The angiotensin II (AngII) type 1 receptor (AT1R), a member of the G protein–coupled receptor (GPCR) family, signals through G proteins and β-arrestins, which act as adaptors to regulate AT1R internalization and mitogen-activated protein kinase (MAPK) ERK1/2 activation. β-arrestin–dependent ERK1/2 regulation is the subject of important studies because its spatiotemporal control remains poorly understood for many GPCRs, including AT1R. To study the link between β-arrestin–dependent trafficking and ERK1/2 signaling, we investigated three naturally occurring AT1R variants that show distinct receptor–β-arrestin interactions: A163T, T282M, and C289W. Using bioluminescence resonance energy transfer (BRET)–based and conformational fluorescein arsenical hairpin–BRET sensors coupled with high-resolution fluorescence microscopy, we show that all AT1R variants form complexes with β-arrestin2 at the plasma membrane and efficiently internalize into endosomes upon AngII stimulation. However, mutant receptors imposed distinct conformations in β-arrestin2 and differentially impacted endosomal trafficking and MAPK signaling. Notably, T282M accumulated in endosomes, but its ability to form stable complexes following internalization was reduced, markedly impairing its ability to co-traffic with β-arrestin2. We also found that despite β-arrestin2 overexpression, T282M's and C289W's residency with β-arrestin2 in endosomes was greatly reduced, leading to decreased β-arrestin–dependent ERK1/2 activation, faster recycling of receptors to the plasma membrane, and impaired AngII-mediated proliferation. Our findings reveal that naturally occurring AT1R variants alter the patterns of receptor/β-arrestin2 trafficking and suggest conformationally dependent β-arrestin–mediated MAPK activation as well as endosomal receptor–β-arrestin complex stabilization in the mitogenic response of AT1R.

中文翻译：

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血管紧张素 II 1 型受体变体改变内体受体-β-抑制蛋白复合物的稳定性和 MAPK 活化。

血管紧张素 II (AngII) 1 型受体 (AT1R) 是 G 蛋白偶联受体 (GPCR) 家族的成员，通过 G 蛋白和 β-抑制蛋白发出信号，它们充当调节 AT1R 内化和丝裂原活化蛋白激酶的接头(MAPK) ERK1/2 激活。β-抑制蛋白依赖性 ERK1/2 调节是重要研究的主题，因为对包括 AT1R 在内的许多 GPCR 对其时空控制仍知之甚少。为了研究 β-抑制蛋白依赖性运输与 ERK1/2 信号传导之间的联系，我们研究了三种天然存在的 AT1R 变体，它们显示出不同的受体-β-抑制蛋白相互作用：A163T、T282M 和 C289W。使用基于生物发光共振能量转移（BRET）的和构象荧光素砷发夹-BRET传感器与高分辨率荧光显微镜相结合，我们表明所有 AT1R 变体在质膜上与 β-arrestin2 形成复合物，并在 AngII 刺激下有效地内化到内体中。然而，突变受体在 β-arrestin2 中施加了不同的构象，并对内体运输和 MAPK 信号传导产生了不同的影响。值得注意的是，T282M 在内体中积累，但其在内化后形成稳定复合物的能力降低，显着削弱了其与 β-arrestin2 共同传输的能力。我们还发现，尽管 β-arrestin2 过度表达，但 T282M 和 C289W 在内体中与 β-arrestin2 的驻留大大减少，导致 β-arrestin 依赖性 ERK1/2 活化减少，受体更快地再循环到质膜，并受损 AngII-介导的增殖。