Department of Biochemistry and Molecular Biology, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, (M.I., F.D.P., J.L.B.); and Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan (A.I.).
Department of Biochemistry and Molecular Biology, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, (M.I., F.D.P., J.L.B.); and Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan (A.I.)
Mol Pharmacol. 2022 Feb;101(2):87-94. doi: 10.1124/molpharm.121.000332. Epub 2021 Dec 1.
G protein-coupled receptors (GPCRs) transduce a diverse variety of extracellular stimuli into intracellular signaling. These receptors are the most clinically productive drug targets at present. Despite decades of research on the signaling consequences of molecule-receptor interactions, conformational components of receptor-effector interactions remain incompletely described. The -adrenergic receptor ( AR) is a prototypical and extensively studied GPCR that can provide insight into this aspect of GPCR signaling thanks to robust structural data and rich pharmacopeia. Using bioluminescence resonance energy transfer -based biosensors, second messenger assays, and biochemical techniques, we characterize the properties of AR-F193A. This single point mutation in extracellular loop 2 of the AR is sufficient to intrinsically bias the AR away from -arrestin interaction and demonstrates altered regulatory outcomes downstream of this functional selectivity. This study highlights the importance of extracellular control of intracellular response to stimuli and suggests a previously undescribed role for the extracellular loops of the receptor and the extracellular pocket formed by transmembrane domains 2, 3, and 7 in GPCR regulation that may contribute to biased signaling at GPCRs. SIGNIFICANCE STATEMENT: The role of extracellular G protein-coupled receptor (GPCR) domains in mediating intracellular interactions is poorly understood. We characterized the effects of extracellular loop mutations on agonist-promoted interactions of GPCRs with G protein and β-arrestin. Our studies reveal that F193 in extracellular loop 2 in the β-adrenergic receptor mediates interactions with G protein and β-arrestin with a biased loss of β-arrestin binding. These results provide new insights on the role of the extracellular domain in differentially modulating intracellular interactions with GPCRs.
G 蛋白偶联受体（GPCR）将各种细胞外刺激转导为细胞内信号。这些受体是目前最具临床生产力的药物靶点。尽管对分子-受体相互作用的信号转导后果进行了几十年的研究，但受体-效应器相互作用的构象成分仍未得到充分描述。β-肾上腺素能受体（β-AR）是一种典型的、经过广泛研究的 GPCR，由于其具有强大的结构数据和丰富的药理学，因此可以深入了解 GPCR 信号转导的这一方面。使用基于生物发光共振能量转移的生物传感器、第二信使测定和生化技术，我们对β-AR-F193A 的特性进行了表征。β-AR 细胞外环 2 中的这一单点突变足以使β-AR 从β-抑制蛋白相互作用中固有地偏向，并证明了这种功能选择性的下游调节结果发生改变。这项研究强调了细胞外控制细胞内对刺激反应的重要性，并提示受体的细胞外环和由跨膜域 2、3 和 7 形成的细胞外环在 GPCR 调节中可能发挥以前未描述的作用，从而导致 GPCR 信号的偏向。
细胞外 G 蛋白偶联受体（GPCR）结构域在介导细胞内相互作用中的作用尚未完全了解。我们研究了细胞外环突变对激动剂促进 GPCR 与 G 蛋白和β-抑制蛋白相互作用的影响。我们的研究表明，β-肾上腺素能受体细胞外环 2 中的 F193 介导与 G 蛋白和β-抑制蛋白的相互作用，并且β-抑制蛋白结合的偏向性丧失。这些结果提供了关于细胞外结构域在调节 GPCR 细胞内相互作用方面的新见解。
