Localization and regulation of SR-BI in membrane rafts of HepG2 cells.
The scavenger receptor class B, type I (SR-BI) mediates cholesteryl esters (CE) selective uptake from low density lipoprotein (LDL) and high-density lipoprotein (HDL) particles. In a number of tissues expressing caveolin, SR-BI is localized in caveolae. We show using detergent-free sucrose gradients that SR-BI is found in membrane rafts devoid of caveolin-1 in the human hepatoma HepG2 cell. Perturbation of the structure of HepG2 cell membrane rafts with cholesterol oxidase or sphingomyelinase decreased LDL-CE association due to selective uptake by 60%, while HDL3-CE selective uptake was increased 2.3-fold by cholesterol oxidase but was not affected by sphingomyelinase. Sequestration of membrane cholesterol with filipin III decreased LDL-CE selective uptake by 25%, while it had no effect on HDL3-CE selective uptake. Extraction of cell membrane cholesterol with beta-cyclodextrin increased LDL- and HDL3-CE selective uptake by 1.6-fold and 3-fold, respectively. We found that CE-selective uptake from both HDL and LDL occurs by a pathway involving retro-endocytosis in HepG2 cells. An analysis of the effect of SR-BI level on the expression of critical lipid sensor and lipid binding proteins was conducted with stable transformants of HepG2 cell overexpressing SR-BI. We found that liver-type fatty acid binding protein expression level is higher in SR-BI-overexpressing cells and that caveolin-1 and sterol response element binding protein-2 levels are reduced. Thus, in this hepatic cell model, SR-BI is associated with membrane rafts devoid of caveolin and its expression affects intracellular lipid binding and lipid sensor proteins. SR-BI-dependent LDL- and HDL-CE selective uptake are affected differently by the integrity of membrane rafts, but both occur by a retroendocytic pathway in HepG2 cells.
Rhainds
David
D
Département des Sciences Biologiques, Université du Québec à Montréal, 1200 Saint-Alexandre, H3B 3H5, Canada.
Bourgeois
Philippe
P
Bourret
Geneviève
G
Huard
Karine
K
Falstrault
Louise
L
Brissette
Louise
L
D018955:CD36 Antigens; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000378:metabolism; D002788:Cholesterol Esters / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004705:Endocytosis; D050556:Fatty Acid-Binding Proteins; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D050356:Lipid Metabolism; D008074:Lipoproteins / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D011971:Receptors, Immunologic / Q000096:biosynthesis*; D051116:Receptors, Scavenger; D051122:Scavenger Receptors, Class B; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology; D047392:beta-Cyclodextrins / Q000378:metabolism
D018955:CD36 Antigens; D002352:Carrier Proteins; D002788:Cholesterol Esters; D003902:Detergents; D050556:Fatty Acid-Binding Proteins; D008074:Lipoproteins; D008077:Lipoproteins, LDL; D011971:Receptors, Immunologic; D051116:Receptors, Scavenger; C495124:SCARB1 protein, human; D051122:Scavenger Receptors, Class B; D047392:beta-Cyclodextrins; D013395:Sucrose; D002784:Cholesterol; D013108:Sphingomyelin Phosphodiesterase; C031215:betadex