Spatially encoded GPCR signaling depends on the precise subcellular positioning of receptor‐containing endosomes

Abstract

G protein‐coupled receptors (GPCRs), which comprise the largest class of drug targets, are critical signaling proteins that translate extracellular stimuli to mediate human physiology. As such, understanding GPCR signaling cascades will provide insight into the molecular processes underlying complex physiologies, how these are perturbed in disease, and inform our ability to design more efficient therapeutics. In recent years, our lab and others have made the paradigm‐shifting discovery that GPCRs can be activated after drug‐induced internalization into endosomal compartments to generate spatially biased responses. We have found that transcriptional signaling downstream of GPCR activation is initiated strictly from intracellular receptors. However, it remains unknown how the endosome can promote such selective signaling outcomes. One possibility is that the biophysical positioning of endosomal GPCRs within the cell, specifically their proximity to the nucleus, may allow for cAMP signaling cascades that result in gene transcription. To address this, I devised a strategy to rapidly redistribute endosomes in intact cells and coupled the approach with two novel microscopy‐based optical transcriptional readouts. Specifically, I developed and optimized a chemically induced heterodimerization system that fuses endosomal embedding proteins to plus‐ or minus‐end directed molecular motors and allows their relocalization ‘on command’. To determine the impact of endosomal redistribution on transcription, I utilized either accumulation of a fluorescent cAMP transcriptional reporter or single‐molecule RNA in situ hybridization of endogenous target genes. With these complementary readouts, I show that redistributing receptor‐containing endosomes toward the cell periphery significantly inhibits GPCR‐mediated gene transcription without perturbing the biochemical composition of the endosome. In an orthogonal approach, I utilized localized optogenetic adenylyl cyclases to demonstrate that cAMP produced at or proximal to the cell periphery similarly failed to induce transcription. Lastly, I uncovered that this critical role of endosomal positioning stems in part from enhanced PDE activity around the plasma membrane. This study supports a key role for endosomal positioning in the transduction of the GPCR‐initiated signal. Ultimately, this work helps dissect what makes the endosome a unique signaling compartment and illuminate the molecular mechanisms underlying spatial selectivity of GPCR signaling.