The μ-opioid receptor (μOR) represents an important target of therapeutic and abused drugs. So far, most understanding of μOR activity has focused on a subset of known signal transducers and regulatory molecules. Yet μOR 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 μOR 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 μOR proximal proteome mediated by endocytosis and endosomal sorting. Moreover, we identify two new μOR 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.

μ-阿片受体（μOR）是治疗药物和滥用药物的重要靶点。到目前为止，对 μOR 活性的大多数理解都集中在已知信号转导器和调节分子的子集上。然而，μOR 信号传导是由激活受体相互作用网络中的其他蛋白质协调的，由于缺乏系统地询问这些网络的技术，这些蛋白质在很大程度上仍然是不可见的。在这里，我们描述了一种蛋白质组学和计算方法，用于绘制激活 μOR 的近端蛋白质组图，并提取 G 蛋白偶联受体 (GPCR) 活性的亚细胞位置、运输和功能伙伴。我们证明不同的阿片类激动剂在胞吞作用和内体分选介导的μOR近端蛋白质组中产生差异。此外，我们还发现了两个新的 μOR 网络组件 EYA4 和 KCTD12，它们是在受体触发的 G 蛋白激活的基础上招募的，可能形成一个以前未被识别的缓冲系统，用于广泛调节细胞 GPCR 信号传导的 G 蛋白活性。