Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells

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 core underlying hypothesis - that different agonists drive GPCRs to engage different cytoplasmic proteins in living cells - remains untested due to the complexity of downstream readouts through which receptor-proximal interactions are typically inferred. Here we describe a scalable cell-based assay to overcome this challenge, based on the use of engineered GPCR-interacting proteins as orthogonal biosensors that are disconnected from endogenous transduction mechanisms. Focusing on opioid receptors, we directly demonstrate differences between protein probe recruitment produced by chemically distinct opioid ligands in living cells. We then show how the selective recruitment applies to GRK2, a biologically relevant opioid receptor regulator protein, through discrete interactions of GRK2 with receptors or with G protein beta-gamma subunits which are differentially promoted by agonists.