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

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.