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

Miriam Stoeber, Damien Jullié, Joy Li, Soumen Chakraborty, Susruta Majumdar, Nevin A Lambert, Aashish Manglik, Mark von Zastrow

eLife 2020 Feb 25

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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. © 2020, Stoeber et al.

Citation

Miriam Stoeber, Damien Jullié, Joy Li, Soumen Chakraborty, Susruta Majumdar, Nevin A Lambert, Aashish Manglik, Mark von Zastrow. Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells. eLife. 2020 Feb 25;9