Abstract
The lipid raft location of mu-opioid receptor (MOR) determines the receptor activities. However, the manner in which MOR is anchored within the lipid rafts is undetermined. Using the targeted proteomic approach and mass spectrometry analyses, we have identified GRIN1 (G protein-regulated inducer of neurite outgrowth 1) can tether MOR with the G protein alpha-subunit and subsequently regulate the receptor distribution within the lipid rafts. Glutathione S-transferase fusion pulldown and receptor mutational analyses indicate that GRIN1-MOR interaction involves a receptor sequence (267)GSKEK(271) within the MOR third intracellular loop that is not involved in Galpha interaction. The GRIN1 domains involved in MOR interaction are also distinct from those involved in Galpha interaction. Pertussis toxin pretreatment reduced the amount of GRIN1 co-immunoprecipitated with MOR but not the amount with Galpha. Furthermore, overexpression 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 pertussis toxin-sensitive neurite outgrowth. In contrast to minimal neurite outgrowth induced by etorphine in control neuroblastoma N2A cells, overexpression of GRIN1 resulted in the increase in etorphine- and non-morphine-induced neurite outgrowth in these cells. Knocking down endogenous GRIN1 by small interfering RNA attenuated the agonist-induced neurite outgrowth. Disrupting lipid raft by methyl-beta-cyclodextrin also blocked neurite outgrowth. Hence, by tethering Galpha with MOR, GRIN1 stabilizes the receptor within the lipid rafts and potentiates the receptor signaling in the neurite outgrowth processes.
Highlights
Grants DA007339, DA016674, DA000564, and DA011806. □S The on-line version of this article contains supplemental Figs
Proteins known to interact with lipid rafts and to have activities influenced by such interaction, include G protein-coupled receptors (GPCRs), tyrosine kinase receptors, GTPases (e.g. H-Ras and ␣-subunits of heterotrimeric G proteins), and nonreceptor tyrosine kinases (e.g. Src and Src family kinases), as well as mitogen-activated protein kinase (MAPK)/ERK kinase and ERK [2,3,4,5,6,7,8]
GRIN1 Promotes MOR Distribution into Lipid Raft cells are located in lipid rafts, and cholesterol depletion of these cells has no effect on the association between -opioid receptors and G protein [19], indicating receptor and G protein occupy different membrane microdomains
Summary
Culturing of N2A Cells Stably Expressing His6-MOR and Transient Transfection of cDNA—The rat MOR-tagged with the His epitope at the N terminus was subcloned in pCDNAmp vector. Proteins were eluted by resuspending the beads in 2 volumes of 2ϫ SDS sample buffer (10 mM Tris, 15 mM SDS, 20 mM DTT, 20% glycerol, 0.02% bromphenol blue, pH 6.8) followed by incubation at 65 °C for 30 min. Proteins were resolved by SDS-PAGE, transferred to Immobilon-P membranes (Millipore, Bedford, MA), followed by immunoblotting with anti-GRIN1 primary antibodies (T116), and detected with anti-rabbit alkaline phosphatase-linked secondary antibodies (Bio-Rad) diluted in Tris-buffered saline containing 5% powdered milk and 0.1% Tween 20 unless indicated otherwise. Protein samples were separated by SDS-polyacrylamide gel and transferred to Immobilon-P membranes (Millipore, Bedford, MA), followed by incubation with in vitro translation product of HA-GRIN1 or premixing of in vitro translation product of HA-GRIN1 and GST-MORi33 or GSTMORi35 by covering the corresponding spot of Immobilon-P membrane with transparency films (3M, St. Paul, MN) for 1 h at room temperature. To normalize the Western analyses from separate runs, the relative percentage of the proteins in each fraction was obtained by dividing each individual gradient fraction versus the sum of the pixels from all fractions
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