BRAG2/GEP100/IQSec1 Interacts with Clathrin and Regulates α5β1 Integrin Endocytosis through Activation of ADP Ribosylation Factor 5 (Arf5)
BRAG2/GEP100/IQSec1 Interacts with Clathrin and Regulates α5β1 Integrin Endocytosis through Activation of ADP Ribosylation Factor 5 (Arf5)
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DOI：
10.1074/jbc.m112.383117
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发表时间：
2012-09-07
影响因子：
4.8
通讯作者：
Casanova, James E.
中科院分区：
文献类型：
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作者：
Moravec, Radim;Conger, Kathryn K.;Casanova, James E.
ADP核糖基化因子（Arfs）是小的GTP结合蛋白，已知其在囊泡运输中的作用，其中它们在载体囊泡形成的位点处使外壳蛋白复合物的组装成核。与其他GTP酶类似，Arfs需要鸟嘌呤核苷酸交换因子来催化GTP加载和活化。ArfGEF的一个亚家族，BRAG，已显示激活Arf 6，其在内吞途径中起作用以控制货物蛋白（包括整联蛋白）的子集的运输。我们先前已经表明BRAG 2通过调节整合素表面表达来调节细胞粘附。在这里，我们表明，除了Arf 6，内源性BRAG 2也激活II类Arf，Arf 4和Arf 5，令人惊讶的是，它是Arf 5介导的整合素内化。我们观察到，在BRAG 2或Arf 5而不是Arf 6的抑制后，纤维连接蛋白上的细胞铺展增强。类似地，BRAG 2耗尽的细胞中的扩散通过快速循环Arf 5突变体（T161 A）的表达而不是通过相应的Arf 6构建体（T157 A）而恢复。我们还表明，BRAG 2结合网格蛋白和AP-2适配器复合物，BRAG 2和Arf 5定位于质膜网格蛋白包被的小坑。与这些观察结果一致，Arf 5的消耗，而不是Arf 6或Arf 4的消耗，减缓了β 1整联蛋白的内化，而不影响转铁蛋白受体的摄取。总之，这些发现表明BRAG 2作用于网格蛋白包被的凹坑，通过激活Arf 5促进整合素内化，并表明Arf 5在网格蛋白介导的特定货物的内吞作用中的先前未被认识的作用。
ADP ribosylation factors (Arfs) are small GTP-binding proteins known for their role in vesicular transport, where they nucleate the assembly of coat protein complexes at sites of carrier vesicle formation. Similar to other GTPases, Arfs require guanine nucleotide exchange factors to catalyze GTP loading and activation. One subfamily of ArfGEFs, the BRAGs, has been shown to activate Arf6, which acts in the endocytic pathway to control the trafficking of a subset of cargo proteins including integrins. We have previously shown that BRAG2 modulates cell adhesion by regulating integrin surface expression. Here, we show that, in addition to Arf6, endogenous BRAG2 also activates the class II Arfs, Arf4 and Arf5, and that surprisingly, it is Arf5 that mediates integrin internalization. We observed that cell spreading on fibronectin is enhanced upon inhibition of BRAG2 or Arf5 but not Arf6. Similarly, spreading in BRAG2-depleted cells is reverted by expression of a rapid cycling Arf5 mutant (T161A) but not by a corresponding Arf6 construct (T157A). We also show that BRAG2 binds clathrin and the AP-2 adaptor complex and that both BRAG2 and Arf5 localize to clathrin-coated pits at the plasma membrane. Consistent with these observations, depletion of Arf5, but not Arf6 or Arf4, slows internalization of beta 1 integrins without affecting transferrin receptor uptake. Together, these findings indicate that BRAG2 acts at clathrin-coated pits to promote integrin internalization by activating Arf5 and suggest a previously unrecognized role for Arf5 in clathrin-mediated endocytosis of specific cargoes.