Regulation of the Ca2+-inhibitable adenylyl cyclase type VI by capacitative Ca2+ entry requires localization in cholesterol-rich domains.
The endogenous Ca(2+)-inhibitable adenylyl cyclase type VI of C6-2B glioma cells is regulated only by capacitative Ca(2+) entry and not by a substantial elevation of [Ca(2+)](i) from either intracellular stores or via ionophore-mediated Ca(2+) entry (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F. (1995) J. Biol. Chem. 270, 1149-1155; Fagan, K. A., Mons, N., and Cooper, D. M. F. (1998) J. Biol. Chem. 273, 9297-9305). The present studies explored the role of cholesterol-rich domains in maintaining this functional association. The cholesterol-binding agent, filipin, profoundly inhibited adenylyl cyclase activity. Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin did not affect forskolin-stimulated adenylyl cyclase activity and did not affect capacitative Ca(2+) entry. However, cholesterol depletion completely ablated the regulation of adenylyl cyclase by capacitative Ca(2+) entry. Repletion of cholesterol restored the sensitivity of adenylyl cyclase to capacitative Ca(2+) entry. Adenylyl cyclase catalytic activity and immunoreactivity were extracted into buoyant caveolar fractions with Triton X-100. The presence of adenylyl cyclase in such structures was eliminated by depletion of plasma membrane cholesterol. Altogether, these data lead us to conclude that adenylyl cyclase must occur in cholesterol-rich domains to be susceptible to regulation by capacitative Ca(2+) entry. These findings are the first indication of regulatory significance for the localization of adenylyl cyclase in caveolae.
Fagan
K A
KA
Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Smith
K E
KE
Cooper
D M
DM
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D003505:Cyclodextrins / Q000494:pharmacology; D005372:Filipin / Q000494:pharmacology; D008854:Microscopy, Electron; D017830:Octoxynol; D051381:Rats; D012995:Solubility; D014407:Tumor Cells, Cultured; D047392:beta-Cyclodextrins
D000067956:Adenylyl Cyclase Inhibitors; D015220:Calcium Channels; D003505:Cyclodextrins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D005576:Colforsin; D005372:Filipin; D017830:Octoxynol; D002784:Cholesterol; D000262:Adenylyl Cyclases; C121100:adenylyl cyclase 6; D002118:Calcium