Upon binding hormones or drugs, many G protein-coupled receptors are internalized, leading to receptor recycling, receptor desensitization, and down-regulation. Much less understood is whether heterotrimeric G proteins also undergo agonist-induced endocytosis. To investigate the intracellular trafficking of Gαs, we developed a functional Gas-green fluorescent protein (GFP) fusion protein that can be visualized in living cells during signal transduction. C6 and MCF-7 cells expressing Gαs-GFP were treated with 10 μM isoproterenol, and trafficking was assessed with fluorescence microscopy. Upon isoproterenol stimulation, Gαs-GFP was removed from the plasma membrane and internalized into vesicles. Vesicles containing Gαs-GFP did not colocalize with markers for early endosomes or late endosomes/lysosomes, revealing that Gαs does not traffic through common endocytic pathways. Furthermore, Gαs-GFP did not colocalize with internalized β2-adrenergic receptors, suggesting that Gαs and receptors are removed from the plasma membrane by distinct endocytic pathways. Nonetheless, activated Gαs-GFP did colocalize in vesicles labeled with fluorescent cholera toxin B, a lipid raft marker. Agonist significantly increased Gαs protein in Triton X-100-insoluble membrane fractions, suggesting that Gas moves into lipid rafts/caveolae after activation. Disruption of rafts/caveolae by treatment with cyclodextrin prevented agonist-induced internalization of Gαs-GFP, as did overexpression of a dominant-negative dynamin. Taken together, these results suggest that receptor-activated Gαs moves into lipid rafts and is internalized from these membrane microdomains. It is suggested that agonist-induced internalization of Gαs plays a specific role in G protein-coupled receptor-mediated signaling and could enable Gαs to traffic into the cellular interior to regulate effectors at multiple cellular sites.
ASJC Scopus subject areas
- Molecular Medicine