Regulation of the Ca2+-inhibitable Adenylyl Cyclase Type VI by Capacitative Ca2+ Entry Requires Localization in Cholesterol-rich Domains*

  1. Dermot M. F. Cooper
  1. From the Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262


The endogenous Ca2+-inhibitable adenylyl cyclase type VI of C6-2B glioma cells is regulated only by capacitative Ca2+ entry and not by a substantial elevation of [Ca2+]i from either intracellular stores or via ionophore-mediated Ca2+ 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-β-cyclodextrin did not affect forskolin-stimulated adenylyl cyclase activity and did not affect capacitative Ca2+entry. However, cholesterol depletion completely ablated the regulation of adenylyl cyclase by capacitative Ca2+ entry. Repletion of cholesterol restored the sensitivity of adenylyl cyclase to capacitative Ca2+ 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 Ca2+ entry. These findings are the first indication of regulatory significance for the localization of adenylyl cyclase in caveolae.

  • Abbreviations:
    capacitative Ca2+ entry
    adenylyl cyclase type VI
    intracellular Ca2+ concentration
    extracellular Ca2+ concentration, TG, thapsigargin
    inositol trisphosphate
    phosphate-buffered saline
    phosphatidylinositol bisphosphate
    epidermal growth factor
    endothelial nitric-oxide synthase
    • Received February 18, 2000.
    • Revision received May 28, 2000.
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    This Article

    1. The Journal of Biological Chemistry 275, 26530-26537.
    1. All Versions of this Article:
      1. M001369200v1
      2. 275/34/26530 (most recent)

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