Department of Psychiatry, University of California, San Francisco, San Francisco, United States.
Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland.
Elife. 2020 Feb 25;9:e54208. doi: 10.7554/eLife.54208.
G protein-coupled receptors (GPCRs) signal through allostery, and it is increasingly clear that chemically distinct agonists can produce different receptor-based effects. It has been proposed that agonists selectively promote receptors to recruit one cellular interacting partner over another, introducing allosteric 'bias' into the signaling system. However, the underlying hypothesis - that different agonists drive GPCRs to engage different cytoplasmic proteins in living cells - remains untested due to the complexity of readouts through which receptor-proximal interactions are typically inferred. We describe a cell-based assay to overcome this challenge, based on GPCR-interacting biosensors that are disconnected from endogenous transduction mechanisms. Focusing on opioid receptors, we directly demonstrate differences between biosensor recruitment produced by chemically distinct opioid ligands in living cells. We then show that selective recruitment applies to GRK2, a biologically relevant GPCR regulator, through discrete interactions of GRK2 with receptors or with G protein beta-gamma subunits which are differentially promoted by agonists.
G 蛋白偶联受体（GPCRs）通过变构信号转导，越来越明显的是，化学性质不同的激动剂可以产生不同的基于受体的效应。有人提出，激动剂选择性地促进受体招募一个细胞相互作用伙伴而不是另一个，从而在信号转导系统中引入变构“偏向”。然而，由于通过受体近端相互作用通常推断的读出的复杂性，这一基本假设——不同的激动剂驱动 GPCR 与活细胞中不同的细胞质蛋白结合——尚未得到验证。我们描述了一种基于细胞的测定方法来克服这一挑战，该方法基于与内源性转导机制分离的 GPCR 相互作用生物传感器。我们专注于阿片受体，直接证明了在活细胞中由化学性质不同的阿片样激动剂产生的生物传感器募集之间的差异。然后，我们通过 GRK2 与受体或与 G 蛋白β-γ亚基的离散相互作用显示选择性募集适用于 GRK2，GRK2 是一种具有生物学意义的 GPCR 调节剂，GRK2 与受体或 G 蛋白β-γ亚基的相互作用通过激动剂被不同地促进。
