An engineered trafficking biosensor reveals a role for DNAJC13 in DOR downregulation

Trafficking of G protein-coupled receptors (GPCRs) through the endo-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 low affinity protein-receptor interactions. Here we show that the engineered enzyme APEX2 is a highly sensitive biosensor for GPCR trafficking to the lysosome, and this trafficking can be monitored through APEX-based activation of fluorogenic substrates such as Amplex UltraRed (AUR). We used GPCR-APEX2/AUR to perform a genome-wide CRISPR interference screen focused on the delta type opioid receptor (DOR), a GPCR which modulates anxiety and pain. We provide a genetic map of components in the endo-lysosomal pathway necessary for DOR trafficking to the lysosome and subsequent downregulation. Using our GPCR-APEX2 pipeline, we demonstrate that a gene identified in our screen, DNAJC13, controls trafficking of DOR to the lysosome by regulating the endosomal proteome and endosomal homeostasis. Overall design: Single guide RNA (sgRNA) enrichment within sorted cell populations based on fluorogenic reaction readout for opioid receptor abundance after receptor agonist stimulation.