induce acute pancreatitis in pancreatic acini. We concluded that helices VI and III of the CCK1R are functionally linked through the CCK1R agonist binding site and that positioning of the C-terminal ends of peptidic agonists toward Phe 330 of helix VI is responsible for extent of phospholipase C activation through G␣ q coupling. Given the potential therapeutic interest of partial agonists such as JMV 180, our structural data will serve for target structure-based design of new CCK1R ligands.

Cholecystokinin and Gastrin Receptors

“…In fact, biological experiments with mutants as well as dynamic simulations of modeled liganded CCK1R support the conclusion that introduction of hydrophobic residues near position 121 determines positioning of the aromatic ring of the Phe of CCK within the binding pocket (145). This was further documented in a study with JMV 180, a partial agonist of the CCK1R, which partly shares its binding site with that of CCK (15). In this study, it was shown that helices III and VI of the CCK1R are functionally linked through the CCK1R agonist binding site and that positioning of the COOH-terminal ends of peptidic agonists towards Phe330 of helix VI is responsible for extent of phospholipase C activation through G␣ q coupling (15).…”

“…This was further documented in a study with JMV 180, a partial agonist of the CCK1R, which partly shares its binding site with that of CCK (15). In this study, it was shown that helices III and VI of the CCK1R are functionally linked through the CCK1R agonist binding site and that positioning of the COOH-terminal ends of peptidic agonists towards Phe330 of helix VI is responsible for extent of phospholipase C activation through G␣ q coupling (15). The molecular basis for CCK1R coupling to adenylyl cyclase was investigated based on the data showing that CCK1R, but not CCK2R, stimulates cAMP formation.…”

Receptor Subtype-specific Docking of Asp6.59 with C-terminal Arginine Residues in Y Receptor Ligands

“…Significant structural rearrangements, however, are required to obtain active GPCRs. Much effort has been made for many small ligand GPCRs, but little is known so far for peptide GPCRs (43)(44)(45). In peptide GPCRs one of the most challenging problems is the identification of the binding mode of agonists (42).…”

How does cholecystokinin stimulate exocrine pancreatic secretion? From birds, rodents, to humans

“…The COOH-terminal portion of the third cytoplasmic loop of CCK1R contains a stretch of charged residues that are thought to form an amphipathic ␣-helical extension of the sixth transmembrane domain in a critical orientation for G protein activation (143). Theoretical models of CCK1R have also been built by others (3,28).…”