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-Adrenergic Receptor Stimulation Promotes G
s Internalization through Lipid Rafts: A Study in Living Cells
Departments of Physiology & Biophysics (J.A.A., J.Z.Y., R.J.D., M.M.R.) and Psychiatry (M.M.R), University of Illinois at Chicago, College of Medicine, Chicago, Illinois
Abstract
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 G
s, we developed a functional Gs-green fluorescent protein (GFP) fusion protein that can be visualized in living cells during signal transduction. C6 and MCF-7 cells expressing Gs-GFP were treated with 10 µM isoproterenol, and trafficking was assessed with fluorescence microscopy. Upon isoproterenol stimulation, Gs-GFP was removed from the plasma membrane and internalized into vesicles. Vesicles containing Gs-GFP did not colocalize with markers for early endosomes or late endosomes/lysosomes, revealing that Gs does not traffic through common endocytic pathways. Furthermore, Gs-GFP did not colocalize with internalized 
2-adrenergic receptors, suggesting that Gs and receptors are removed from the plasma membrane by distinct endocytic pathways. Nonetheless, activated Gs-GFP did colocalize in vesicles labeled with fluorescent cholera toxin B, a lipid raft marker. Agonist significantly increased Gs protein in Triton X-100 –insoluble membrane fractions, suggesting that Gs moves into lipid rafts/caveolae after activation. Disruption of rafts/caveolae by treatment with cyclodextrin prevented agonist-induced internalization of Gs-GFP, as did overexpression of a dominant-negative dynamin. Taken together, these results suggest that receptor-activated Gs moves into lipid rafts and is internalized from these membrane microdomains. It is suggested that agonist-induced internalization of Gs plays a specific role in G protein-coupled receptor-mediated signaling and could enable Gs to traffic into the cellular interior to regulate effectors at multiple cellular sites.
Address correspondence to: Dr. Mark M. Rasenick, Department of Physiology and Biophysics (MC 901), College of Medicine, University of Illinois at Chicago (UIC), 835 South Wolcott Avenue, Chicago, IL 60612–7342. E-mail: raz{at}uic.edu
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