Received for publication October 18, 2004.
Revised February 8, 2005.
Accepted for publication February 9, 2005.

-Adrenergic Receptor Stimulation Promotes G_s Internalization Through Lipid Rafts: A Study in Living Cells

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 Gs, we developed a functional Gs-GFP fusion protein which can be visualized in living cells during signal transduction. C6 and MCF-7 cells expressing Gs-GFP were treated with 10µM 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 receptor 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 GPCR mediated signaling, and could enable Gs to traffic into the cellular interior to activate effectors at multiple cellular sites.

Key words: Adrenergic, Gs family, cAMP, G protein regulation, Receptor synthesis/trafficking, Lipid rafts/microdomains, Sequestration/Internalization