Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607, USA.
J Biol Chem. 2011 Mar 4;286(9):7171-81. doi: 10.1074/jbc.M110.199695. Epub 2010 Dec 28.
Membrane trafficking is dictated by dynamic molecular interactions involving discrete determinants in the cargo proteins and the intracellular transport machineries. We have previously reported that cell surface expression of GPR15, a G protein-coupled receptor (GPCR) that serves as a co-receptor for HIV, is correlated with the mode III binding of 14-3-3 proteins to the receptor C terminus. Here we provide a mechanistic basis for the role of 14-3-3 in promoting the cell surface expression of GPR15. The Ala mutation of penultimate phospho-Ser (S359A) that abolishes 14-3-3 binding resulted in substantially reduced O-glycosylation and the cell surface expression of GPR15. The surface membrane protein CD8 fused with the C-terminal tail of GPR15(S359A) mutant was re-localized in the endoplasmic reticulum (ER). In the context of S359A mutation, the additional mutations in the upstream stretch of basic residues (RXR motif) restored O-glycosylation and the cell surface expression. The RXR motif was responsible for the interaction with coatomer protein I (COPI), which was inversely correlated with the 14-3-3 binding and cell surface expression. These results suggest that 14-3-3 binding promotes cell surface expression of GPR15 by releasing the receptor from ER retrieval/retention pathway that is mediated by the interaction of RXR motif and COPI. Moreover, 14-3-3 binding substantially increased the stability of GPR15 protein. Thus 14-3-3 proteins play multiple roles in biogenesis and trafficking of an HIV co-receptor GPR15 to control its cell surface density in response to the phosphorylation signal.
膜运输受货物蛋白和细胞内运输机制中离散决定因素的动态分子相互作用支配。我们之前报道过，G 蛋白偶联受体（GPCR）GPR15 的细胞表面表达与 14-3-3 蛋白与受体 C 末端的模式 III 结合有关。在这里，我们为 14-3-3 促进 GPR15 细胞表面表达的作用提供了一个机制基础。倒数第二个磷酸丝氨酸（S359A）的 Ala 突变消除了 14-3-3 的结合，导致 GPR15 的 O-糖基化和细胞表面表达大大减少。与 GPR15（S359A）突变体 C 末端尾部融合的表面膜蛋白 CD8 在内质网（ER）中重新定位。在 S359A 突变的情况下，上游碱性残基（RXR 基序）的额外突变恢复了 O-糖基化和细胞表面表达。RXR 基序负责与衣壳蛋白 I（COPI）相互作用，这与 14-3-3 结合和细胞表面表达呈负相关。这些结果表明，14-3-3 结合通过释放受体从由 RXR 基序和 COPI 介导的 ER 回收/保留途径促进 GPR15 的细胞表面表达。此外，14-3-3 结合大大增加了 GPR15 蛋白的稳定性。因此，14-3-3 蛋白在 HIV 共受体 GPR15 的生物发生和运输中发挥多种作用，以响应磷酸化信号控制其细胞表面密度。
