Abstract
Endocytosis mediated by both LDL receptors (LDLRs) and transferrin receptors (TfRs) occurs in clathrin-coated pits and requires specific tyrosine-based internalization sequences located in the cytoplasmic domain of these receptors. Internalization of these receptors is mediated by endocytic proteins that interact with the internalization domains. We previously showed that macrophage colony-stimulating factor (M-CSF) rapidly increases LDLR-dependent uptake and metabolism of LDL. To study the mechanism by which M-CSF regulates LDL uptake, we compared the effect of M-CSF on the internalization of LDL and transferrin (Tf). Our results show that M-CSF substantially increased the rate of LDLR internalization without increasing LDLR localization on the cell surface. In contrast, M-CSF treatment of macrophages rapidly increased the localization of TfR to the cell surface but did not alter the relative rate of Tf internalization. Moreover, M-CSF regulated TfR and LDLR via the activation of distinct signaling pathways. Recruitment of TfR to the cell surface was attenuated by phosphatidylinositol 3-kinase inhibitors, whereas stimulated LDL uptake was inhibited by the serine/threonine phosphatase inhibitor okadaic acid. Taken together, our results indicate that M-CSF differentially regulates receptors that undergo endocytosis and that increased LDL uptake results from a selective increase in the rate of LDLR internalization.
Highlights
Endocytosis mediated by both LDL receptors (LDLRs) and transferrin receptors (TfRs) occurs in clathrin-coated pits and requires specific tyrosine-based internalization sequences located in the cytoplasmic domain of these receptors
To determine whether macrophage colony-stimulating factor (M-CSF) induced a similar increase in cell surface localization of the low density lipoprotein receptor (LDLR), J774 macrophages were preincubated with M-CSF and subsequent ligand binding to cell surface receptors was determined at 4ЊC (Fig. 2)
M-CSF enhances the rate of LDLR internalization An increase in LDLR-mediated ligand association without a parallel increase in binding to receptors on the cell surface suggests that M-CSF increases the rate of LDLR internalization
Summary
Endocytosis mediated by both LDL receptors (LDLRs) and transferrin receptors (TfRs) occurs in clathrin-coated pits and requires specific tyrosine-based internalization sequences located in the cytoplasmic domain of these receptors. Interactions between the endocytic motif of the LDLR and clathrin adaptor proteins permit constitutive receptor internalization at a rate that is not altered by ligand binding [16,17,18,19,20]. Because measurements of total Tf association at 25ЊC include both surface and internalized ligand, we used an acid-wash protocol to remove surface-bound ligand, thereby allowing determination of internalized [125I]Tf. Incubation of cells with M-CSF for 10 min resulted in a 2.3- Ϯ 0.3-fold (n ϭ 3) increase in the amount of [125I]Tf bound to the surface of J774 macrophages (Fig. 1B).
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