β‐arrestins (βarrs) are key regulators of G protein‐coupled receptor (GPCR) signaling and trafficking, and their knockdown typically leads to a decrease in agonist‐induced ERK1/2 MAP kinase activation. Interestingly, for some GPCRs, knockdown of βarr1 augments agonist‐induced ERK1/2 phosphorylation although a mechanistic basis for this intriguing phenomenon is unclear. Here, we use selected GPCRs to explore a possible correlation between the spatial positioning of receptor phosphorylation sites and the contribution of βarr1 in ERK1/2 activation. We discover that engineering a spatially positioned double‐phosphorylation‐site cluster in the bradykinin receptor (B₂R), analogous to that present in the vasopressin receptor (V₂R), reverses the contribution of βarr1 in ERK1/2 activation from inhibitory to promotive. An intrabody sensor suggests a conformational mechanism for this role reversal of βarr1, and molecular dynamics simulation reveals a bifurcated salt bridge between this double‐phosphorylation site cluster and Lys²⁹⁴ in the lariat loop of βarr1, which directs the orientation of the lariat loop. Our findings provide novel insights into the opposite roles of βarr1 in ERK1/2 activation for different GPCRs with a direct relevance to biased agonism and novel therapeutics.

中文翻译：

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GPCR 中的关键磷酸化位点协调 β-Arrestin 1 在 ERK1/2 激活中的作用。

β-arrestins (βarrs) 是 G 蛋白偶联受体 (GPCR) 信号传导和运输的关键调节因子，它们的敲低通常会导致激动剂诱导的 ERK1/2 MAP 激酶活化减少。有趣的是，对于一些 GPCR，βarr1 的敲低增强了激动剂诱导的 ERK1/2 磷酸化，尽管这种有趣现象的机制基础尚不清楚。在这里，我们使用选定的 GPCR 来探索受体磷酸化位点的空间定位与 βarr1 在 ERK1/2 激活中的贡献之间可能存在的相关性。_{我们发现，在缓激肽受体 (B 2} R)中设计了一个空间定位的双磷酸化位点簇，类似于加压素受体 (V ₂R)，将 βarr1 在 ERK1/2 激活中的作用从抑制转变为促进。体内传感器表明了 βarr1 的这种作用逆转的构象机制，分子动力学模拟揭示了这种双磷酸化位点簇和 βarr1 套索环中的 Lys ²⁹⁴之间的分叉盐桥，它指导套索环的方向。我们的研究结果为不同 GPCR 的 ERK1/2 激活中 βarr1 的相反作用提供了新的见解，与偏向激动和新疗法直接相关。