NOSTRIN functions as a homotrimeric adaptor protein facilitating internalization of eNOS
NOSTRIN functions as a homotrimeric adaptor protein facilitating internalization of eNOS
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DOI：
10.1242/jcs.02620
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发表时间：
2005-11-01
影响因子：
4
通讯作者：
Schilling, K
中科院分区：
文献类型：
--
作者：
Icking, A;Matt, S;Schilling, K
内皮型一氧化氮合酶(ENOS)在不同隔室之间的细胞内转运尚不完全清楚。最近，我们描述了一种新的eNOS相互作用蛋白，NOSTRIN，它在过度表达时驱动eNOS离开质膜进入细胞内。NOSTRIN和Pacsins/Syndapins的序列相似性提示了NOSTRIN在内吞作用中的作用。因此，我们在这里证明了NOSTRIN通过其SH3结构域与大的GTPase Dynamin和肌动蛋白成核促进因子N-WASP相互作用，SH3结构域也代表eNOS的对接位置。NOSTRIN通过蛋白质C-末端的卷曲线圈区域进行寡聚，主要形成三聚体，这将允许与SH3结构域的多个结合伙伴同时相互作用。与这一概念一致，只有在NOSTRIN存在的情况下，在稳定表达eNOS(CHO-eNOS)的CHO细胞中表达Dynamin-2-GFP会导致eNOS募集到Dynamin阳性结构中。同样，当N-WASP-GFP和NOSTRIN在CHO-eNOS细胞中共表达时，这两种蛋白都与eNOS强烈共定位，并被招募到沿着肌动蛋白细丝运行的结构中。然而，如果肌动蛋白细胞骨架被细胞松弛素D解聚，NOSTRIN和eNOS与细胞外围的延伸结构相关，可能无法离开质膜。这些结果表明，NOSTRIN可能通过协调Dynamin和N-WASP的功能来促进eNOS的内吞作用。
Intracellular trafficking of endothelial nitric oxide synthase (eNOS) between different compartments is incompletely understood. Recently, we described a novel eNOS-interacting protein, NOSTRIN, which upon overexpression drives eNOS away from the plasma membrane towards intracellular compartments. Sequence similarity of NOSTRIN and pacsins/syndapins suggested a role for NOSTRIN in endocytosis. Accordingly, we show here that NOSTRIN interacts with the large GTPase dynamin and the actin nucleation promoting factor N-WASP by means of its SH3 domain, which also represents the docking site for eNOS. Via a coiled-coil region in the C-terminal portion of the protein, NOSTRIN oligomerizes, mainly forming trimers, which would allow simultaneous interaction with multiple binding partners of the SH3 domain. Consistent with this notion, expression of dynamin-2-GFP in CHO cells stably expressing eNOS (CHO-eNOS) results in recruitment of eNOS to dynamin-positive structures, only when NOSTRIN is present as well. Similarly, when N-WASP-GFP and NOSTRIN are co-expressed in CHO-eNOS cells, both proteins strongly co-localize with eNOS and are recruited to structures running along actin filaments. If, however, the actin cytoskeleton is depolymerized by cytochalasin D, NOSTRIN and eNOS are associated with extended structures in the cell periphery, possibly being unable to leave the plasma membrane. Together, these results indicate that NOSTRIN may facilitate endocytosis of eNOS by coordinating the function of dynamin and N-WASP.