PT  - JOURNAL ARTICLE
AU  - John A. Allen
AU  - Jiang Z. Yu
AU  - Robert J. Donati
AU  - Mark M. Rasenick
TI  - β-Adrenergic Receptor Stimulation Promotes Gαs Internalization through Lipid Rafts: A Study in Living Cells
AID  - 10.1124/mol.104.008342
DP  - 2005 May 01
TA  - Molecular Pharmacology
PG  - 1493--1504
VI  - 67
IP  - 5
4099  - http://molpharm.aspetjournals.org/content/67/5/1493.short
4100  - http://molpharm.aspetjournals.org/content/67/5/1493.full
SO  - Mol Pharmacol2005 May 01; 67
AB  - 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 Gαs-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 Gαs 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.