Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
Sci Signal. 2018 Aug 21;11(544):eaar7084. doi: 10.1126/scisignal.aar7084.
Biased agonists of G protein-coupled receptors (GPCRs), which selectively activate either G protein- or β-arrestin-mediated signaling pathways, are of major therapeutic interest because they have the potential to show improved efficacy and specificity as drugs. Efforts to understand the mechanistic basis of this phenomenon have focused on the hypothesis that G proteins and β-arrestins preferentially couple to distinct GPCR conformations. However, because GPCR kinase (GRK)-dependent receptor phosphorylation is a critical prerequisite for the recruitment of β-arrestins to most GPCRs, GRKs themselves may play an important role in establishing biased signaling. We showed that an alanine mutant of the highly conserved residue tyrosine 219 (Y219A) in transmembrane domain five of the β-adrenergic receptor (βAR) was incapable of β-arrestin recruitment, receptor internalization, and β-arrestin-mediated activation of extracellular signal-regulated kinase (ERK), whereas it retained the ability to signal through G protein. We found that the impaired β-arrestin recruitment in cells was due to reduced GRK-mediated phosphorylation of the βAR Y219A C terminus, which was recapitulated in vitro with purified components. Furthermore, in vitro ligation of a synthetically phosphorylated peptide onto the C terminus of βAR Y219A rescued both the initial recruitment of β-arrestin and its engagement with the intracellular core of the receptor. These data suggest that the Y219A mutation generates a G protein-biased state primarily by conformational selection against GRK coupling, rather than against β-arrestin. Together, these findings highlight the importance of GRKs in modulating the biased agonism of GPCRs.
G 蛋白偶联受体（GPCR）的偏倚激动剂选择性地激活 G 蛋白或β-arrestin 介导的信号通路，具有重要的治疗意义，因为它们有可能作为药物显示出更好的疗效和特异性。为了理解这一现象的机制基础，人们的注意力集中在以下假设上：即 G 蛋白和β-arrestin 优先与不同的 GPCR 构象偶联。然而，由于 GPCR 激酶（GRK）依赖性受体磷酸化是大多数 GPCR 募集β-arrestin 的关键前提，因此 GRK 本身可能在建立偏倚信号中发挥重要作用。我们发现，β肾上腺素能受体（βAR）跨膜域五的高度保守残基酪氨酸 219（Y219）的丙氨酸突变体不能募集β-arrestin、受体内化和β-arrestin 介导的细胞外信号调节激酶（ERK）激活，而保留了通过 G 蛋白信号转导的能力。我们发现，细胞中β-arrestin 募集受损是由于 GRK 介导的βAR Y219A C 端磷酸化减少所致，这在体外用纯化的成分得到了重现。此外，体外将合成磷酸化肽连接到βAR Y219A 的 C 端可挽救β-arrestin 的初始募集及其与受体细胞内核心的结合。这些数据表明，Y219A 突变主要通过与 GRK 偶联的构象选择而不是与β-arrestin 偶联，产生 G 蛋白偏倚状态。总之，这些发现强调了 GRK 在调节 GPCR 偏倚激动剂中的重要性。
