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

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.
Competing Interests: Competing interests: M.K. is a co-scientific founder of Montara Therapeutics, serves on the scientific advisory boards of Engine Biosciences, Casma Therapeutics, Cajal Neuroscience, Alector and Montara Therapeutics and is an advisor to Modulo Bio and Recursion Therapeutics. M.K. is an inventor on US patent 11,254,933 related to CRISPRi and CRISPRa screening and on a US patent application on in vivo screening methods. The other authors declare no competing interests.
(© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)