Filamin A Binds to CCR2B and Regulates Its Internalization
Filamin A Binds to CCR2B and Regulates Its Internalization
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DOI：
10.1371/journal.pone.0012212
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发表时间：
2010-08-17
期刊：
影响因子：
3.7
通讯作者：
Aragay, Anna M.
中科院分区：
文献类型：
--
作者：
Minsaas, Laura;Planaguma, Jesus;Aragay, Anna M.
趋化因子（C-C基序）受体2B （CCR2B）是单核细胞趋化蛋白-1 （CCL2）受体的两种异构体之一，CCL2是单核细胞的主要趋化剂，参与一系列慢性炎症疾病。利用酵母双杂交系统，我们鉴定了肌动蛋白结合蛋白filamin A （FLNa）是与CCR2B的羧基末端尾部相关的蛋白。共免疫沉淀实验和体外拉下实验表明，FLNa与CCR2B构成性结合。内源性CCR2B和丝蛋白A在THP-1细胞表面和内化囊泡中共定位。此外，CCR2B和FLNa在表达CCR2B的A7细胞板足结构中共定位。在丝蛋白缺乏的M2细胞中表达该受体，并在HEK293细胞中进行siRNA敲除FLNa的实验，表明FLNa缺乏会延迟受体的内化。此外，通过RNA干扰减少THP-1单核细胞中的FLNa，减少了细胞响应MCP-1的迁移。因此，FLNa成为控制CCR2B受体在不同动态膜结构中内化和空间定位的重要蛋白。
The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), the major chemoattractant for monocytes, involved in an array of chronic inflammatory diseases. Employing the yeast two-hybrid system, we identified the actin-binding protein filamin A (FLNa) as a protein that associates with the carboxyl-terminal tail of CCR2B. Co-immunoprecipitation experiments and in vitro pull down assays demonstrated that FLNa binds constitutively to CCR2B. The colocalization of endogenous CCR2B and filamin A was detected at the surface and in internalized vesicles of THP-1 cells. In addition, CCR2B and FLNa were colocalized in lamellipodia structures of CCR2B-expressing A7 cells. Expression of the receptor in filamin-deficient M2 cells together with siRNA experiments knocking down FLNa in HEK293 cells, demonstrated that lack of FLNa delays the internalization of the receptor. Furthermore, depletion of FLNa in THP-1 monocytes by RNA interference reduced the migration of cells in response to MCP-1. Therefore, FLNa emerges as an important protein for controlling the internalization and spatial localization of the CCR2B receptor in different dynamic membrane structures.