Beta-adrenergic receptor stimulation promotes G alpha s internalization through lipid rafts: a study in living cells.
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 alpha s, we developed a functional G alpha s-green fluorescent protein (GFP) fusion protein that can be visualized in living cells during signal transduction. C6 and MCF-7 cells expressing G alpha s-GFP were treated with 10 microM isoproterenol, and trafficking was assessed with fluorescence microscopy. Upon isoproterenol stimulation, G alpha s-GFP was removed from the plasma membrane and internalized into vesicles. Vesicles containing G alpha s-GFP did not colocalize with markers for early endosomes or late endosomes/lysosomes, revealing that G alpha s does not traffic through common endocytic pathways. Furthermore, G alpha s-GFP did not colocalize with internalized beta2-adrenergic receptors, suggesting that G alpha s and receptors are removed from the plasma membrane by distinct endocytic pathways. Nonetheless, activated G alpha s-GFP did colocalize in vesicles labeled with fluorescent cholera toxin B, a lipid raft marker. Agonist significantly increased G alpha s protein in Triton X-100 -insoluble membrane fractions, suggesting that G alpha s moves into lipid rafts/caveolae after activation. Disruption of rafts/caveolae by treatment with cyclodextrin prevented agonist-induced internalization of G alpha s-GFP, as did overexpression of a dominant-negative dynamin. Taken together, these results suggest that receptor-activated G alpha s moves into lipid rafts and is internalized from these membrane microdomains. It is suggested that agonist-induced internalization of G alpha s plays a specific role in G protein-coupled receptor-mediated signaling and could enable G alpha s to traffic into the cellular interior to regulate effectors at multiple cellular sites.
Allen
John A
JA
Department of Physiology and Biophysics, University of Illinois at Chicago (UIC), 60612-7342, USA.
Yu
Jiang Z
JZ
Donati
Robert J
RJ
Rasenick
Mark M
MM
D000818:Animals; D045744:Cell Line, Tumor; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D021962:Membrane Microdomains / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*
D011943:Receptors, Adrenergic, beta; D019205:GTP-Binding Protein alpha Subunits, Gs; D007545:Isoproterenol