Profiling the proximal proteome of the mu opioid receptor identifies novel regulators of receptor signaling and trafficking

Abstract

G protein-coupled receptors (GPCRs) regulate many aspects of physiology and represent actionable targets for drug discovery. These receptors translate ligand-dependent input into a cellular response by remodeling protein interaction networks that are exquisitely organized both spatially and temporally. APEX-based proximity biotin labeling combined with quantitative mass spectrometry has emerged as a powerful approach for capturing all aspects of this dynamic ligand-dependent cellular re-organization. However, a major challenge in this approach is the extraction of salient information from these highly complex datasets. Here, we describe a computational framework for analyzing proximity labeling datasets that reliably predicts the subcellular location of GPCRs and deconvolutes spatial and temporal components of protein interaction network organization. Focusing on the mu-opioid receptor and its activation with distinct agonist drugs, we demonstrate the applicability of this framework to resolve agonist-specific differences in receptor localization and trafficking as well as the protein interaction networks it engages. Using this approach, we discover two novel protein network components which produce widespread, yet distinct, effects on G protein-dependent signaling. While the framework is broadly applicable to proximity biotin labeling datasets, it holds specific promise for further rationalizing GPCR drug discovery efforts by providing an unbiased assessment of ligand-dependent effects, and by enabling the discovery of additional network components for consideration as novel therapeutic targets.

Endosomal Chemokine Receptor Signalosomes Regulate Central Mechanisms Underlying Cell Migration

“…Another interesting signaling protein enriched by CCR7 activation includes members of the eyes absent (EYA) subfamily of proteins, EYA2. These proteins have been shown to interact with and regulate Gαi and Gαz subunits 27,28 , and EYA4, a member of the same family, was recently identified in a similar APEX2-based study of the μ-opioid receptor 29 . We also detected three novel interaction partners GRIP2, MARK4, and EI24 as being among the highest levels regardless of the activation ligand or time (Fig 5A and S5B).…”

“…This interaction prevents their translocation to the nucleus, and thus, block their role as transcription co-factors 34 . Coincidently, EYA4 was recently identified in a similar APEX2-based study of the μ-opioid receptor, and thus, might be more involved in downstream signaling responses of G i/o -coupled receptors such as chemokine receptors than currently appreciated 36 .…”

Ligand-directed labeling of opioid receptors for covalent attachment of fluorophores or small-molecule probes

“… 8 pCDNA3.1_SSFMORwt Polacco et al. 9 pOG44 Invitrogen Cat# V600520 Software and algorithms FlowJo™ v10.8.1 BD Life Sciences Fiji (ImageJ) On GitHub License GPLv3+ QCapture v2.98.2 QImaging Other Falcon TM Tissue Culture Treated Flasks, 175 cm 2 Corning Cat# 353112 Falcon TM Tissue Culture Treated Flasks, 75 cm 2 Corning Cat# 353110 Vibratome Leica Biosystems Leica VT1200S …”

ACKR3 Proximity Labeling Identifies Novel G protein- and β-arrestin-independent GPCR Interacting Proteins

“…ACKR3, as a receptor that (1) does not activate G proteins, (2) recruits β-arrestin, but (3) demonstrates functionality independent of β-arrestin, serves as a unique model system to identify noncanonical GPCR signaling partners. Prior studies have successfully demonstrated the use of proximity labeling to interrogate protein:GPCR interactions at different receptors, using different ligands, and even at different subcellular locations (38)(39)(40)(41)(42)(43). These prior studies have laid the foundation for our understanding of the GPCR "interactome".…”