Title: 14-3-3ζ Protein regulates anterograde transport of the human κ-opioid receptor (hKOPR).
Authors: Li, Jian-Guo; Chen, Chongguang; Huang, Peng; Wang, Yujun; Liu-Chen, Lee-Yuan
Published In J Biol Chem, (2012 Nov 02)
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.
PubMed ID: 22989890
MeSH Terms: 14-3-3 Proteins/genetics; 14-3-3 Proteins/metabolism*; Amino Acid Motifs; Amino Acid Sequence; Animals; Cell Line, Tumor; Cells, Cultured; Coatomer Protein/metabolism; Endoplasmic Reticulum/metabolism; Female; Immunoblotting; Immunoprecipitation; Male; Mice; Microscopy, Fluorescence; Mutation; Pregnancy; Protein Binding; Protein Transport; Proteomics/methods*; RNA Interference; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa/genetics; Receptors, Opioid, kappa/metabolism*; Signal Transduction*