Trafficking of G protein-coupled receptors (GPCRs) through the endosomal–lysosomal pathway is critical to homeostatic regulation of GPCRs following activation with agonist. Identifying the genes involved in GPCR trafficking is challenging due to the complexity of sorting operations and the large number of cellular proteins involved in the process. Here, we developed a high-sensitivity biosensor for GPCR expression and agonist-induced trafficking to the lysosome by leveraging the ability of the engineered peroxidase APEX2 to activate the fluorogenic substrate Amplex UltraRed (AUR). We used the GPCR–APEX2/AUR assay to perform a genome-wide CRISPR interference screen focused on identifying genes regulating expression and trafficking of the δ-opioid receptor (DOR). We identified 492 genes consisting of both known and new regulators of DOR function. We demonstrate that one new regulator, DNAJC13, controls trafficking of multiple GPCRs, including DOR, through the endosomal–lysosomal pathway by regulating the composition of the endosomal proteome and endosomal homeostasis.

G 蛋白偶联受体 (GPCR) 通过内体-溶酶体途径的运输对于激动剂激活后 GPCR 的稳态调节至关重要。由于分选操作的复杂性以及该过程中涉及大量细胞蛋白，识别参与 GPCR 运输的基因具有挑战性。在这里，我们利用工程过氧化物酶 APEX2 激活荧光底物 Amplex UltraRed (AUR) 的能力，开发了一种高灵敏度生物传感器，用于 GPCR 表达和激动剂诱导的溶酶体运输。我们使用 GPCR-APEX2/AUR 检测进行全基因组 CRISPR 干扰筛选，重点是识别调节 δ-阿片受体 (DOR) 表达和运输的基因。我们鉴定了 492 个由已知和新的 DOR 功能调节因子组成的基因。我们证明了一种新的调节因子DNAJC13，通过调节内体蛋白质组的组成和内体稳态，通过内体-溶酶体途径控制多种GPCR（包括DOR）的运输。