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 β₂-adrenergic receptor (β₂AR) 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 β₂AR 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 β₂AR 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.

选择性激活G蛋白或β-arrestin介导的信号通路的G蛋白偶联受体（GPCR）的偏置激动剂具有重要的治疗意义，因为它们有可能显示出改善的药效和特异性。努力理解这种现象的机理基础的努力集中在以下假设上：G蛋白和β-arrestin优先结合不同的GPCR构象。但是，由于依赖GPCR激酶（GRK）的受体磷酸化是将β-arrestin募集到大多数GPCR的关键先决条件，因此GRK本身可能在建立偏向信号中起重要作用。我们表明，在β的跨膜结构域5的高度保守的残基酪氨酸219（Y219A）的丙氨酸突变体₂肾上腺素能受体（β₂ AR）无法进行β-arrestin募集，受体内在化和β-arrestin介导的细胞外信号调节激酶（ERK）的激活，而保留了通过G蛋白发出信号的能力。我们发现，β抑制蛋白募集细胞中受损的是由于β的减小GRK介导的磷酸₂ AR Y219A C末端，将其在体外与概括纯化组分。此外，在体外合成地磷酸化的肽上的β的C末端的结扎₂AR Y219A挽救了β-arrestin的最初募集及其与受体细胞内核心的结合。这些数据表明，Y219A突变主要通过针对GRK偶联而非针对β-arrestin的构象选择而产生G蛋白偏倚状态。总之，这些发现凸显了GRK在调节GPCR的偏向激动性中的重要性。