Inducible Inhibition of G Reveals Localization-dependent Functions at the Plasma Membrane and Golgi
Inducible Inhibition of G Reveals Localization-dependent Functions at the Plasma Membrane and Golgi
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DOI：
10.1074/jbc.m116.750430
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发表时间：
2017-02-03
影响因子：
4.8
通讯作者：
Wedegaertner, Philip B.
中科院分区：
文献类型：
--
作者：
Klayman, Lauren M.;Wedegaertner, Philip B.
异源三聚体G蛋白除了在质膜（PM）的细胞质表面处的典型功能之外，还在各种内膜位置处发出信号，在质膜（PM）的细胞质表面处，它们被细胞表面G蛋白偶联受体激活。在这里，我们专注于信号在高尔基体，激活信号级联，最终导致囊泡分裂从trans-Golgi网络（TGN）。为了开发一种新的分子工具，抑制内源性的时空方式，我们利用广泛使用的抑制剂GRK 2ct（GRK 2ct-KERE）和FRB/FKBP异源二聚化系统的脂质缔合突变体。我们表明，GRK 2ct-KERE不能抑制功能时，在细胞中表达，但招聘到一个特定的膜位置恢复GRK 2ct-KERE抑制信号传导的能力。PM募集的GRK 2ct-KERE抑制溶血磷脂酸诱导的Akt磷酸化，而高尔基体募集的GRK 2ct-KERE抑制从TGN到PM的货物运输。此外，我们发现，高尔基体招募GRK 2ct-KERE抑制模型basolateralized有针对性的，但不是顶端有针对性的货物交付，为PM-注定和分泌货物，提供第一个证据的货物运输方面的选择性调节。最后，我们表明，由ilimaquinone和nocodazole诱导的高尔基体碎片被抑制阻断，表明这是影响高尔基体形态的多个途径的关键调节因子。因此，我们已经开发了一种新的分子工具，可招募GRK 2ct-KERE，在特定的亚细胞位置调节信号，我们证明了新的货物选择性调节TGN PM运输和一个新的作用，在介导高尔基体片段。
Heterotrimeric G proteins signal at a variety of endomembrane locations, in addition to their canonical function at the cytoplasmic surface of the plasma membrane (PM), where they are activated by cell surface G protein-coupled receptors. Here we focus on signaling at the Golgi, where activates a signaling cascade, ultimately resulting in vesicle fission from the trans-Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous in a spatial-temporal manner, we take advantage of a lipid association mutant of the widely used inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system. We show that GRK2ct-KERE cannot inhibit function when expressed in cells, but recruitment to a specific membrane location recovers the ability of GRK2ct-KERE to inhibit signaling. PM-recruited GRK2ct-KERE inhibits lysophosphatidic acid-induced phosphorylation of Akt, whereas Golgi-recruited GRK2ct-KERE inhibits cargo transport from the TGN to the PM. Moreover, we show that Golgi-recruited GRK2ct-KERE inhibits model basolaterally targeted but not apically targeted cargo delivery, for both PM-destined and secretory cargo, providing the first evidence of selectivity in terms of cargo transport regulated by . Last, we show that Golgi fragmentation induced by ilimaquinone and nocodazole is blocked by inhibition, demonstrating that is a key regulator of multiple pathways that impact Golgi morphology. Thus, we have developed a new molecular tool, recruitable GRK2ct-KERE, to modulate signaling at specific subcellular locations, and we demonstrate novel cargo selectivity for regulation of TGN to PM transport and a novel role for in mediating Golgi fragmentation.