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,Savvas Christoforidis

doi:10.1074/jbc.m413927200

Abstract

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.

Highlights

CD39 is a plasma membrane ecto-enzyme that regulates purinergic receptor signaling by controlling the levels of extracellular nucleotides
Using polarized MadinDarby canine kidney (MDCK) cells and caveolin-1 genedisrupted mice, we show that caveolae are not essential either for the enzymatic activity of CD39 or for its targeting to plasma membrane
We used the in vitro model system of polarized MDCK cells. When these cells are grown on polycarbonate filters they become fully polarized and contain caveolae exclusively on the basolateral side (23, 24). This system allowed us to test whether CD39 can be targeted to a membrane that lacks caveolae, and whether this enzyme remains active in the absence of these plasmalemma invaginations

Summary

Introduction

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. 3.6.1.5) is a plasma membrane ecto-nucleotidase that hydrolyzes tri- and diphosphonucleosides (including ATP and ADP) but not monophosphonucleosides It plays an important role in regulating the extracellular levels of nucleotides, thereby controlling purinergic receptor signaling (1–3). Caveolar pits are formed by polymerization of caveolin-1 with lipid-assemblies, called rafts, which are cholesterol- and glycosphingolipid-enriched microdomains (12, 13) Both caveolae and rafts depend on the presence of cholesterol, because removal of this component from membranes disrupts their structure and function (11, 12). Despite the importance of caveolae for various cellular functions, there are cells that lack these organelles In this case, it is believed that rafts take over their role in signaling. Caveolae are exclusively found basolaterally (23, 24), whereas apical rafts are responsible for formation of microvilli (25, 26)

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