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 (β2-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. Summary G 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 compartments[1][1]. While the endosome-associated receptor pool was classically presumed to be functionally inactive, it is now clear that receptors can also signal from endosomes[2][2]-[4][3]. 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 receptors[5][4], [6][5]. Since then, compartmentalized signaling has been implicated in the transduction of distinct phosphosignaling[7][6], [8][7] and in the coordination of unique physiologies and drug actions[8][7]-[17][8]. Yet, how the endosome selectively facilitates these responses compared to other subcellular compartments remains unclear. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2 [3]: #ref-4 [4]: #ref-5 [5]: #ref-6 [6]: #ref-7 [7]: #ref-8 [8]: #ref-17