LIPOPROTEIN LIPASE-MEDIATED UPTAKE OF LIPOPROTEIN IN HUMAN FIBROBLASTS - EVIDENCE FOR AN LDL RECEPTOR-INDEPENDENT INTERNALIZATION PATHWAY

Lipoprotein lipase (LPL), a key enzyme in lipoprotein triglyceride met abolism, produces a marked increase in the retention and uptake of all classes of lipoproteins by cultured cells. It was previously shown th at two different receptors are involved in mediating the LPL effects: heparan sulfate proteoglycans (HSPG) and the low density lipoprotein ( LDL) receptor-related protein/alpha(2) macroglobulin receptor (LRP). B y immunofluorescence we show here that cell sur face-bound LPL display s a pattern that corresponds to the previously described distribution of cell surface HSPG. No evident relation to the distribution of bound activated cr macroglobulin (alpha(2)M) or to LRP was observed. By im munoelectron microscopy we found that after 30 min at 37 degrees C mos t of the detected alpha(2)M (70% of the total gold particles) was ins ide the cells and associated with endosomal vesicles. However, at the same time, 76% of the LPL remained at the cell surface, suggesting tha t LPL is internalized by a slow endocytic process. Binding of triglyce ride-rich lipoproteins (TRL) or LDL together with LPL led to a spectac ular increase in bound lipoproteins, which completely colocalized with LPL. After incubation at 37 degrees C, LPL and -dioctadecyl-3,3,3',3' -tetramethylindocarbocyanine (DiI)-TRL formed large clusters on the ce ll surface. Immunofluorescence and quantitative immunoelectron microsc opy provided evidence of co-internalization of LPL and apoE-containing TRL by a slow endocytic process. In the absence of LPL, the fibroblas ts rapidly internalized DiI-LDL and showed fluorescence in central, ly sosome-like vesicles. In contrast, when LPL was present, internalizati on of DiI-LDL involved small, widely distributed vesicles. This patter n slowly changed to one consisting of large perinuclear vesicles. LDL receptor-deficient fibroblasts internalized DiI-LDL, either with or wi thout LPL, into small widely distributed vesicles and no central vesic les were seen. Chloroquine-treated normal fibroblasts internalized DiI -LDL in a pattern similar to that of receptor-deficient fibroblasts. T aken together our results suggest an alternative receptor-independent endocytosis pathway for LDL. This pathway is potentiated by LPL and is characterized by a slow uptake involving small vesicles that graduall y reach lysosomes. We suggest that, through its interaction with HSPG, LPL provides high capacity binding sites for lipoproteins and a indep endent internalization pathway.