Chemical Biology Approaches for Genome‐Wide Screening of GPCR Trafficking

G protein‐coupled receptor (GPCRs) are the largest family of membrane‐bound signal transduction molecules. Many of these receptors have a complex lifecycle which includes residence at various intracellular organelles: endoplasmic reticulum, Golgi, plasma membrane, endosomes, and lysosomes. Movement of the receptor between these organelles is essential for GPCR function and mis‐trafficking can alter, blunt, or entirely disrupt GPCR signaling. Given the complexity of GPCR trafficking, we sought to develop chemical biology methods which would allow forward genetic screens to be performed in mammalian cells. Here we describe a novel approach, based on a genetically encoded peroxidase called APEX, allowing for quantitative fluorescent detection of GPCR expression. The fluorogenic substrate used in this method is membrane permeable while the fluorescent oxidation product is stably deposited into cells, allowing for subsequent single‐cell analysis and sorting. We demonstrate that APEX is protease‐labile and its activity is rapidly quenched upon trafficking of the GPCR‐APEX transgene to the lysosome. Specific chemical or genetic perturbation of GPCR trafficking to lysosomes preserves the enzymatic activity of APEX, which opens the door to forward genetic analysis. We combine this chemical biology method with genome‐wide CRISPRi screening to identify genes involved in the trafficking of the delta opioid receptor from ER (synthesis) to lysosome (degradation). Together with our previous work using APEX for high‐resolution proximity labeling, we present a comprehensive pipeline for proteomic and genomic characterization of GPCR cell biology. Support or Funding Information NIH DA043607