G(s) signaling is intact after disruption of lipid rafts.
Membrane microdomains enriched in cholesterol and sphingolipids modulate a number of signal transduction pathways and provide a residence for heterotrimeric G proteins, their receptors, and their effectors. We investigated whether signaling through G(s) was dependent on these membrane domains, characterized by their resistance to detergents, by depleting cells of cholesterol and sphingolipids. For cholesterol depletion, rat salivary epithelial A5 cells were cultured under low-cholesterol conditions, and then treated with the cholesterol chelator methyl-beta-cyclodextrin. For sphingolipid depletion, LY-B cells, a mutant CHO cell line that is unable to synthesize sphingolipids, were incubated under low-sphingolipid conditions. Depletion of cholesterol or sphingolipid led to a loss or decrease, respectively, in the amount of Galpha(s) from the detergent-resistant membranes without any change in the cellular or membrane-bound amounts of Galpha(s). The cAMP accumulation in response to a receptor agonist was intact and the level slightly increased in cells depleted of cholesterol or sphingolipids compared to that in control cells. These results indicate that localization of Galpha(s) to detergent-resistant membranes was not required for G(s) signaling. Analysis of the role of lipid rafts on the kinetics of protein associations in the membrane suggests that compartmentalization in lipid rafts may be more effective in inhibiting protein interactions and, depending on the pathway, ultimately inhibit or promote signaling.
Miura
Y
Y
Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Hanada
K
K
Jones
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TL
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002784:Cholesterol / Q000302:isolation & purification / Q000378:metabolism; D006224:Cricetinae; D003505:Cyclodextrins; D003902:Detergents; D004351:Drug Resistance; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; D003902:Detergents; D011943:Receptors, Adrenergic, beta; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; D019205:GTP-Binding Protein alpha Subunits, Gs