title: 14-3-3ζ Protein regulates anterograde transport of the human κ-opioid receptor (hKOPR).
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.
authors.affiliation: Department of Pharmacology and Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
authors.forename: Jian-Guo
authors.initials: JG
authors.lastname: Li
authors.forename: Chongguang
authors.initials: C
authors.lastname: Chen
authors.forename: Peng
authors.initials: P
authors.lastname: Huang
authors.forename: Yujun
authors.initials: Y
authors.lastname: Wang
authors.forename: Lee-Yuan
authors.initials: LY
authors.lastname: Liu-Chen
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D020756:Coatomer Protein / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005260:Female; D015151:Immunoblotting; D047468:Immunoprecipitation; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D011247:Pregnancy; D011485:Protein Binding; D021381:Protein Transport; D040901:Proteomics / Q000379:methods*; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D017473:Receptors, Opioid, kappa / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D048948:14-3-3 Proteins; D020756:Coatomer Protein; D017473:Receptors, Opioid, kappa