Getting Up Close and Personal: What Makes an Endosome Special in GPCR Signaling

Abstract

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 assessed the biophysical positioning of receptor-containing endosomes as an alternative salient mechanism coordinating the transduction of spatially-biased responses. We focused on the beta2-adrenergic receptor (β2AR), a prototypical GPCR that signals from early endosomes via cyclic AMP (cAMP) production. We examined the role of endosome positioning in the context of gene transcription as a representative signaling readout, because endosomal β2ARs are well-known to selectively stimulate transcriptional reprogramming. First, we developed subcellular-localized cAMP and protein kinase A (PKA) sensors to enable selective quantification of endosomal GPCR-mediated activity. We also generated two complementary optical readouts that enable robust measurements of bulk- and gene-specific GPCR/cAMP-dependent transcription with single-cell resolution. We next overcame a technical challenge that has hindered the direct assessment of the functional role of endosome positioning by devising a strategy to selectively and rapidly redistribute receptor-containing endosomes ‘on command’ in intact cells without perturbing their biochemical composition. By combining these readouts with rapid endosome relocalization, we established that disruption of native endosome positioning inhibits the initiation of the endosome-dependent responses. Lastly, utilizing the cAMP and PKA sensors, we demonstrated a prominent mechanistic role of local PKA activity and phosphodiesterase (PDE)-mediated cAMP hydrolysis in this process. This dissertation work, therefore, illuminates a novel mechanism regulating GPCR function by identifying endosome positioning as the principal mediator of spatially-selective receptor signaling.