G protein-coupled receptors mediate two functionally distinct pathways of tyrosine phosphorylation in rat 1a fibroblasts - Shc phosphorylation and receptor endocytosis correlate with activation of Erk kinases
G protein-coupled receptors mediate two functionally distinct pathways of tyrosine phosphorylation in rat 1a fibroblasts - Shc phosphorylation and receptor endocytosis correlate with activation of Erk kinases
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DOI：
10.1074/jbc.272.50.31648
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发表时间：
1997-12-12
影响因子：
4.8
通讯作者：
Lefkowitz, RJ
中科院分区：
文献类型：
--
作者：
Luttrell, LM;Daaka, Y;Lefkowitz, RJ
G 蛋白偶联受体 (GPCR) 对 Erk 激酶的 Ras 依赖性激活被认为涉及对接蛋白的酪氨酸磷酸化，对接蛋白充当 Ras 鸟嘌呤核苷酸交换因子（例如 Grb2-mSos 复合物）质膜募集的支架。我们研究了两种 GPCR 调节的酪氨酸磷酸化蛋白 p125(FAK) (FAK) 和 Shc 在 Ras 1a 成纤维细胞中内源表达的 GPCR 引起的 Erk 激酶依赖的 Ras 激活中的作用。多条证据表明 FAK 和 Shc 的酪氨酸磷酸化是独立调节的。溶血磷脂酸 (LPA)、凝血酶和铃蟾肽的 GPCR 介导 FAK 酪氨酸磷酸化和 FAK-Grb2 关联的等效增加。相比之下，只有 LPA 和凝血酶受体显着刺激 Shc 酪氨酸磷酸化和 Shc-Grb2 复合物形成。 FAK 的酪氨酸磷酸化对百日咳毒素不敏感，可以被钙离子载体模拟，并且可以通过解聚肌动蛋白细胞骨架的细胞松弛素 D 处理来抑制。相反，Shc 的酪氨酸磷酸化可以通过百日咳毒素处理来抑制，不受钙离子载体的诱导，并且对细胞松弛素 D 不敏感。在每种情况下，Erk 1/2 的快速刺激与 Shc 的酪氨酸磷酸化相关，但与 FAK 无关，FAK-Grb2 复合物形成与受体介导的 Erk 1/2 激活的解离表明，Grb2-mSos 向质膜的募集不足以介导快速 Erk 激活，使用网格蛋白介导的内吞作用的四种机制不同的抑制剂、刀豆球蛋白 A、高渗介质、细胞内钾的消耗和monodansylcadaverine，我们发现 GPCR 介导的 Erk 1/2 激活也是内吞作用依赖性的。因此，我们提出，成纤维细胞中 Erk 激酶的快速、Ras 依赖性激活需要涉及囊泡介导的内吞作用的额外步骤。
The Ras-dependent activation of Erk kinases by G protein-coupled receptors (GPCRs) is thought to involve tyrosine phosphorylation of docking proteins that serve as scaffolds for the plasma membrane recruitment of Ras guanine nucleotide exchange factors, such as the Grb2-mSos complex. We have investigated the role of two GPCR-regulated tyrosine phosphoproteins, p125(FAK) (FAK) and Shc, in the Ras-dependent activation of Erk kinases by endogenously expressed GPCRs in Rat 1a fibroblasts, Several lines of evidence suggest that tyrosine phosphorylation of FAK and Shc are independently regulated. The GPCRs for lysophosphatidic acid (LPA), thrombin, and bombesin mediate equivalent increases in FAK tyrosine phosphorylation and FAK-Grb2 association, In contrast, only LPA and thrombin receptors significantly stimulate Shc tyrosine phosphorylation and Shc-Grb2 complex formation. Tyrosine phosphorylation of FAK is pertussis toxin-insensitive, can be mimicked by calcium ionophore, and is inhibited by treatment with cytochalasin D, which depolymerizes the actin cytoskeleton, In contrast, tyrosine phosphorylation of Shc is inhibited by pertussis toxin treatment, is not induced by calcium ionophore, and is insensitive to cytochalasin D, In each case, the rapid stimulation of Erk 1/2 correlates with tyrosine phosphorylation of Shc but not of FAK, The dissociation of FAK-Grb2 complex formation from receptor-mediated activation of Erk 1/2 indicates that recruitment of Grb2-mSos to the plasma membrane is not sufficient to mediate rapid Erk activation, Using four mechanistically distinct inhibitors of clathrin-mediated endocytosis, concanavalin A, hypertonic medium, depletion of intracellular potassium, and monodansylcadaverine, we find that GPCR-mediated Erk 1/2 activation is also endocytosis-dependent. Thus, we propose that an additional step involving vesicle-mediated endocytosis is required for the rapid, Ras-dependent activation of Erk kinases in fibroblasts.