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.
Allen
John A
JA
Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago (UIC), Chicago, IL 60612-7342, USA.
Yu
Jiang Z
JZ
Dave
Rahul H
RH
Bhatnagar
Anushree
A
Roth
Bryan L
BL
Rasenick
Mark M
MM
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D051242:Caveolin 1 / Q000502:physiology*; D045744:Cell Line, Tumor; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D055785:Gene Knockdown Techniques / Q000379:methods; D008297:Male; D021962:Membrane Microdomains / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D021381:Protein Transport / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*
D000067956:Adenylyl Cyclase Inhibitors; D051242:Caveolin 1; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases
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