Activation and targeting of extracellular signal-regulated kinases by β-arrestin scaffolds
Activation and targeting of extracellular signal-regulated kinases by β-arrestin scaffolds
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DOI：
10.1073/pnas.041604898
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发表时间：
2001-02-27
影响因子：
11.1
通讯作者：
Lefkowitz, RJ
中科院分区：
文献类型：
--
作者：
Luttrell, LM;Roudabush, FL;Lefkowitz, RJ
利用共聚焦免疫荧光显微镜和生化方法，我们研究了β -阻滞蛋白在刺激血管紧张素II 1a型受体（AT1aR）后，在细胞外信号调节激酶2 （ERK2）的激活和靶向中的作用。在表达血凝素标记AT1aR的HEK-293细胞中，血管紧张素刺激触发β -阻滞蛋白2与受体结合并内化AT1aR-阻滞蛋白复合物。使用红色荧光蛋白标记的ERK2来追踪ERK2的亚细胞分布，我们发现血管紧张素治疗导致活化的ERK2重新分布到含有at1ar - β -阻滞蛋白复合物的内体囊泡中。ERK2的靶向反应了多种蛋白复合物的形成，这些蛋白复合物包含AT1aR、β -阻滞蛋白-2和ERK级联的组成激酶、craft -1、MEK1和ERK2。myc标记的caff -1、MEK1和绿色荧光蛋白标记的ERK2与flag标记的β -arrestin-2在转染的COS-7细胞中共沉淀。craft -1与β -arrestin-2的共沉淀不依赖于MEK1和ERK2，而在过表达的craft -1存在下，MEK1和ERK2与β -arrestin-2的共沉淀显著增强，这表明craft -1与β -arrestin-2的结合促进了craft -1、MEK1、ERK2复合物的组装。caff -1的共表达显著增强了β -抑制蛋白复合物中ERK2的磷酸化，这种作用被MEK1的催化失活显性抑制突变体的表达所阻断。血管紧张素刺激增加了caff -1和ERK2与β -arrestin-2的结合，以及β -arrestin-2、caff -1和ERK2与AT1aR的关联。这些数据表明β -阻滞蛋白既可以作为支架增强caff -1和mek依赖性ERK2的激活，也可以作为靶向蛋白将活化的ERK引导到特定的亚细胞位置。
Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of beta -arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinin-tagged AT1aR, angiotensin stimulation triggered beta -arrestin-2 binding to the receptor and internalization of AT1aR-beta -arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-beta -arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, beta -arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged beta -arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with beta -arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with beta -arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to beta -arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in beta -arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to beta -arrestin-2, and the association of beta -arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that beta -arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.