Cholesterol-dependent lipid assemblies regulate the activity of the ecto-nucleotidase CD39.
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.
Papanikolaou
Agathi
A
Laboratory of Biological Chemistry, Medical School, University of Ioannina, 45110 Ioannina, Greece.
Papafotika
Alexandra
A
Murphy
Carol
C
Papamarcaki
Thomais
T
Tsolas
Orestes
O
Drab
Marek
M
Kurzchalia
Teymuras V
TV
Kasper
Michael
M
Christoforidis
Savvas
S
D000199:Actins / Q000737:chemistry; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D001081:Apyrase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism; D006224:Cricetinae; D003902:Detergents / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000378:metabolism; D005372:Filipin / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007150:Immunohistochemistry; D008055:Lipids / Q000737:chemistry*; D008168:Lung / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D009761:Nystatin / Q000494:pharmacology; D010920:Placenta / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D010974:Platelet Aggregation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013154:Spleen / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology
D000199:Actins; D015703:Antigens, CD; C495540:CAV1 protein, human; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D003902:Detergents; D008055:Lipids; D009761:Nystatin; D013395:Sucrose; D005372:Filipin; D002784:Cholesterol; D001081:Apyrase; C067869:CD39 antigen