Approaches to Dissect the Role of Endosome Positioning in Spatially Encoded GPCR Signaling

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, my lab and others have made the paradigm-shifting discovery that GPCRs can be activated after drug-induced internalization from the plasma membrane into endosomal compartments to generate spatially biased responses. We have found that transcriptional responses to GPCR activation are initiated strictly from intracellular receptors. However, it remains unknown how the endosome promotes these selective signaling outcomes. One possibility is that the biophysical proximity of endosomal GPCRs to the nucleus allow for cAMP signaling cascades that result in gene transcription. To address this, I have devised a strategy to rapidly redistribute endosomes towards the cell periphery and coupled it with microscopy-based transcriptional readouts. Specifically, I have optimized a chemically induced heterodimerization system that fuses endosomal embedding proteins to plus- or minus-end directed molecular motors. To determine the impact of endosomal redistribution on transcription, an endosomal selective GPCR response, I monitor accumulation of a fluorescent cAMP transcriptional reporter and use in situ RNA hybridization to examine transcription of endogenous cAMP target genes. I will discuss recent progress made with these approaches to dissect how spatial organization of endosomes influences downstream GPCR signaling. Ultimately, this work will help dissect what makes the endosome a unique signaling compartment and illuminate the molecular mechanisms underlying spatial selectivity of GPCR signaling.