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

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

doi:10.7554/elife.54208

Abstract

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.

Highlights

G protein-coupled receptors (GPCRs) comprise nature’s largest family of signaling receptors and an important class of therapeutic drug targets
The ability of agonists to impose selectivity on protein recruitment by GPCRs has been proposed for many years and is a core hypothesis underlying the present concept of biased agonism (Schmid et al, 2017; Smith et al, 2018; Urban et al, 2007), but testing this hypothesis in an intact cellular environment has remained challenging due to the complexity of cellular transduction and regulatory pathways that GPCRs typically engage (Kenakin, 2019)
The present study describes a direct, reductionist approach to this problem based on the application of engineered proteins that bind activated receptors but are not known to bind other cellular proteins

Summary

Introduction

G protein-coupled receptors (GPCRs) comprise nature’s largest family of signaling receptors and an important class of therapeutic drug targets. This was followed by the demonstration of agonist-selective control of opioid receptor endocytosis, leading to the identification of functional selectivity among agonists defined by differences in relative ability to drive receptor engagement of G protein relative to betaarrestin-dependent cellular pathways (Keith et al, 1998; Keith et al, 1996; Whistler et al, 1999; Whistler and von Zastrow, 1998) This concept further evolved to the present view of biased receptor recruitment of G proteins relative to beta-arrestins, with receptor-proximal selectivity calculated by fitting quantitative measures of downstream pathway or protein response to operational models of receptor-effector coupling (Schmid et al, 2017). We delineate a cellbased method to assess selective protein recruitment by opioid receptors at the receptorproximal level, taking advantage of two engineered protein folds established to bind agonist-activated GPCRs in intact cells without requiring or engaging other known cellular proteins (Stoeber et al, 2018; Wan et al, 2018). We show how the principle of receptor-proximal protein selection applies in a more complex manner to GRK2, a biologically relevant regulator

Results

Discussion

Materials and methods

Funding Funder National Institutes of Health