Distinct CCK-2 Receptor Conformations Associated with β-Arrestin-2 Recruitment or Phospholipase-C Activation Revealed by a Biased Antagonist

Abstract

Seven-transmembrane receptors (7TMRs), also termed G protein-coupled receptors (GPCRs), form the largest class of cell surface membrane receptors, involving several hundred members in the human genome. Nearly 30% of marketed pharmacological agents target 7TMRs. 7TMRs adopt multiple conformations upon agonist binding. Biased agonists, in contrast to non-biased agonists, are believed to stabilize conformations preferentially activating either G-protein- or β-arrestin-dependent signaling pathways. However, proof that cognate conformations of receptors display structural differences within their binding site where biased agonism initiates, are still lacking. Here, we show that a non-biased agonist, cholecystokinin (CCK) induces conformational states of the CCK2R activating Gq-protein-dependent pathway (CCK2R(G)) or recruiting β-arrestin2 (CCK2R(β)) that are pharmacologically and structurally distinct. Two structurally unrelated antagonists competitively inhibited both pathways. A third ligand (GV150013X) acted as a high affinity competitive antagonist on CCK2R(G) but was nearly inefficient as inhibitor of CCK2R(β). Several structural elements on both GV150013X and in CCK2R binding cavity, which hinder binding of GV150013X only to the CCK2R(β) were identified. At last, proximity between two conserved amino acids from transmembrane helices 3 and 7 interacting through sulfur-aromatic interaction was shown to be crucial for selective stabilization of the CCK2R(β) state. These data establish structural evidence for distinct conformations of a 7TMR associated with β-arrestin-2 recruitment or G-protein coupling and validate relevance of the design of biased ligands able to selectively target each functional conformation of 7TMRs.

Comparison of cell-based assays for the identification and evaluation of competitive CXCR4 inhibitors

“…A compound shown to physically interact with the ligand binding pocket of CXCR4 effectively inhibited the response in all signal transduction and functional receptor assays tested with consistent relative potencies, while compounds that did not interfere with agonist binding showed no or only very limited effect (Fig 5). Biased GPCR ligands, including antagonists that preferentially inhibit one functional or signaling pathway over the other have been described for other GPCRs [30, 31] and raise general interest since they might evoke less side effects in clinical settings [32]. Our data, however, indicate that compounds commonly used to interrupt CXCL12/CXCR4 interaction display no bias in activity when tested in functional assays relevant for CXCR4.…”

Internalization and desensitization of the human glucose-dependent-insulinotropic receptor is affected by N-terminal acetylation of the agonist

“…As a consequence, N-acetyl-GIP has a less number of interactions with helices 6 and 7 compared to GIP. These results, together with data showing that helices 6 and 7 in G-protein coupled receptors are essential for stabilization of the active conformation of receptors, may explain atypical activity of N-acetyl-GIP analogue (Hollenstein et al, 2014;Audet and Bouvier, 2012;Magnan et al, 2013). So, it is plausible that the two agonists may stabilize different conformations of the GIPR, each of which having distinct ability to trigger signals.…”

Multiple Binding Sites for Small-Molecule Antagonists at the CC Chemokine Receptor 2

“…Multiple Binding Sites at the Chemokine Receptor CCR2 Magnan et al, 2013). Notably, it has been reported that some CCR2 antagonists are capable of discriminating between different functional states of the receptor (Kredel et al, 2011).…”