The DHR1 domain of DOCK180 binds to SNX5 and regulates cation-independent mannose 6-phosphate receptor transport
The DHR1 domain of DOCK180 binds to SNX5 and regulates cation-independent mannose 6-phosphate receptor transport
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DOI：
10.1091/mbc.e08-03-0314
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发表时间：
2008-09-01
影响因子：
3.3
通讯作者：
Matsuda, Michiyuki
中科院分区：
文献类型：
--
作者：
Hara, Shigeo;Kiyokawa, Etsuko;Matsuda, Michiyuki
DOCK180是DOCK180家族小GTPases Rac1和Cdc42的鸟嘌呤核苷酸交换因子的原型。dock180家族蛋白共享两个保守结构域，称为DOCK同源区(DHR)-1和-2。虽然DHR2的功能是激活Rac1，但DHR1是与磷酸肌苷结合所必需的。为了更好地了解DHR1的功能，我们通过直接纳米流液相色谱/串联质谱法寻找了它的结合伙伴，并鉴定了分选连接蛋白（SNX） 1、2、5和6，它们组成了一个多聚体蛋白复合物，介导了阳离子非依赖性甘糖6-磷酸受体（CI-MPR）的内体到反式高尔基网络（TGN）的逆行运输。在这些SNX蛋白中，SNX5与DOCK180共免疫沉淀效果最好。与这一观察结果一致，DOCK180在核内体上与SNX5共定位。RNA干扰介导的SNX5和DOCK180的敲低，而不是Rac1的敲低，导致CI-MPR从TGN重新分布到核内体。此外，DOCK180 DHR1结构域的表达足以恢复DOCK180敲低细胞中受干扰的CI-MPR分布。这些数据表明DOCK180通过SNX5调节CI-MPR运输，并且该功能独立于其对Rac1的鸟嘌呤核苷酸交换因子活性。
DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.