Co-localization of t 2 l-Epidermal Growth Factor and Ferritin-low Density Lipoprotein in Coated Pits : A Quantitative Electron Microscopic Study in Normal Mutant Human Fibroblasts and

LOW density lipoprotein (LDL) and epidermal growth factor (EGF) bind to receptors on the surface of human fibroblasts and are internalized in coated vesicles. Each of the ligands has been studied separately by electron microscopy in human fibroblasts using ferritin-LDL as one visual probe and 1251-EGF as a second visual probe. A mutant strain of human fibroblasts (J.D.) has been described in which LDL does not localize to coated pits and hence is not internalized. Because LDL and EGF do not compete with each other for binding, in the current studies we coincubated the two ligands with normal and mutant cells to visualize their cellular fates. In normal fibroblasts ferritin-LDL and 12~I-EGF both bound preferentially to coated pits at 4°C and both ligands were internalized into endocytotic vesicles and lysosomes. Quantitative studies in normal cells showed that 75% of the coated pits and vesicles that contained 12SI-EGF also contained ferritin-LDL, indicating that both ligands enter the cell through the same endocytotic vesicles. In the LDL internalization-mutant J.D. cells, ferritin-LDL did not localize in coated pits and was not internalized, but ~2SI-EGF bound to coated pits and was internalized just as in normal fibroblasts. Low density lipoprotein (LDL) and epidermal growth factor (EGF) bind to specific receptors on the surface of human fibroblasts and are subsequently internalized by receptor-mediated endocytosis. When morphologic probes of the two ligands are studied separately in the form of ferritin-LDL (1-3) I~5I-LDL (4), or ~25I-EGF (5) at 4°C, both ligands localize preferentially to coated pits on the cell surface, and at 37°C both are internalized by the cells. By contrast, a mutant strain of fibroblasts (J.D.) binds LDL, but the ligand is not internalized because its receptors are not localized in coated pits (4, 6). Since a variety of different ligands appear to be internalized by receptor-mediated endocytosis, often by coated pits (7), it is of interest to know whether these receptors function through common or separate coated regions of membrane. By fluorescence microscopy, it has been reported that a2-macroglobulin ]-HE JOURNAL OF CELL BIOLOGY VOLUME 95 OCTOBER 1982 73-77 © The Rockefe l ler Un ivers i t y Press • 0021 -9525 /82 /10 /0073 /05 $1.00 and EGF are internalized by a common endocytotic vesicle pathway (8). Electron microscopy studies have shown that a2macroglobulin and vesicular stomatitis-virus particles localize to the same coated pits and endocytotic vesicles (9). Similar observations were reported for az-macroglobulin and the lysosomal enzyme fl-galactosidase (10). Although these studies suggest that receptors for different ligands are not segregated into separate coated pits and vesicles, quantitative data on the extent of co-localization was not provided. In this study, we have taken advantage of the unique properties of the mutant J.D. fibroblasts together with the well established quantitative methodology offered by ferritin binding and 12~1 autoradiography to address three questions about the distribution of binding sites for LDL-ferritin and 125I-EGF in the same cell: (a) What is the distribution of EGF binding 73 on M ay 1, 2017 D ow nladed fom Published October 1, 1982