The Lysosome Surface is an Unappreciated Hub for Vasopressin V2 Receptor Signaling

G protein-coupled receptors (GPCRs) have traditionally been understood to signal through heterotrimeric G proteins exclusively from the cell surface followed by {beta}-arrestin ({beta}arr)-mediated desensitization and receptor internalization into endosomes. However, this view has evolved significantly with growing evidence showing that some GPCRs continue to signal from endosomes after their internalization as well as from other intracellular organelles. The vasopressin V2 receptor (V2R) exemplifies this paradigm shift as it promotes robust endosomal G protein before being sorted to lysosomes for degradation. Intriguingly, recent observations suggest that the lysosomal surface itself holds a substantial pool of heterotrimeric G proteins, raising the possibility that GPCRs such as the V2R may stimulate signaling from this subcellular region. To investigate this, we here employed a NanoBiT bystander approach to track intracellular V2R trafficking and transducer activation in real-time. Our results show that activated V2R is trafficked relatively fast to lysosomes where it retains the ability to couple to both G proteins and {beta}arrs. Applying nanobody/intrabody biosensors, we further demonstrated that the V2R activates endogenous G proteins and {beta}arrs at the lysosomal surface and that inhibition of V2R translocation to endolysosomal compartments blunts its ability to stimulate G protein signaling. Together, these findings suggest that the lysosomal surface serves as an unappreciated hub for signaling by some GPCRs before they eventually are engulfed into the lysosomal lumen for degradation. One-sentence summaryUpon activation, the vasopressin V2 receptor is internalized and sorted to the lysosomal membrane, where it activates G proteins and {beta}-arrestins.