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

Area of Science:

• Cell Biology
• Molecular Pharmacology
• Genomics

Background:

• G protein-coupled receptor (GPCR) trafficking via the endosomal-lysosomal pathway is crucial for cellular homeostasis.
• Identifying genes that regulate GPCR trafficking is complex due to the intricate nature of cellular sorting mechanisms.

Purpose of the Study:

• To develop a sensitive biosensor for monitoring GPCR expression and lysosomal trafficking.
• To conduct a genome-wide screen to identify novel genes involved in δ-opioid receptor (DOR) regulation.

Main Methods:

• Development of a high-sensitivity biosensor combining engineered peroxidase APEX2 with fluorogenic substrate Amplex UltraRed (AUR) for GPCR-lysosomal trafficking.
• Genome-wide CRISPR interference (CRISPRi) screen to identify genes regulating DOR expression and trafficking.

Main Results:

• Identification of 492 genes, including known and novel regulators, influencing DOR function.
• Discovery of DNAJC13 as a novel regulator controlling trafficking of multiple GPCRs, including DOR.
• Demonstration that DNAJC13 modulates endosomal proteome composition and homeostasis, thereby affecting GPCR trafficking.

Conclusions:

• The novel GPCR-APEX2/AUR biosensor assay is effective for large-scale genetic screens of GPCR trafficking.
• DNAJC13 plays a significant role in GPCR endosomal-lysosomal trafficking and homeostasis.
• This study expands the understanding of genetic regulators governing GPCR trafficking and cellular signaling.