High-density lipoprotein 3 receptor-dependent endocytosis pathway in a human hepatoma cell line (HepG2).
The internalization of HLD3 into HepG2 cells at 37 degrees C was precisely measured, taking advantage of the previously observed rapid dissociation of HDL3 from its two binding sites [Barbaras, R., et al. (1994) Biochemistry 33, 2335-2340]. We observed a high level of HDL3 internalization (100 ng/mg of cell protein, corresponding to 45.5% of the total HDL3 associated to the cells at 37 degrees C) reaching a plateau at 15 min. Apolipoprotein A-I (the main HDL3 apolipoprotein) associated with dimyristoylphosphatidylcholine (DMPC) complexes was also internalized by HepG2 cells, at levels comparable to those obtained with HDL3 lipid-free apolipoprotein A-I, which can bind only to the HDL3 high-affinity binding site, and displayed a weak internalization (5 ng internalized/mg of cell protein compared to 250 ng/mg for apolipoprotein A-I complexed with DMPC). Clathrin-coated vesicle purification following HDL3 or LDL internalization at 37 degrees C showed radioactivity associated with these vesicles, and further content analysis evidenced the presence of radiolabeled apoA-I and apoB, respectively. Treatment of the cells either by saccharose hypertonic shock or by potassium depletion, in order to block clathrin-coated vesicle formation, completely inhibited HDL3 internalization, as also observed with LDL. Altogether, these observations clearly demonstrate that HDL3 internalization into HepG2 cells occurs through an endocytosis pathway involving an interaction between apolipoprotein A-I and a cell surface protein, leading to the formation of clathrin-coated vesicles.
Garcia
A
A
INSERM U 326, Hôpital Purpan, Toulouse, France.
Barbaras
R
R
Collet
X
X
Bogyo
A
A
Chap
H
H
Perret
B
B
D016632:Apolipoprotein A-I / Q000378:metabolism; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002352:Carrier Proteins; D002966:Clathrin / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000378:metabolism*; D053440:Lipoproteins, HDL3; D011485:Protein Binding; D016601:RNA-Binding Proteins; D018110:Receptors, Lipoprotein / Q000378:metabolism*; D013696:Temperature; D014407:Tumor Cells, Cultured
D016632:Apolipoprotein A-I; D002352:Carrier Proteins; D002966:Clathrin; D008075:Lipoproteins, HDL; D053440:Lipoproteins, HDL3; D016601:RNA-Binding Proteins; D018110:Receptors, Lipoprotein; C031616:high density lipoprotein receptors; C075197:high density lipoprotein binding protein; D004134:Dimyristoylphosphatidylcholine