Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling

Abstract

SUMMARY G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in humans, and they regulate processes ranging from neurotransmission to cardiovascular biology. Although GPCRs have been studied for decades, current methods for tracking GPCR signaling often suffer from low throughput, modification or overexpression of effector proteins, and low temporal resolution. Here, we introduce a new approach using peroxidase-catalyzed proximity labeling to track GPCR signaling and internalization in living cells. Combination of this technique with isobaric labeling and triple-stage mass spectrometry enables precise, quantitative, and time-resolved measurement of thousands of receptor-proximal proteins at their native levels to comprehensively track GPCR agonist response. Using this technique, we examine the response of the angiotensin II type 1 receptor to both balanced and biased agonists. In addition, we extend the approach to the β2 adrenergic receptor, underscoring the generalizability of this technology.

How Ligands Illuminate GPCR Molecular Pharmacology

“…The delineation of these two signaling pathways has led to a major reconceptualization of how GPCRs mediate their actions in both normal physiology and disease and has ushered in a new era of GPCR drug discovery to identify agonists biased for either G protein (White et al, 2015) or arrestin (Allen et al, 2011b) signaling with the promise of improved therapeutic properties and reduced side-effects (DeWire et al, 2011). It should be noted, however, that identification and characterization of GPCR effectors remains an active area of investigation (Paek et al, 2017), and that there are likely many more unidentified intracellular proteins that interact with signaling complexes and modulate GPCR signaling (Paek et al, 2017). …”

“…Similar ligand binding properties between the Neuropeptide S receptor (NPS) and the orphan GPCR GPR37L1 identified the NPS receptor ligand SHA68 as a novel ligand to interrogate GPR37L1 function. The figure is extensively modified from Ngo et al (2017) with permission.…”

Proximity labeling to detect RNA–protein interactions in live cells

“…Although previous studies have reported that proximity‐dependent labeling has a high background in mammalian cells , we have achieved a very good signal‐to‐noise ratio in this transient transfection experiment. Proximity labeling biotinylates proteins in a way that relies on proximal distance and duration time.…”

From atoms to physiology: what it takes to really understand inflammasomes

“…; Paek et al . ). These tools thus seem suitable to define the cellular proteins that interact with assembling, signalling or degrading inflammasomes.…”