Tsvetanova, N. | bioRxiv Science

bioRxiv2022

A genome-wide CRISPR interference screen using an engineered trafficking biosensor reveals a role for RME-8 in opioid receptor regulation

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.

cell biology↗

bioRxiv2022

Endosome positioning coordinates spatially selective GPCR signaling

G protein-coupled receptors (GPCRs), a class of critical regulators of mammalian physiology, can initiate unique functional responses depending on the subcellular compartment of their activation. Yet, how endosomal receptors transduce location-biased outcomes remains poorly understood. Efforts to uncover the mechanistic basis of compartmentalized GPCR signaling have largely focused on the biochemical aspect of this regulation through dissection of the relevant factors. Here, we assess the biophysical positioning of receptor-containing endosomes as an alternative salient mechanism coordinating the transduction of spatially biased responses. We focus on the prototypical beta2-adrenergic receptor ({beta}2-AR), which preferentially mediates transcriptional reprogramming via cyclic AMP (cAMP) production from early endosomes. We overcome a technical challenge that has hindered the direct assessment of the role of endosome positioning in this paradigm by devising a strategy to selectively and rapidly redistribute endosomes on command in intact cells without perturbing their biochemical composition. Next, we present two complementary optical readouts that enable robust measurements of bulk- and gene-specific GPCR/cAMP-dependent transcription with single-cell resolution. We then combine these readouts with rapid endosome relocalization to establish that increasing endosome distance from the nucleus inhibits the initiation of the endosome-dependent response. Lastly, we demonstrate a prominent mechanistic role of phosphodiesterase (PDE)-mediated cAMP hydrolysis in this process. Our study, therefore, illuminates a novel mechanism regulating GPCR function by identifying endosome positioning as a principal mediator of spatially selective receptor signaling. SummaryG protein-coupled receptors (GPCRs) orchestrate essential aspects of mammalian physiology. GPCR function is tightly controlled by endocytic trafficking, where the ligand-activated receptor engages arrestins and clathrin machinery and is subsequently internalized into endosomal compartments1. While the endosome-associated receptor pool was classically presumed to be functionally inactive, it is now clear that receptors can also signal from endosomes2-4. Moreover, endosomal receptors can initiate cellular responses that are distinct from those activated at the plasma membrane. Transcriptional reprogramming was one of the first location-biased GPCR responses to be identified and shown to be stimulated from intracellular receptors5, 6. Since then, compartmentalized signaling has been implicated in the transduction of distinct phosphosignaling7, 8 and in the coordination of unique physiologies and drug actions8-17. Yet, how the endosome selectively facilitates these responses compared to other subcellular compartments remains unclear.

cell biology↗