Caveolin-1 and lipid microdomains regulate Gs trafficking and attenuate Gs/adenylyl cyclase signaling.

Lipid rafts and caveolae are specialized membrane microdomains implicated in regulating G protein-coupled receptor signaling cascades. Previous studies have suggested that rafts/caveolae may regulate beta-adrenergic receptor/Galpha(s) signaling, but underlying molecular mechanisms are largely undefined. Using a simplified model system in C6 glioma cells, this study disrupts rafts/caveolae using both pharmacological and genetic approaches to test whether caveolin-1 and lipid microdomains regulate G(s) trafficking and signaling. Lipid rafts/caveolae were disrupted in C6 cells by either short-term cholesterol chelation using methyl-beta-cyclodextrin or by stable knockdown of caveolin-1 and -2 by RNA interference. In imaging studies examining Galpha(s)-GFP during signaling, stimulation with the betaAR agonist isoproterenol resulted in internalization of Galpha(s)-GFP; however, this trafficking was blocked by methyl-beta-cyclodextrin or by caveolin knockdown. Caveolin knockdown significantly decreased Galpha(s) localization in detergent insoluble lipid raft/caveolae membrane fractions, suggesting that caveolin localizes a portion of Galpha(s) to these membrane microdomains. Methyl-beta-cyclodextrin or caveolin knockdown significantly increased isoproterenol or thyrotropin-stimulated cAMP accumulation. Furthermore, forskolin- and aluminum tetrafluoride-stimulated adenylyl cyclase activity was significantly increased by caveolin knockdown in cells or in brain membranes obtained from caveolin-1 knockout mice, indicating that caveolin attenuates signaling at the level of Galpha(s)/adenylyl cyclase and distal to GPCRs. Taken together, these results demonstrate that caveolin-1 and lipid microdomains exert a major effect on Galpha(s) trafficking and signaling. It is suggested that lipid rafts/caveolae are sites that remove Galpha(s) from membrane signaling cascades and caveolins might dampen globally Galpha(s)/adenylyl cyclase/cAMP signaling.

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.