High-density lipoprotein 3 receptor-dependent endocytosis pathway in a human hepatoma cell line (HepG(2))

The internalization of HDL(3) into HepG(2) cells at 37 degrees C was precisely measured, taking advantage of the previously observed rapid dissociation of HDL(3) from its two binding sites [Barbaras, R., et al. (1994) Biochemistry 33, 2335-2340]. We observed a high level of HDL(3) internalization (100 ng/mg of cell protein, corresponding to 45.5% of the total HDL(3) associated to the cells at 37 degrees C) reaching a plateau at 15 min. Apolipoprotein A-I (the main HDL(3) apolipoprotein) associated with dimyristoylphosphatidylcholine (DMPC) complexes was also internalized by HepG(2) cells, at levels comparable to those obtained with HDL(3) lipid-free apolipoprotein A-I, which can bind only to the HDL(3) 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 HDL(3) 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 HDL(3) internalization, as also observed with LDL. Altogether, these observations clearly demonstrate that HDL(3) internalization into HepG(2) 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.