Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University, Beijing 100191, China.
Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing 100191, China.
Proc Natl Acad Sci U S A. 2021 Sep 14;118(37). doi: 10.1073/pnas.2026491118.
Arrestins were initially identified for their role in homologous desensitization and internalization of G protein-coupled receptors. Receptor-bound arrestins also initiate signaling by interacting with other signaling proteins. Arrestins scaffold MAPK signaling cascades, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. In particular, arrestins facilitate ERK1/2 activation by scaffolding ERK1/2 (MAPK), MEK1 (MAP2K), and Raf (MAPK3). However, the structural mechanism underlying this scaffolding remains unknown. Here, we investigated the mechanism of arrestin-2 scaffolding of cRaf, MEK1, and ERK2 using hydrogen/deuterium exchange-mass spectrometry, tryptophan-induced bimane fluorescence quenching, and NMR. We found that basal and active arrestin-2 interacted with cRaf, while only active arrestin-2 interacted with MEK1 and ERK2. The ATP binding status of MEK1 or ERK2 affected arrestin-2 binding; ATP-bound MEK1 interacted with arrestin-2, whereas only empty ERK2 bound arrestin-2. Analysis of the binding interfaces suggested that the relative positions of cRaf, MEK1, and ERK2 on arrestin-2 likely facilitate sequential phosphorylation in the signal transduction cascade.
最初， arrestins 因其在 G 蛋白偶联受体同源脱敏和内化中的作用而被识别。受体结合的 arrestins 还通过与其他信号蛋白相互作用来启动信号转导。Arrestins 支架 MAPK 信号级联，MAPK 激酶激酶（MAP3K），MAPK 激酶（MAP2K）和 MAPK。特别是，arrestins 通过支架 ERK1/2（MAPK），MEK1（MAP2K）和 Raf（MAPK3）来促进 ERK1/2 的激活。然而，这种支架的结构机制仍然未知。在这里，我们使用氢/氘交换 - 质谱法，色氨酸诱导的双马来酰亚胺荧光猝灭和 NMR 研究了 arrestin-2 对接构 cRaf，MEK1 和 ERK2 的机制。我们发现基础和活性 arrestin-2 与 cRaf 相互作用，而只有活性 arrestin-2 与 MEK1 和 ERK2 相互作用。MEK1 或 ERK2 的 ATP 结合状态影响 arrestin-2 的结合；ATP 结合的 MEK1 与 arrestin-2 相互作用，而只有空 ERK2 结合 arrestin-2。结合界面的分析表明，arrestin-2 上 cRaf、MEK1 和 ERK2 的相对位置可能有利于信号转导级联中的顺序磷酸化。
