Subcellular compartment determines signal dominance: Endosomal cAMP overrides concurrent plasma membrane RhoA activation from a single receptor

Cells convey information not only via the identity of activated signaling pathways but also through the subcellular location where those signals originate. Whether spatially distinct signals from a single activated receptor compete to determine net cellular output has remained unclear. Here, we demonstrate this principle for a G-protein-coupled receptor (GPCR), showing that endosomal and plasma membrane signals from a single activated receptor are not merely distinct but functionally opposed, with endosomal signaling predominating to reverse the net cellular output. Using the adhesion GPCR GPR64/ADGRG2 as a model system, we show that ligand activation engages two spatially distinct G-protein pathways that are not just additive but functionally opposed: plasma membrane G ₁₃ /RhoA activation and Gs/cAMP production that persists from endosomes following receptor internalization. The endosomal cAMP pool actively overrides concurrent plasma membrane RhoA activation, resulting in net RhoA inactivation, an outcome that is completely reversed by pharmacological blockade of endocytosis or endosomal G _s inactivation. In parathyroid cells, where GPR64 is highly expressed, this endosomal signal dominance suppresses parathyroid hormone secretion independently of extracellular Ca ²⁺ levels. These findings support a model in which the subcellular compartment, rather than pathway identity, determines the dominant signal and the overall cellular response of a single activated receptor, with implications for understanding the complexity of GPCR signaling.