Profiling the diversity of agonist-selective effects on the proximal proteome environment of G protein-coupled receptors

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. ### Competing Interest Statement The Krogan Laboratory has received research support from Vir Biotechnology and F. Hoffmann-La Roche. Nevan Krogan has consulting agreements with the Icahn School of Medicine at Mount Sinai, New York, Maze Therapeutics and Interline Therapeutics. He is a shareholder in Tenaya Therapeutics, Maze Therapeutics and Interline Therapeutics, and a financially compensated Scientific Advisory Board Member for GEn1E Lifesciences, Inc.