Luttrell L M, Roudabush F L, Choy E W, Miller W E, Field M E, Pierce K L, Lefkowitz R J
The Geriatrics Research, Education and Clinical Center, Durham Veterans Affairs Medical Center, Durham, NC 27705, USA.
Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2449-54. doi: 10.1073/pnas.041604898. Epub 2001 Feb 20.
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.
利用共聚焦免疫荧光显微镜和生化方法，我们研究了β-抑制蛋白在1a型血管紧张素受体（AT1aR）刺激后细胞外信号调节激酶2（ERK2）激活和靶向中的作用。在表达血凝素标记的AT1aR的HEK-293细胞中，血管紧张素刺激引发β-抑制蛋白-2与受体结合以及AT1aR-β-抑制蛋白复合物的内化。使用红色荧光蛋白标记的ERK2追踪ERK2的亚细胞分布，我们发现血管紧张素处理导致活化的ERK2重新分布到也含有AT1aR-β-抑制蛋白复合物的内体囊泡中。ERK2的这种靶向反映了包含AT1aR、β-抑制蛋白-2以及ERK级联反应的组成激酶cRaf-1、MEK1和ERK2的多蛋白复合物的形成。Myc标记的cRaf-1、MEK1和绿色荧光蛋白标记的ERK2与转染的COS-7细胞中Flag标记的β-抑制蛋白-2共沉淀。cRaf-1与β-抑制蛋白-2的共沉淀不依赖于MEK1和ERK2，而在过表达cRaf-1的情况下，MEK1和ERK2与β-抑制蛋白-2的共沉淀显著增强，这表明cRaf-1与β-抑制蛋白的结合促进了cRaf-1、MEK1、ERK2复合物的组装。cRaf-1的共表达显著增强了β-抑制蛋白复合物中ERK2的磷酸化，并且这种作用被MEK1的催化无活性显性抑制突变体的表达所阻断。血管紧张素刺激增加了cRaf-1和ERK2与β-抑制蛋白-2的结合，以及β-抑制蛋白-2、cRaf-1和ERK2与AT1aR的结合。这些数据表明，β-抑制蛋白既作为支架增强cRaf-1和MEK依赖型ERK2的激活，又作为靶向蛋白将活化的ERK引导至特定的亚细胞位置。
