Regulation of the Ca-inhibitable Adenylyl Cyclase Type VI by Capacitative Ca Entry Requires Localization in Cholesterol-rich

The endogenous Ca-inhibitable adenylyl cyclase type VI of C6-2B glioma cells is regulated only by capacitative Ca entry and not by a substantial elevation of [Ca]i from either intracellular stores or via ionophoremediated Ca 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 methylb-cyclodextrin did not affect forskolin-stimulated adenylyl cyclase activity and did not affect capacitative Ca entry. However, cholesterol depletion completely ablated the regulation of adenylyl cyclase by capacitative Ca entry. Repletion of cholesterol restored the sensitivity of adenylyl cyclase to capacitative Ca 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 entry. These findings are the first indication of regulatory significance for the localization of adenylyl cyclase in caveolae.