Significant advancements in the cellular biology of G protein-coupled receptors (GPCRs) about a novel biosensor shed light on the endosomal proteome associated with the δ-opioid receptor (DOR). This study, published in Nature Chemical Biology, highlights the role of the chaperone protein DNAJC13 in the endosomal-lysosomal pathway for DOR.
The research team, led by Brandon Novy and colleagues, utilized an innovative APEX2/AUR biosensor that allows scientists to monitor the agonist-induced trafficking of DOR to the lysosome with high precision. By employing this biosensor, the research team identified 492 genes that significantly influence DOR function, with DNAJC13 emerging as a standout candidate along with WDR91 and SNX24. The findings suggest that DNAJC13 is crucial to the trafficking of DOR, facilitating its downregulation and influencing cellular signaling pathways essential for pain and anxiety regulation.
How does this new biosensor work?
The critical player is Apurinic/Apyrimidinic Endodeoxyribonuclease 2 (APEX2). APEX2 is a peroxidase enzyme that catalyzes the oxidation of specific substrates in the presence of hydrogen peroxide (H2O2). Therefore, it’s a great molecular tool for proximity labeling approaches in living cells.
In this study, the DOR receptor was genetically fused to APEX2, creating a DOR-APEX2 construct. Upon agonist activation, the DOR-APEX2 complex is internalized and trafficked to the lysosome. During this process, APEX2 can label proteins in the vicinity, allowing the capture of a snapshot of the endosomal proteome associated with the DOR receptor.
APEX2 biosensor utilizes a fluorogenic substrate called Amplex UltraRed (AUR). When AUR is oxidized by APEX2 in the presence of H2O2, it produces a fluorescent product that can be detected and quantified. This fluorescence serves as a readout for the activity of APEX2 and, by extension, the trafficking of the GPCR of interest. After the labeling reaction, the researchers purified and biotinylated proteins (those that have been labeled by APEX2) and analyzed them using quantitative proteomics such as tandem mass tags. Finally, this analysis provided insights into the molecular composition of the endosomal environment and the proteins involved in the trafficking of the DORs.
The development of molecular tools to study GPCR trafficking in real-time opens new avenues for understanding drug tolerance and resistance, particularly in the context of opioid therapies. As the opioid crisis continues to challenge public health, insights gained from this research could inform the development of safer analgesics that minimize the risk of addiction while maintaining their efficacy.
The implications of this research extend beyond basic science; understanding the role of DNAJC13 in GPCR trafficking could profoundly affect the development of therapeutic strategies for conditions related to other GPCR-mediated pathways. As the field continues to evolve, this study represents a crucial step toward unraveling the understanding of GPCR regulation and its potential to revolutionize human health.
For further details, refer to the full article: DOI: 10.1038/s41589-024-01705-2
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