| Abstract No.: | B-B2038 |
| Country: | Canada |
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| Title: | DELTA OPIOID RECEPTOR (DOR) DESENSITIZATION IS ENHANCED BY SRC INTERFERENCE WITH RECEPTOR RECYCLING |
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| Authors/Affiliations: | 2 Elodie Archer-Lahlou*; 2 Nicolas Audet; 1 Mohammad Gholi Amraei; 1 Karine Huard; 2 Mélanie Paquin-Gobeil; 3 Graciela Pineyro;
1 Centre de recherche Fernand-Seguin, Montreal, QC, Canada; 2 Centre de recherche Fernand-Seguin; Département de Pharmacologie, Université de Montréal, QC, Canada; 3 Centre de recherche Fernand-Seguin; Départements de Pharmacologie et de Psychiatrie, Université de Montréal, QC, Canada
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| Content: | Opiates are the most effective analgesics presently known, but their therapeutic use is restricted by development of tolerance, dependence and their potential for abuse. Though very different in presentation, these undesirable effects are thought to arise from a similar adaptive mechanism in which neurons from distinct brain circuits adapt to counteract the effects of sustained opiate exposure. Development of analgesic tolerance is tightly linked to receptors desensitization, a process that classically involves opioid receptor uncoupling from the G protein, its internalization and eventually its down regulation. In the case of delta opioid receptors (DOR), desensitization is especially swift because receptors are rapidly internalized and are poorly recycled to the membrane. In the present study we investigated whether Src activity contributed to this sorting pattern and to functional desensitization of DORs. Wild-type DORs were expressed in HEK293 cells and a first series of DOR immunopurification assays allowed to establish that Src spontaneously interacted with the receptor and was released following activation by the DOR agonist DPDPE (1 µM; 30 min). cAMP accumulation assays were then used to evaluate Src contribution to the regulation of DOR signalling efficacy. Pre-treatment with Src inhibitor PP2 (20 µM; 1 h) or transfection with a dominant negative Src mutant did not modify acute inhibition of cAMP production by DPDPE, but protected DORs from the desensitization caused by a pre-incubation with this same ligand (1 µM; 30 min). This protection was afforded without interfering with endocytosis, but suboptimal internalization interfered with PP2 ability to preserve DOR signalling, suggesting a post-endocytic site of action for the kinase. This assumption was confirmed by demonstrating that Src inhibition by PP2 or its silencing by siRNA increased membrane recovery of internalized DORs and was further corroborated by showing that inhibition of recycling by monensin or dominant negative Rab11 (Rab11S25N) abolished the ability of Src blockers to prevent desensitization. Finally, Src inhibitors accelerated recovery of DOR-Gαi3 coupling after desensitization. Taken together, these results indicate that Src dynamically regulates DOR recycling and by doing so contributes to desensitization of these receptors. This provides new insights into the molecular mechanism regulating DOR desensitization. In addition, since our studies have so far been carried out exclusively on HEK cells, a new set of experiments is now conducted on primary cultures of neuronal cells. By studying endogenous opioid receptors, we want to establish the physiological significance of our findings. This could provide key information for the development of new analgesic opiates with a better clinical profile. |
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