A spatiotemporally resolved GPCR interactome reveals novel mediators of receptor agonism

Cellular signaling by membrane G protein-coupled receptors (GPCRs) is orchestrated by a complex and diverse array of mechanisms. The dynamics of a GPCR interactome as it evolves over time and space in response to an agonist can offer a unique window on pleiotropic signaling decoding and functional selectivity at a cellular level. In this study, we employed proximity-based APEX2 proteomics to interrogate the interaction network of the GPCR for luteinizing hormone (LHR) on a minute-to-minute timescale. We developed an analytical approach integrating quantitative multiplexed proteomics and temporal reference profiles, providing a platform to identify the proteomic environment of APEX2-tagged LHR at the nanometer scale. LHR activity is exquisitely regulated at a spatial level, leading to identification of novel putative interactors including the Ras-related GTPase RAP2B that modulate both receptor signaling and post-endocytic trafficking, and providing a resource for spatiotemporal nanodomain mapping of LHR interactors across subcellular compartments. SignificanceG-protein coupled receptors (GPCRs) are the largest family of membrane proteins in the human body. GPCRs have established themselves as key drug targets, playing central roles in health and disease. The activity of this superfamily of receptors is highly dynamic, integrating diverse signaling pathways over time and across multiple subcellular compartments. Quantitative proximity proteomics using engineered ascorbate peroxidase (APEX2) has emerged as a powerful tool to map GPCR-protein interaction landscapes in both time and space. However, resolving these networks at a sub-minute timescale remains challenging, particularly for poorly characterized GPCRs with limited pharmacological tools or complex trafficking patterns. In this study, we applied APEX2 proximity proteomics to resolve at nanoscale the agonist (LH)-induced interactome of the luteinizing hormone receptor (LHR), a key GPCR in human reproduction. LHR undergoes rapid endocytosis upon LH stimulation, trafficking to a poorly understood compartment termed the very early endosome (VEE), where it engages in G protein signaling and post-endocytic sorting. Harnessing the temporal profile of GIPC1 protein, a key known interactor of LHR in the VEE pathway, we developed an analytical pipeline to identify novel endocytic interactors of LHR. Using our platform, we identified RAP2B and RAB38 as potential modulators of LHR signaling and trafficking. Overall, we present an APEX2 pipeline optimized for interrogating complex datasets, particularly for GPCRs, and potentially other membrane receptors, with limited known interactome data and minimal pharmacological tools. Our findings highlight both the complexity of GPCR interaction networks and an effective strategy to deconvolute them, providing a comprehensive resource to advance understanding of this large family of receptors. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/599010v2_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@13badf7org.highwire.dtl.DTLVardef@11a5182org.highwire.dtl.DTLVardef@dd5453org.highwire.dtl.DTLVardef@cb2c27_HPS_FORMAT_FIGEXP M_FIG C_FIG