Department of Pharmacology and Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
J Biol Chem. 2012 Nov 2;287(45):37778-92. doi: 10.1074/jbc.M112.359679. Epub 2012 Sep 18.
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.
通过蛋白质组学分析，我们发现 14-3-3ζ 是从稳定表达 FLAG-hKOPR（N2A-FLAG-hKOPR 细胞）的可溶性 Neuro2A 细胞提取物中与人类 κ-阿片受体（hKOPR）共免疫沉淀的蛋白质之一。14-3-3 蛋白是真核细胞中一组保守的调节分子，它们参与信号转导、代谢和膜蛋白运输。14-3-3ζ 在 N2A 细胞中与 hKOPR 共定位。hKOPR C 尾在大鼠脑提取物中与 14-3-3ζ 相互作用，并通过下拉技术证明直接结合纯化的 14-3-3ζ。14-3-3ζ siRNA 通过免疫印迹、配体结合和流式细胞术将 N2A-FLAG-hKOPR 细胞和培养的 E19 大鼠原代皮质神经元中的 hKOPR 表达降低约 25%。14-3-3ζ siRNA 的作用被 14-3-3ζ 的过表达逆转。表达 14-3-3 清除蛋白 pGpLI-R18 也降低了 hKOPR 的表达。14-3-3ζ siRNA 不会改变 N2A 细胞中 hDOPR 和 rMOPR 的表达。脉冲追踪研究表明，14-3-3ζ siRNA 降低了成熟 hKOPR 的量，但不改变 hKOPR 蛋白的成熟率或稳定性。hKOPR C 尾中假定的 14-3-3 相互作用模体（354）RQSTS（358）中 R354A/S358A 突变降低了 hKOPR 与 14-3-3ζ 的相互作用，并消除了 14-3-3ζ 敲低对 hKOPR 表达的影响。hKOPR C 尾中内质网保留模体（359）RVR 紧邻 14-3-3 相互作用模体的突变降低了 COPI 与 hKOPR 的相互作用，并消除了 14-3-3ζ 介导的 hKOPR 表达调节。14-3-3ζ siRNA 敲低增加了 COPI 与 hKOPR 的结合。这些结果表明，14-3-3ζ 通过抑制 COPI 和 RVR 基序介导的内质网定位机制促进 hKOPR 的表达。
