Abstract
Gα(s) is classically known for mediating G protein-coupled receptor (GPCR) signaling at the plasma membrane (PM), but it is now established that Gα(s) also supports a second wave of signaling from internalized GPCRs within early endosomes. However, the mechanisms underlying Gα(s) trafficking remain unclear. Here, using live-cell confocal microscopy and bioluminescence resonance energy transfer (BRET) assays, we investigated Gα(s)-GFP dynamics following activation of class A (β(2)AR) and class B (V(2)R) receptors, which exhibit different level of endosomal signaling. Our findings demonstrate that Gα(s) rapidly ( < 2 min) translocates to late (Rab7) and slow recycling (Rab11) endosomes, bypassing the classical endocytic route and displaying only transient colocalization with receptors. This trafficking depends on Gα(s) activation at the PM, its release from the membrane, and an intact palmitoylation site, but occurs independently of receptor internalization. This work shed light on non-canonical route for Gα(s) endosomal trafficking, with important implications for endosomal GPCR signaling.