Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195;
Departamento Fisiologia e Biofisica, ICB Universidade Federal de Minas Gerais, Belo Horizonte, MG 31270-901, Brazil.
Proc Natl Acad Sci U S A. 2017 Jul 11;114(28):E5579-E5588. doi: 10.1073/pnas.1700331114. Epub 2017 Jun 26.
Binding of agonists to G-protein-coupled receptors (GPCRs) activates heterotrimeric G proteins and downstream signaling. Agonist-bound GPCRs are then phosphorylated by protein kinases and bound by arrestin to trigger desensitization and endocytosis. Arrestin plays another important signaling function. It recruits and regulates activity of an extracellular signal-regulated kinase (ERK) cascade. However, molecular details and timing of ERK activation remain fundamental unanswered questions that limit understanding of how arrestin-dependent GPCR signaling controls cell functions. Here we validate and model a system that tracks the dynamics of interactions of arrestin with receptors and of ERK activation using optical reporters. Our intermolecular FRET measurements in living cells are consistent with β-arrestin binding to M muscarinic acetylcholine receptors (MRs) in two different binding modes, transient and stable. The stable mode persists for minutes after agonist removal. The choice of mode is governed by phosphorylation on key residues in the third intracellular loop of the receptor. We detect a similar intramolecular conformational change in arrestin in either binding mode. It develops within seconds of arrestin binding to the M receptor, and it reverses within seconds of arrestin unbinding from the transient binding mode. Furthermore, we observed that, when stably bound to phosphorylated MR, β-arrestin scaffolds and activates MEK-dependent ERK. In contrast, when transiently bound, β-arrestin reduces ERK activity via recruitment of a protein phosphatase. All this ERK signaling develops at the plasma membrane. In this scaffolding hypothesis, a shifting balance between the two arrestin binding modes determines the degree of ERK activation at the membrane.
激动剂与 G 蛋白偶联受体（GPCRs）的结合激活异三聚体 G 蛋白和下游信号转导。然后，被激动剂结合的 GPCR 被蛋白激酶磷酸化，并与 arrestin 结合以触发脱敏和内吞作用。arrestin 发挥着另一个重要的信号功能。它募集并调节细胞外信号调节激酶（ERK）级联的活性。然而，ERK 激活的分子细节和时间仍然是基本未解决的问题，限制了对 arrestin 依赖性 GPCR 信号如何控制细胞功能的理解。在这里，我们使用光学报告物验证和建模了一个跟踪 arrestin 与受体相互作用和 ERK 激活动力学的系统。我们在活细胞中的分子内 FRET 测量结果与β-arrestin 以两种不同的结合模式（瞬时和稳定）与 M 毒蕈碱乙酰胆碱受体（MRs）结合的结果一致。稳定模式在激动剂去除后持续数分钟。模式的选择受受体第三细胞内环中关键残基的磷酸化控制。我们在两种结合模式中的 arrestin 中检测到类似的分子内构象变化。它在 arrestin 与 M 受体结合后的几秒钟内发展，并在 arrestin 从瞬时结合模式解离后的几秒钟内逆转。此外，我们观察到，当稳定地与磷酸化的 MR 结合时，β-arrestin 支架并激活 MEK 依赖性 ERK。相比之下，当瞬时结合时，β-arrestin 通过募集蛋白磷酸酶来降低 ERK 活性。所有这些 ERK 信号都在质膜上发展。在这个支架假说中，两种 arrestin 结合模式之间的平衡变化决定了膜上 ERK 激活的程度。
