| Abstract No.: | B-F2177 |
| Country: | Canada |
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| Title: | BRET ASSAYS REVEAL LIGAND-SPECIFIC CONFORMATIONAL CHANGES WITHIN PREFORMED SIGNALLING COMPLEXES CONTAINING DELTA OPIOID RECEPTOR (DOR) AND HETEROTRIMERIC G PROTEINS. |
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| Authors/Affiliations: | 4 Nicolas Audet*; 2 Caroline Galas; 4 Melodie Archer-Lalhou; 1 Marc Valliares; 3 Peter W. Schiller; 4 Michel Bouvier; 4 Graciela Pineyro;
1 Centre de recherche Fernand-Seguin; 2 INSERM; 3 IRCM; 4 Universite de Montreal, QC, Canada
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| Content: | OBJECTIVES: Opiate drugs produce their effects by acting upon G protein coupled receptors (GPCRs) and although they are among the most effective analgesics available, their clinical use is restricted by unwanted side effects such as tolerance, physical dependence, respiratory depression, nausea and constipation. As a class, opiates share a common profile of unwanted effects but there are also significant differences in ligand liability for producing these actions that could be applied to improve opioid side effects profile. Differences among ligand actions seem to be related to the existence of multiple active conformations that allow receptors to distinctively interact with intracellular signalling partners depending on the ligand bound. In this study we use BRET technology in combination with ligands of different functional profiles to provide in vivo physical evidence of conformational diversity of delta opioid receptors (DORs). METHODS: BRET is a proximity-based assay that allows measuring protein-protein interactions in living cells. To do so it relies on the transfer of energy between an energy donor (luciferase enzyme, luc) and an energy acceptor (green fluorescent protein, GFP), a property that was used herein to monitor interactions between DORs and different G protein subunits. For this purpose the donor luc was fused to the human DOR or to the αi1 subunit of the G protein while the acceptor GFP was fused to DOR or to γ2 subunit. BRET pairs DOR-GFP/αi1-Luc or DOR-Luc/γ2-GFP were then transiently transfected onto HEK293 cells and the latter exposed (10 µM; 2 min) to DOR ligands. BRET results were also compared to cAMP and MAPK signalling ability of each drug. RESULTS: Results showed that DORs and heterotrimeric G-proteins formed a constitutive complex that underwent structural reorganization upon ligand binding. We also observed that ligand rank order of efficacy to modify energy transfer was not maintained across all BRET pairs tested, confirming the existence of ligand-specific conformations. Finally, we showed that ligand ability to modify BRET did not systematically correlate with their signalling efficacy at the cyclase and MAPK cascades, indicating that conformations stabilized by different ligands distinctively interact with different effectors. CONCLUSIONS: DORs adopt ligand-specific conformations and conformational diversity encoded by the receptor is conveyed to the intracellular signalling partners. The existence of multiple active receptor states adds unprecedented diversity to receptor signalling and has implications for the way we conceive specificity of signal transduction. Thus, a ligand that preferentially recognizes a receptor conformation within a particular type of signalling complex would confine modulation of receptor signalling to cells that express that specific type of array. Alternatively, stabilization of a conformation which allows activation of a specific subset of complexes containing a definite type of effector would restrict consequences of receptor activation to a distinct signalling pathway, and to the vital functions it may regulate. Exploiting this signalling diversity could prove effective in developing therapeutic analgesics with reduced side effects. Work funded by the CIHR and NSERC. |
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