title: Caveolin-1 and lipid microdomains regulate Gs trafficking and attenuate Gs/adenylyl cyclase signaling.
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 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.
authors.affiliation: Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago (UIC), Chicago, IL 60612-7342, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Allen
authors.forename: Jiang Z
authors.initials: JZ
authors.lastname: Yu
authors.forename: Rahul H
authors.initials: RH
authors.lastname: Dave
authors.forename: Anushree
authors.initials: A
authors.lastname: Bhatnagar
authors.forename: Bryan L
authors.initials: BL
authors.lastname: Roth
authors.forename: Mark M
authors.initials: MM
authors.lastname: Rasenick
mesh_terms: 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*
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D051242:Caveolin 1; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases
references.citation: Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10339-43
references.pmid: 7479780
references.citation: J Biol Chem. 2002 Sep 13;277(37):34280-6
references.pmid: 12097322
references.citation: Biochemistry. 2007 Jul 3;46(26):7665-77
references.pmid: 17559193
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: Am J Physiol Heart Circ Physiol. 2006 Aug;291(2):H677-86
references.pmid: 16603689
references.citation: Proc Natl Acad Sci U S A. 1989 Oct;86(20):7900-3
references.pmid: 2554294
references.citation: J Biol Chem. 1996 Nov 15;271(46):29427-35
references.pmid: 8910609
references.citation: Biol Psychiatry. 2005 Dec 15;58(12):955-62
references.pmid: 16095566
references.citation: Biochemistry. 1994 Nov 1;33(43):12852-9
references.pmid: 7947691
references.citation: J Biol Chem. 2006 Sep 8;281(36):26391-9
references.pmid: 16818493
references.citation: J Biol Chem. 2005 Sep 2;280(35):31036-44
references.pmid: 15961389
references.citation: Nature. 1981 Dec 10;294(5841):560-2
references.pmid: 7312044
references.citation: J Biol Chem. 2000 Aug 25;275(34):26530-7
references.pmid: 10843990
references.citation: J Biol Chem. 2004 Aug 13;279(33):34614-23
references.pmid: 15190056
references.citation: J Cell Biol. 1992 Dec;119(5):1297-307
references.pmid: 1280272
references.citation: J Neurochem. 1999 Sep;73(3):1114-20
references.pmid: 10461902
references.citation: Biochem J. 1993 Jul 15;293 ( Pt 2):325-8
references.pmid: 8393657
references.citation: J Biol Chem. 2001 Jun 29;276(26):23945-53
references.pmid: 11294873
references.citation: Science. 2001 Sep 28;293(5539):2449-52
references.pmid: 11498544
references.citation: J Neurochem. 1984 Nov;43(5):1447-54
references.pmid: 6436438
references.citation: J Biol Chem. 2009 Apr 17;284(16):10462-72
references.pmid: 19237344
references.citation: J Cell Sci. 2005 Mar 15;118(Pt 6):1099-102
references.pmid: 15764592
references.citation: Mol Biol Cell. 1996 Aug;7(8):1225-33
references.pmid: 8856666
references.citation: Mol Pharmacol. 1989 Nov;36(5):803-8
references.pmid: 2511428
references.citation: J Biol Chem. 1990 Jan 25;265(3):1239-42
references.pmid: 2104835
references.citation: Physiol Rev. 2004 Oct;84(4):1341-79
references.pmid: 15383654
references.citation: Adv Cyclic Nucleotide Res. 1979;10:35-55
references.pmid: 222124
references.citation: Proc Natl Acad Sci U S A. 1995 Sep 12;92(19):8655-9
references.pmid: 7567992
references.citation: Physiology (Bethesda). 2006 Dec;21:430-9
references.pmid: 17119156
references.citation: Br J Pharmacol. 2004 Sep;143(2):235-45
references.pmid: 15289291
references.citation: Nature. 1997 Nov 6;390(6655):88-91
references.pmid: 9363896
references.citation: Structure. 2008 Jun;16(6):897-905
references.pmid: 18547522
references.citation: Mol Pharmacol. 2005 May;67(5):1493-504
references.pmid: 15703379
references.citation: Neuropsychopharmacology. 2005 Jul;30(7):1238-45
references.pmid: 15726116
references.citation: J Biol Chem. 2001 Nov 9;276(45):42063-9
references.pmid: 11533056
references.citation: Physiol Rev. 2007 Jul;87(3):965-1010
references.pmid: 17615394
references.citation: Mol Pharmacol. 1996 Oct;50(4):966-76
references.pmid: 8863843
references.citation: J Neurosci. 2009 Mar 18;29(11):3590-602
references.pmid: 19295163
references.citation: Mol Biol Cell. 2001 Mar;12(3):685-98
references.pmid: 11251080
references.citation: Annu Rev Pharmacol Toxicol. 2008;48:359-91
references.pmid: 17914930
references.citation: J Biol Chem. 2004 Oct 15;279(42):44101-12
references.pmid: 15297467
references.citation: Nat Rev Mol Cell Biol. 2005 Feb;6(2):112-26
references.pmid: 15687999
references.citation: Cell Mol Neurobiol. 2003 Jun;23(3):305-14
references.pmid: 12825829
references.citation: J Biol Chem. 2001 Oct 12;276(41):38121-38
references.pmid: 11457855
references.citation: J Biol Chem. 2008 Sep 5;283(36):24659-72
references.pmid: 18566454
references.citation: J Biol Chem. 2000 Dec 29;275(52):41447-57
references.pmid: 11006286
references.citation: J Neurosci. 2008 Mar 19;28(12):3042-50
references.pmid: 18354007
references.citation: Nat Rev Neurosci. 2007 Feb;8(2):128-40
references.pmid: 17195035
references.citation: Biochim Biophys Acta. 2008 Jun;1783(6):964-73
references.pmid: 18373982
references.citation: Mol Pharmacol. 2002 Feb;61(2):352-9
references.pmid: 11809860
references.citation: Biochemistry. 2001 Dec 18;40(50):15418-23
references.pmid: 11735426
references.citation: Mol Endocrinol. 2004 Jul;18(7):1768-77
references.pmid: 15105436