Profiling the proximal proteome of the activated -opioid receptor

The mu-opioid receptor (mu OR) represents an important target of therapeutic and abused drugs. So far, most understanding of mu OR activity has focused on a subset of known signal transducers and regulatory molecules. Yet mu OR signaling is coordinated by additional proteins in the interaction network of the activated receptor, which have largely remained invisible given the lack of technologies to interrogate these networks systematically. Here we describe a proteomics and computational approach to map the proximal proteome of the activated mu OR and to extract subcellular location, trafficking and functional partners of G-protein-coupled receptor (GPCR) activity. We demonstrate that distinct opioid agonists exert differences in the mu OR proximal proteome mediated by endocytosis and endosomal sorting. Moreover, we identify two new mu OR network components, EYA4 and KCTD12, which are recruited on the basis of receptor-triggered G-protein activation and might form a previously unrecognized buffering system for G-protein activity broadly modulating cellular GPCR signaling. A proteomics and computational approach was developed to map the proximal proteome of the activated mu-opioid receptor and to extract subcellular location, trafficking and functional partners of G-protein-coupled receptor activity.