Growth factor receptor binding protein 2-mediated recruitment of the RING domain of Cbl to the epidermal growth factor receptor is essential and sufficient to support receptor endocytosis
Growth factor receptor binding protein 2-mediated recruitment of the RING domain of Cbl to the epidermal growth factor receptor is essential and sufficient to support receptor endocytosis
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DOI：
10.1091/mbc.e04-09-0832
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发表时间：
2005-03-01
影响因子：
3.3
通讯作者：
Sorkin, A
中科院分区：
文献类型：
--
作者：
Huang, FT;Sorkin, A
通过 RNA 干扰敲低生长因子受体结合蛋白 2 (Grb2) 会强烈抑制网格蛋白介导的表皮生长因子受体 (EGFR) 内吞作用。为了深入了解 Grb2 在 EGFR 内吞作用中的功能，我们生成了细胞系，其中内源性 Grb2 被生理水平表达的黄色荧光蛋白 (YFP) 标记的 Grb2 取代。在这些细胞中，Grb2-YFP完全逆转了Grb2敲低对EGFR内吞作用的抑制作用，而且在内吞作用期间与EGFR一起运输。 Grb2 结合蛋白 c-Cbl 的过度表达并不能恢复 Grb2 耗尽的细胞中的内吞作用。然而，在 Grb2 耗尽的细胞中，EGFR 内吞作用被由与 c-Cbl 融合的 Grb2 的 Src 同源 (SH) 2 结构域组成的嵌合蛋白拯救。 “敲低和救援”分析表明，在没有 Grb2 的情况下，含有 Cb1 RING 指结构域的 Cb`-Grb2/SH2 融合体的表达恢复了 EGFR 的正常泛素化和内化，这与 RING 结构域在 EGFR 内吞作用中的重要作用一致。相反，当 Cb1 的羧基末端结构域连接到 Grb2 SH2 结构域时，与含有 RING 的嵌合体相比，内吞作用救援效应小 4 倍。总之，数据表明 Cb1 羧基末端与 CIN85 的相互作用在 EGFR 内化中具有次要且多余的作用。我们得出的结论是，Grb2 介导的 Cb1 功能性 RING 结构域向 EGFR 的募集对于支持受体内吞作用至关重要且足以支持。
Knockdown of growth factor receptor binding protein 2 (Grb2) by RNA interference strongly inhibits clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR). To gain insights into the function of Grb2 in EGFR endocytosis, we have generated cell lines in which endogenous Grb2 was replaced by yellow fluorescent protein (YFP)-tagged Grb2 expressed at the physiological level. In these cells, Grb2-YFP fully reversed the inhibitory effect of Grb2 knockdown on EGFR endocytosis and, moreover, trafficked together with EGFR during endocytosis. Overexpression of Grb2-binding protein c-Cbl did not restore endocytosis in Grb2-depleted cells. However, EGFR endocytosis was rescued in Grb2-depleted cells by chimeric proteins consisting of the Src homology (SH) 2 domain of Grb2 fused to c-Cbl. The "knockdown and rescue" analysis revealed that the expression of Cb`-Grb2/SH2 fusions containing RING finger domain of Cb1 restores normal ubiquitylation and internalization of the EGFR in the absence of Grb2, consistent with the important role of the RING domain in EGFR endocytosis. In contrast, the carboxy-terminal domain of Cb1, when attached to Grb2 SH2 domain, had 4 times smaller endocytosis-rescue effect compared with the RING-containing chimeras. Together, the data suggest that the interaction of Cb1 carboxy terminus with CIN85 has a minor and a redundant role in EGFR internalization. We concluded that Grb2-mediated recruitment of the functional RING domain of Cb1 to the EGFR is essential and sufficient to support receptor endocytosis.