Novel binding partners of Mu-opioid receptor and their regulatory roles.
2009-12
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Novel binding partners of Mu-opioid receptor and their regulatory roles.
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2009-12
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G-protein-coupled receptors (GPCRs) represent a super-family of proteins in the human genome (>900), which include at least one third of current drug targets. Associated proteins of GPCRs consist of the down-stream signaling pathway, which convert the external stimulus to the final signal of cellular function change. Some useful methods have been used to explore the sophisticated network of GPCR-associated proteins, such as yeast-two hybridization and GST fusion protein pull-down assay. However, in both methods, only one or two domains of the receptor were used to construct a fusion protein for identifying scaffolding proteins. This cannot reflect the conditions in which an agonist/antagonist mediates the opioid receptors' conformational change that leads to protein recruitment. To identify the binding partners of MOR, a member of GPCR rhodopsin subfamily that clarified to be important in regulating drug tolerance and addiction, we purified MOR complexes from (His)6-tagged MOR stably expressing neuroblastma neuro2A (N2A) cells. Combine with Mass Spectrometry and LC MS/MS (Liquid chromatography-electrospray ionization tandem mass spectrometry), some novel MOR binding partners were found. Two of them are studied further about their important roles in regulating MOR functions.
Ribophorin I (RPNI), a component of the oligosaccharidetransferase complex, could directly interact with MOR. RPNI can be shown to participate in MOR export by the intracellular retention of the receptor after siRNA knocking-down of endogenous RPNI. Over-expression of RPNI rescued the surface expression of the MOR 344KFCTR348 deletion mutant (C2) independent of calnexin. Furthermore, RPNI regulation of MOR trafficking is dependent on the glycosylation state of the receptor, as reflected in the inability of over-expression of RPNI to affect the trafficking of the N-glycosylation deficient mutants, or GPCR such as k-opioid receptor that has minimal glycosylation sites. Hence, this novel RPNI chaperone activity is a consequence of N-glycosylation-dependent direct interaction with MOR.
G protein-regulated inducer of neurite outgrowth 1 (GRIN1) can influence MOR lipid raft location by tethering the receptor with the heterotrimeric G protein ?-subunit. GST fusion pull-down and receptor mutational analyses indicated the 267GSKEK271 sequence within the MOR 3rd intracellular loop was involved in interacting with the GRIN1 sequence distinct from that participated in the G? binding. The uncoupling of G? from MOR with PTX reduced the amount of GRIN1 co-immunoprecipitated with MOR while the amount of GRIN1 coimmunoprecipitated with G? was unchanged. Furthermore, over-expression of GRIN1 significantly enhanced the amount of MOR in lipid raft and the receptor signaling magnitude as measured by Src kinase activation. Such increase in MOR signaling was demonstrated further by determining the GRIN1-dependent neurite outgrowth. In contrast to minimal neurite outgrowth induced by etorphine in control cells, over-expression of GRIN1 and the increase in GRIN1-MOR interaction resulted in the increase in etorphine- and not-morphine-induced neurite outgrowth in the neuroblastoma N2A cell that was PTX sensitive. Knocking-down of endogenous GRIN1 by siRNA attenuated the agonist-induced neurite outgrowth. Disrupting lipid raft by M?CD also blocked neurite outgrowth. Hence, by serving as a tether between G? and MOR, GRIN1 stabilizes the receptor within the lipid rafts and potentiates the receptor signaling in the neurite outgrowth process.
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University of Minnesota Ph.D. dissertation. December 2009. Major: Pharmacology. Advisor: Ping-Yee Law. 1 computer file (PDF); ix, 108 pages.
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Ge, Xin. (2009). Novel binding partners of Mu-opioid receptor and their regulatory roles.. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/58342.
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