G-protein-coupled receptors (GPCRs) can initiate unique functional responses depending on the subcellular site of activation. Efforts to uncover the mechanistic basis of compartmentalized GPCR signaling have concentrated on the biochemical aspect of this regulation. Here we assess the biophysical positioning of receptor-containing endosomes as an alternative salient mechanism. We devise a strategy to rapidly and selectively redistribute receptor-containing 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/cyclic AMP (cAMP)-dependent transcriptional signaling with single-cell resolution. With these, we establish that disruption of native endosome positioning inhibits the initiation of the endosome-dependent transcriptional responses. Finally, we demonstrate a prominent mechanistic role of PDE-mediated cAMP hydrolysis and local protein kinase A activity in this process. Our study, therefore, illuminates a new mechanism regulating GPCR function by identifying endosome positioning as the principal mediator of spatially selective receptor signaling.

G 蛋白偶联受体 (GPCR) 可以根据激活的亚细胞位点启动独特的功能反应。揭示区室化 GPCR 信号传导机制基础的努力集中在该调节的生化方面。在这里，我们评估了含有受体的内体作为替代显着机制的生物物理定位。我们设计了一种策略，可以在完整细胞中“按命令”快速、选择性地重新分配含有受体的内体，而不扰乱其生化组成。接下来，我们提出了两种互补的光学读数，能够以单细胞分辨率对批量和基因特异性 GPCR/环 AMP (cAMP) 依赖性转录信号进行稳健测量。通过这些，我们确定天然内体定位的破坏抑制了内体依赖性转录反应的启动。最后，我们证明了 PDE 介导的 cAMP 水解和局部蛋白激酶 A 活性在此过程中的显着机制作用。因此，我们的研究通过识别内体定位作为空间选择性受体信号传导的主要介质，阐明了调节 GPCR 功能的新机制。