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J. Biol. Chem., Vol. 280, Issue 28, 26406-26414, July 15, 2005

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Cholesterol-dependent Lipid Assemblies Regulate the Activity of the Ecto-nucleotidase CD39^*

Agathi Papanikolaou, Alexandra Papafotika, Carol Murphy, Thomais Papamarcaki, Orestes Tsolas, Marek Drab¶||, Teymuras V. Kurzchalia¶, Michael Kasper**, and Savvas Christoforidis

From the Laboratory of Biological Chemistry, Medical School, University of Ioannina, 45110 Ioannina, Greece, the Biomedical Research Institute/Foundation for Research and Technology Hellas, 45110 Ioannina, Greece, the ^¶Max Planck Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany, and the ^**Institute of Anatomy, Medical Faculty, Technical University of Dresden, Fetscher-Strasse 74, 01307 Dresden, Germany

CD39 (ecto-nucleoside triphosphate diphosphohydrolase-1; E-NTPDase1) is a plasma membrane ecto-enzyme that regulates purinergic receptor signaling by controlling the levels of extracellular nucleotides. In blood vessels this enzyme exhibits a thromboregulatory role through the control of platelet aggregation. CD39 is localized in caveolae, which are plasma membrane invaginations with distinct lipid composition, similar to dynamic lipid microdomains, called rafts. Cholesterol is enriched together with sphingolipids in both rafts and caveolae, as well as in other specialized domains of the membrane, and plays a key role in their function. Here, we examine the potential role of cholesterol-enriched domains in CD39 function. Using polarized Madin-Darby canine kidney (MDCK) cells and caveolin-1 gene-disrupted mice, we show that caveolae are not essential either for the enzymatic activity of CD39 or for its targeting to plasma membrane. On the other hand, flotation experiments using detergent-free or detergent-based approaches indicate that CD39 associates, at least in part, with distinct lipid assemblies. In the apical membrane of MDCK cells, which lacks caveolae, CD39 is localized in microvilli, which are also cholesterol and raft-dependent membrane domains. Interfering with cholesterol levels using drugs that either deplete or sequester membrane cholesterol results in a strong inhibition of the enzymatic and anti-platelet activity of CD39. The effects of cholesterol depletion are completely reversed by replenishment of membranes with pure cholesterol, but not by cholestenone. These data suggest a functional link between the localization of CD39 in cholesterol-rich domains of the membrane and its role in thromboregulation.

Received for publication, December 10, 2004 , and in revised form, April 27, 2005.

^* This work was supported by the EMBO Young Investigator Program, the European Commission (Marie Curie Individual Fellowship, HPMF-CT-1999-00360), the General Secretariat for Research and Technology, Ministry of Development, Greece, and the Pythagoras II program of the Operational Programme for Education and Initial Vocational Training, Ministry of National Education and Religious Affairs co-funded by the European Union. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Present address: Max Planck Institute for Infection Biology, 10119 Berlin, Germany.

To whom correspondence should be addressed. Tel.: 30-26510-97808; Fax: 30-26510-97868; E-mail: schristo{at}cc.uoi.gr.

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