The effect of arrestin conformation on the recruitment of c-Raf1, MEK1, and ERK1/2 activation.
The effect of arrestin conformation on the recruitment of c-Raf1, MEK1, and ERK1/2 activation.
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DOI：
10.1371/journal.pone.0028723
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Gurevich VV
中科院分区：
文献类型：
--
作者：
Coffa S;Breitman M;Hanson SM;Callaway K;Kook S;Dalby KN;Gurevich VV
抑制蛋白是多功能信号转导衔接子，最初被发现为通过G蛋白偶联受体（GPCR）“抑制”G蛋白活化的蛋白质。最近，GPCR与抑制蛋白的复合物已被提出激活G蛋白非依赖性信号通路。特别地，已经证明了细胞外信号调节激酶1/2（ERK 1/2）的抑制蛋白依赖性激活。在这里，我们已经进行了体外结合试验与纯蛋白质，以证明第一次，ERK 2直接结合游离arrestin-2和-3，以及受体相关的arrestins-1，-2，和-3。此外，我们发现在COS-7细胞中，arrestin-2和arrestin-3与β2-肾上腺素能受体（β2AR）的结合显著增强了ERK 2的结合，但对arrestin与上游激酶c-Raf 1和MEK 1的相互作用几乎没有影响。抑制蛋白存在于三种构象状态：游离、受体结合和微管相关。使用构象偏向的arrestin突变体，我们发现ERK 2优先结合其中的两种：“组成型失活”arrestin-Δ7模拟微管结合状态和arrestin-3A，受体结合构象的模拟物。两者都在arrestin-2/3双敲除成纤维细胞中拯救arrestin介导的ERK 1/2/活化。我们还发现，arrestin-2-c-Raf 1相互作用增强受体结合，而arrestin-3-c-Raf 1相互作用不是。
Arrestins are multifunctional signaling adaptors originally discovered as proteins that “arrest” G protein activation by G protein-coupled receptors (GPCRs). Recently GPCR complexes with arrestins have been proposed to activate G protein-independent signaling pathways. In particular, arrestin-dependent activation of extracellular signal-regulated kinase 1/2 (ERK1/2) has been demonstrated. Here we have performed in vitro binding assays with pure proteins to demonstrate for the first time that ERK2 directly binds free arrestin-2 and -3, as well as receptor-associated arrestins-1, -2, and -3. In addition, we showed that in COS-7 cells arrestin-2 and -3 association with β2-adrenergic receptor (β2AR) significantly enhanced ERK2 binding, but showed little effect on arrestin interactions with the upstream kinases c-Raf1 and MEK1. Arrestins exist in three conformational states: free, receptor-bound, and microtubule-associated. Using conformationally biased arrestin mutants we found that ERK2 preferentially binds two of these: the “constitutively inactive” arrestin-Δ7 mimicking microtubule-bound state and arrestin-3A, a mimic of the receptor-bound conformation. Both rescue arrestin-mediated ERK1/2/activation in arrestin-2/3 double knockout fibroblasts. We also found that arrestin-2-c-Raf1 interaction is enhanced by receptor binding, whereas arrestin-3-c-Raf1 interaction is not.
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