Abstract
By proteomic analysis, we found that 14-3-3ζ was one of the proteins co-immunoprecipitated with human κ-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. 14-3-3ζ co-localized with the hKOPR in N2A cells. The hKOPR C-tail interacted with 14-3-3ζ in rat brain extracts and bound directly to purified 14-3-3ζ as demonstrated by pulldown techniques. 14-3-3ζ 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 14-3-3ζ siRNA was reversed by overexpression of 14-3-3ζ. Expression of the 14-3-3 scavenger protein pGpLI-R18 also decreased hKOPR expression. 14-3-3ζ siRNA did not change expressions of the hDOPR and rMOPR in N2A cells. Pulse-chase study showed that 14-3-3ζ 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 14-3-3ζ and abolished the effect of 14-3-3ζ 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 14-3-3ζ-mediated regulation of hKOPR expression. 14-3-3ζ knockdown increased association of COPI with the hKOPR. These results suggest that 14-3-3ζ promotes expression of the hKOPR by inhibiting COPI and RVR motif-mediated endoplasmic reticulum localization machinery.
Highlights
Regulation of export of 7TMRs is not well understood
We found that 14-3-3 was one of the proteins co-immunoprecipitated with human -opioid receptor from extracts of solubilized Neuro2A cells stably expressing FLAG-human KOPR (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
Proteomic Analysis of Proteins Co-immunoprecipitated with the hKOPR—Fig. 1A shows staining of a representative SDSPAGE gel of proteins immunoprecipitated with anti-FLAG antibody from control N2A cells and N2A-FLAG-hKOPR cells
Summary
Regulation of export of 7TMRs is not well understood. Results: 14-3-3 binds hKOPR C-tail and facilitates hKOPR export by inhibiting COPI- and RVR motif-mediated ER localization. Mutation of the endoplasmic reticulum retention motif 359RVR 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. Studies have shown that 7TMRs interact with many proteins in addition to G proteins These proteins directly participate in signaling of the receptor and act as part of a scaffolding complex to modulate receptor signaling or regulate receptor trafficking, localization, and pharmacological characteristics (for review, see Ref. 4).
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