Class A and B GPCRs trigger rapid Gαs translocation to late and slow recycling endosomes.

Gαs is classically known for mediating G protein-coupled receptor (GPCR) signaling at the plasma membrane (PM), but it is now established that Gαs also supports a second wave of signaling from internalized GPCRs within early endosomes. However, the mechanisms underlying Gαs trafficking remain unclear. Here, using live-cell confocal microscopy and bioluminescence resonance energy transfer (BRET) assays, we investigated Gαs-GFP dynamics following activation of class A (β2AR) and class B (V2R) receptors, which exhibit different level of endosomal signaling. Our findings demonstrate that Gαs 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 Gαs 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 Gαs endosomal trafficking, with important implications for endosomal GPCR signaling.