Abstract
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 β-adrenergic receptor/Gαs 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 Gs trafficking and signaling. Lipid rafts/caveolae were disrupted in C6 cells by either short-term cholesterol chelation using methyl-β-cyclodextrin or by stable knockdown of caveolin-1 and -2 by RNA interference. In imaging studies examining Gαs-GFP during signaling, stimulation with the βAR agonist isoproterenol resulted in internalization of Gαs-GFP; however, this trafficking was blocked by methyl-β-cyclodextrin or by caveolin knockdown. Caveolin knockdown significantly decreased Gαs localization in detergent insoluble lipid raft/caveolae membrane fractions, suggesting that caveolin localizes a portion of Gαs to these membrane microdomains. Methyl-β-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 Gαs/adenylyl cyclase and distal to GPCRs. Taken together, these results demonstrate that caveolin-1 and lipid microdomains exert a major effect on Gαs trafficking and signaling. It is suggested that lipid rafts/caveolae are sites that remove Gαs from membrane signaling cascades and caveolins might dampen globally Gαs/adenylyl cyclase/cAMP signaling.
- GPCR, G protein-coupled receptor
- RNAi, RNA interference
- GFP, green fluorescent protein
- DMEM, Dulbecco's modified Eagle's medium
- TSH, thyrotropin
- TSHR, thyrotropin receptor
- DTT, dithiothreitol
- BF, buoyant fraction
- HF, heavy fraction
- PBS, phosphate-buffered saline
- PAGE, polyacrylamide gel electrophoresis
- IBMX, 3-isobutyl-1-methylxanthine
- AC VI, adenylyl cyclase type 6
- AC, adenylyl cyclase
- WT, wild-type
- AR, adrenergic receptor
- PTX, pertussis toxin.
Footnotes
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This work was supported by the National Institutes of Health National Institute of Mental Health [Grants R01-MH78200, R01-MH39595, R01-MH61887]; National Institutes of Health National Institute on Drug Abuse [Grant R21-DA20568]; National Institutes of Health National Heart, Lung, and Blood Institute [Grant T32-HL07692]; National Institutes of Health National Institute of Child Health & Human Development [Grant T32-HD040127]; and the UNC Neurodevelopmental Disorders Research Center.
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The study has been previously presented in preliminary form: Allen JA, Yu JZ, Bhatnagar A, Roth BB, and Rasenick MM (2006) Agonist induced internalization of G alpha s regulates adenylyl cyclase (Abstract). FASEB J 20: A694; and Rasenick MM (2007) Regulation of G protein signaling by cytoskeletal components and membrane microdomains. Experimental Biology; April 28–May 2, 2007; Washington DC.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.060160
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ABBREVIATIONS:
- Received August 6, 2009.
- Accepted August 18, 2009.
- © 2009 The American Society for Pharmacology and Experimental Therapeutics
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