14-3-3? Protein regulates anterograde transport of the human ?-opioid receptor (hKOPR).
Ontology highlight
ABSTRACT: By proteomic analysis, we found that 14-3-3? was one of the proteins co-immunoprecipitated with human ?-opioid receptor (hKOPR) from extracts of solubilized Neuro2A cells stably expressing FLAG-hKOPR (N2A-FLAG-hKOPR cells). 14-3-3 proteins are a family of conserved regulatory molecules in eukaryotic cells, where they participate in signal transduction, metabolism, and membrane protein transport. 14-3-3? co-localized with the hKOPR in N2A cells. The hKOPR C-tail interacted with 14-3-3? in rat brain extracts and bound directly to purified 14-3-3? as demonstrated by pulldown techniques. 14-3-3? siRNA decreased expression of the hKOPR in N2A-FLAG-hKOPR cells and cultured primary cortical neurons of E19 rats by ~25% as determined by immunoblotting, ligand binding, and flow cytometry. The effect of 14-3-3? siRNA was reversed by overexpression of 14-3-3?. Expression of the 14-3-3 scavenger protein pGpLI-R18 also decreased hKOPR expression. 14-3-3? siRNA did not change expressions of the hDOPR and rMOPR in N2A cells. Pulse-chase study showed that 14-3-3? siRNA decreased the amount of mature hKOPR but did not change the rate of maturation or stability of hKOPR protein. Mutations of R354A/S358A in the putative 14-3-3 interaction motif (354)RQSTS(358) in the hKOPR C-tail reduced interaction of the hKOPR with 14-3-3? and abolished the effect of 14-3-3? knockdown on hKOPR expression. Mutation of the endoplasmic reticulum retention motif (359)RVR adjacent to the 14-3-3 interaction motif in the hKOPR C-tail decreased interaction of coatomer protein I (COPI) with the hKOPR and abolished 14-3-3?-mediated regulation of hKOPR expression. 14-3-3? knockdown increased association of COPI with the hKOPR. These results suggest that 14-3-3? promotes expression of the hKOPR by inhibiting COPI and RVR motif-mediated endoplasmic reticulum localization machinery.
SUBMITTER: Li JG
PROVIDER: S-EPMC3488053 | biostudies-literature | 2012 Nov
REPOSITORIES: biostudies-literature
ACCESS DATA
