G protein-coupled receptors (GPCRs) are the largest family of membrane-bound signaling molecules. Activity of these receptors is critically regulated by their trafficking through the endo-lysosomal pathway. Identifying the genes involved in GPCR trafficking is challenging due the complexity of sorting operations and low affinity protein-protein interactions. Here we present a chemical biology fluorescence-based technique to interrogate GPCR trafficking. 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 this approach to perform a genome-wide CRISPR interference screen focused on the delta type opioid receptor (DOR), a GPCR which modulates anxiety, depression, and pain. The screen identified 492 genes including known- and novel-regulators of DOR expression and trafficking. We demonstrate that one of the novel genes, RME-8, localizes to early endosomes and plays a critical role in regulating DOR trafficking to the lysosome. Together, our data demonstrate that GPCR-APEX2/AUR is a flexible and highly sensitive chemical biology platform for genetic interrogation of receptor trafficking.
Membrane protein trafficking is essential for cellular function and mistrafficking can alter or disrupt membrane protein activity. Here we describe a highly sensitive chemical biology method which allows for quantitative single‐cell analysis of membrane protein expression and trafficking. This approach is based on the engineered peroxidase called APEX2 and activation of a fluorogenic substrate. When genetically fused to a membrane protein, we demonstrate that the catalytic activity of APEX2 is rapidly quenched upon trafficking to the lysosome. This characteristic allows APEX2‐based genetic screening of the preceding trafficking steps including movement through the secretory, endocytic, and endosomal pathways. We applied this approach to understand the mechanism by which the delta opioid receptor traffics to the lysosome. Using a genome‐wide CRISPR interference screen, we identified novel genes regulating the trafficking of the delta opioid receptor including receptor‐mediated endocytosis 8 (RME‐8). We show that RME‐8 plays a critical role in the post‐endocytic trafficking of the delta opioid receptor. Together, this chemical biology approach for studying membrane protein trafficking provides a rapid and highly sensitive way to interrogate the critical processes which mediate movement of membrane proteins between organelles.
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