Linking Non-peptide Ligand Binding Mode to Activity at the Human Cholecystokinin-2 Receptor

Abstract

Given the importance of G-protein-coupled receptors as pharmacological targets in medicine, efforts directed at the understanding the molecular mechanism by which pharmacological compounds regulate their activity is of paramount importance. Here, we investigated at an atomic level the mechanism of inverse agonism and partial agonism of two high affinity, high selectivity very similar non-peptide ligands of the cholecystokinin-2 receptor (CCK2R) which differ by the absence or presence of a methyl group on their indole moiety. Using in silico, site-directed mutagenesis and pharmacological experiments, we demonstrated that these functionally different activities are due to differing anchoring modes of the two compounds to a residue of helix II (Thr-2.61) in the inactive state of the CCK2R. The binding mode of the inverse agonist allows the ligand to interact through its phenyl moiety with a key amino acid for CCK2R activation (Trp-6.48), preventing rotation of helix VI and, thus, CCK2R activation, whereas the partial agonist binds deeper into the binding pocket and closer to helix V, so that CCK2R activation is favored. This study on the molecular mechanism of ligand action opens the possibility of target-based optimization of G protein-coupled receptor non-peptide ligands.G protein-coupled receptors (GPCRs) 3 are membrane-embedded proteins having seven transmembrane domains, responsible for communication between the cell and its environment (1). GPCRs represent, therefore, a major focus in functional genomics programs and drug development research (2).GPCRs naturally exist in different conformations which correspond to both active and inactive states. Depending on their propensity to bind preferentially with one of these receptor conformations or to identically interact with both, synthetic ligands can behave as agonists, inverse agonists, or neutral antagonists. To date, three three-dimensional GPCR structures have been precisely determined by x-ray crystallography, namely that of rhodopsin and ␤2-and ␤1-adrenergic receptors (3-6). In this context, refinement of modeled GPCR structures in both active and inactive conformations and progress in the understanding of the precise mechanisms, which govern their activation, are of paramount importance for drug design strategies (7).The cholecystokinin-2 (CCK-2) receptor (CCK2R) belongs to family A of rhodopsin-like GPCRs. It binds both cholecystokinin and gastrin with a similar, high affinity (8, 9). CCK2R mediates a wide spectrum of CCK-and gastrininduced biological effects in the central nervous system and in periphery including anxiety, pain perception, and gastric acid secretion as well as controlling growth and differentiation of the gastric mucosa. The CCK2R and/or a constitutively active variant of this receptor may contribute to human diseases (10, 11). This has recently generated considerable interest in the identification, among the large panel of synthetic ligands, of CCK2R antagonists having an inverse agonist activity (12,13).The CCK2R appears to be an ex...

Regulation of Membrane Cholecystokinin-2 Receptor by Agonists Enables Classification of Partial Agonists as Biased Agonists

“…HEK 293 cells stably expressing CCK2R (Flp-In TM CCK2R-293) were directly plated onto 24-well plates. Approximately 24 h after transfer to 24-well plates, binding assays were performed using 125 I-CCK according to the protocol previously described in detail (16). Receptor density (B max ) and K d were calculated from homologous 125 I-CCK competition binding experiments using Ligand software (Kell, Cambridge, UK).…”

“…HEK 293 cells stably expressing CCK2R (Flp-In TM CCK2R-293) were directly plated onto 24-well plates. Approximately 24 h after transfer to 24-well plates and after overnight incubation in DMEM containing 2 Ci/ml myo- [2-3 H]inositol (specific activity, 10 -25 Ci/mmol; PerkinElmer Life Sciences), inositol phosphate production was determined as described previously (16).…”

“…In previous works, we investigated the intrinsic activation mechanism of the CCK2R and the regulation of this activity by different ligands (15)(16)(17). This led us to understand how two structurally close nonpeptide ligands display opposite activities (partial agonist and inverse agonist) (16).…”

“…This led us to understand how two structurally close nonpeptide ligands display opposite activities (partial agonist and inverse agonist) (16). Moreover, we delineated the role and mechanism of action of RGS-2 (regulator of G-protein signaling-2) in CCK2R-induced inositol phosphate production (17).…”

Molecular Basis for Binding and Subtype Selectivity of 1,4-Benzodiazepine Antagonist Ligands of the Cholecystokinin Receptor

“…Although residue 2.61 appears to play a critically important role in the CCK2R, this is most likely indirect, as it does not make substantial contact with the benzodiazepine ligand in this model. It should be noted that, again based on an interpretation of indirect mutagenesis data, residue 2.61 in the CCK2R has been suggested to play an anchoring role for relatively large non-peptide ligands based on a dibenzobicyclo[2.2.2]octane skeleton (JB93,182 and JB93,242), but these are not structurally similar to benzodiazepines and may more closely reflect peptide binding to this receptor (51).…”