Evidence for a Direct Interaction between the Penultimate Aspartic Acid of Cholecystokinin and Histidine 207, Located in the Second Extracellular Loop of the Cholecystokinin B Receptor

Silvente-Poirot, Sandrine; Escrieut, Chantal; Galès, Céline; Fehrentz, Jean‐Alain; Escherich, Achim; Wank, Stephen A.; Martinez, Jean; Moröder, Luis; Maigret, Bernard; Bouisson, Michelle; Vaysse, Nicole; Fourmy, Daniel

doi:10.1074/jbc.274.33.23191

Cited by 42 publications

(98 citation statements)

References 39 publications

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“…It was further postulated that the designed ligands should mimic the two aromatic rings of gastrin, which are crucial for the binding affinity of the tetrapeptide. This view agrees with our data indicating that Trp and Phe of CCK and interacting residues in the CCK2R are involved in CCK2R activation (14,18). However, in our modeled CCK2R⅐CCK complex, CCK did not adopt a 3 10 helix conformation, and the distance between Trp and Phe was twice that observed in water (26).…”

Section: Discussionsupporting

confidence: 81%

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

Foucaud

Marco

Escrieut

et al. 2008

Journal of Biological Chemistry

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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...

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Section: Discussionsupporting

confidence: 81%

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

Foucaud

Marco

Escrieut

et al. 2008

Journal of Biological Chemistry

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“…In subsequent mutagenesis studies, the authors showed that within this five-amino acid sequence, an His is crucial for CCK recognition and interacts with the penultimate aspartic acid of CCK (471,472). Furthermore, several amino acids and key regions that interact with the amidated COOH-terminal phenylalanine of CCK were identified in transmembrane helices IV and VI (161, 264).…”

Section: A Localization Of Ligand Binding Sitesmentioning

confidence: 99%

Cholecystokinin and Gastrin Receptors

Dufresne

Seva²,

Fourmy³

2006

Physiological Reviews

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417

418

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Cholecystokinin and gastrin receptors (CCK1R and CCK2R) are G protein-coupled receptors that have been the subject of intensive research in the last 10 years with corresponding advances in the understanding of their functioning and physiology. In this review, we first describe general properties of the receptors, such as the different signaling pathways used to exert short- and long-term effects and the structural data that explain their binding properties, activation, and regulation. We then focus on peripheral cholecystokinin receptors by describing their tissue distribution and physiological actions. Finally, pathophysiological peripheral actions of cholecystokinin receptors and their relevance in clinical disorders are reviewed.

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“…Total IP accumulation was measured after stimulation by the indicated concentration of CCK-9 for 1 h at 371C as described previously (Silvente-Poirot et al, 1999;Gales et al, 2000). EC 50 was calculated using the GraphPad Prism Program.…”

Section: Measurement Of Total Inositol Phosphate Accumulationmentioning

confidence: 99%

High tumorigenic potential of a constitutively active mutant of the cholecystokinin 2 receptor

et al. 2003

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The cholecystokinin 2 receptor (CCK2R) increases proliferation of normal and neoplastic gastrointestinal cells and activates various mitogenic signaling pathways when stimulated by gastrin. To study the incidence of permanent activation of this receptor in tumorigenicity, a constitutively active mutant was generated by replacing residue Glu151 in the conserved E/DRY motif by Ala. Expression of the E151A-CCK2R mutant in NIH-3T3 cells causes ligand-independent activation of phospholipase C and ornithine decarboxylase, two enzymes critical for mitogenesis. Strikingly, the constitutive activity of this mutant was associated with dramatic alteration of NIH-3T3 cell morphology, enhanced cell proliferation and invasion. Moreover, injection of cells expressing E151A-CCK2R in nude mice resulted in the development of large and rapidly growing tumors. By contrast, none of these effects was observed with cells expressing the wildtype CCK2R, indicating that the tumorigenic properties of the E151A-CCK2R mutant is the result of its constitutive activation. To date, this is the first report that provides evidence for the high tumorigenic effect of a constitutively active CCK2R mutant, thus raising a potential role of the CCK2R in human cancer.

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Evidence for a Direct Interaction between the Penultimate Aspartic Acid of Cholecystokinin and Histidine 207, Located in the Second Extracellular Loop of the Cholecystokinin B Receptor

Cited by 42 publications

References 39 publications

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

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

Cholecystokinin and Gastrin Receptors

High tumorigenic potential of a constitutively active mutant of the cholecystokinin 2 receptor

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