Identification of a VLDL-induced, FDNPVY-independent internalization mechanism for the LDLR

Abstract

The low-density lipoprotein (LDL) receptor (LDLR) binds to and internalizes lipoproteins that contain apolipoproteinB100 (apoB100) or apolipoproteinE (apoE). Internalization of the apoB100 lipoprotein ligand, LDL, requires the FDNPVY(807) sequence on the LDLR cytoplasmic domain, which binds to the endocytic machinery of coated pits. We show here that inactivation of the FDNPVY(807) sequence by mutation of Y807 to cysteine prevented the uptake of LDL; however, this mutation did not prevent LDLR-dependent uptake of the apoE lipoprotein ligand, beta-VLDL. Comparison of the surface localization of the LDLR-Y807C using LDLR-immunogold, LDL-gold and beta-VLDL-gold probes revealed enrichment of LDLR-Y807C-bound beta-VLDL in coated pits, suggesting that beta-VLDL binding promoted the internalization of the LDLR-Y807C. Consistent with this possibility, treatment with monensin, which traps internalized LDLR in endosomes, resulted in the loss of surface LDLR-Y807C only when beta-VLDL was present. Reconstitution experiments in which LDLR variants were introduced into LDLR-deficient cells showed that the HIC(818) sequence is involved in beta-VLDL uptake by the LDLR-Y807C. Together, these experiments demonstrate that the LDLR has a very low-density lipoprotein (VLDL)-induced, FDNPVY-independent internalization mechanism.

Figure 1

JD fibroblasts can internalize β-VLDL but not LDL. Normal, JD, ARH^−/− and LDLR^−/− fibroblasts were cultured in lipoprotein poor media for 48 h. (A) Cells were treated with 20 μg/ml Alexa546-labeled LDL or 10 μg/ml DiI-labeled β-VLDL for 2 h at 37°C and visualized by epifluorescent microscopy. (B, C), cells were treated with 10 μg/ml Alexa546-labeled LDL (B) or 5 μg/ml DiI-labeled β-VLDL (C) for 1 h at 4°C, and then shifted to 37°C for the times indicated. Cells were washed, suspended and fixed at 4°C. Mean fluorescent uptake of lipoproteins was determined by flow cytometry and is reported as a percentage of normal cell uptake at 4 h.

Figure 2

β-VLDL-gold but not LDLR-immunogold or LDL-gold is present in the clathrin-coated pits of JD fibroblasts. Normal, JD, ARH^−/− and LDLR^−/− fibroblasts were cultured in lipoprotein-poor media for 48 h and then processed for LDLR-immunogold, LDL-gold or β-VLDL-gold surface labeling as described in Materials and methods. Arrowheads indicate coated pits. Quantification of the gold particle distributions is presented below the electron micrographs.

Figure 3

β-VLDL can drive uptake of the LDLR-Y807C of JD fibroblasts. Normal, JD and ARH^−/− fibroblasts were cultured in lipoprotein-poor media for 48 h and then treated with monensin alone for 0, 5, 15 or 60 min, or in combination with 20 μg/ml LDL for 5, 15 or 60 min, or in combination with 10 μg/ml βVLDL for 5, 15 or 60 min. Cells were then surface biotinylated and lysed. The biotinylated proteins were isolated from the whole-cell lysates by neutravidin agarose precipitation. (A) Biotinylated proteins were run on 5–17% SDS–PAGE gels, transferred to nylon membranes and immunoblotted for the presence of LDLR or for CD44. (B) The whole-cell lysates were run on 5–17% SDS–PAGE gels, transferred to nylon membranes and immunoblotted for the presence of LDLR.

Figure 4

JD fibroblasts can target β-VLDL but not LDL to late endosomes. Normal, JD, ARH^−/− and LDLR^−/− fibroblasts were cultured in lipoprotein-poor media for 48 h and then treated with LDL-gold or β-VLDL-gold for 90 min at 37°C. Arrows indicate multivesicular bodies, a morphologically distinctive late endosome.

Figure 5

The HIC⁸¹⁸ sequence is required for β-VLDL uptake by the LDLR-Y807C. LDLR^−/− fibroblasts were transfected with plasmids that direct the expression of GFP (A), GFP-LDLR (B), GFP-LDLR-W792X (C), GFP-LDLR-Y807C (D), GFP-LDLR-Y807C+E812X (E), GFP-LDLR-Y807C+I817X (F), GFP-LDLR-Y807C+G823X (G), GFP-LDLR-Y807C+R829X (H), GFP-LDLR-Y807C+EDE⁸¹⁴>AAA (I), GFP-LDLR-Y807C+EVH⁸¹⁶>AAA (J), GFP-LDLR-Y807C+HIC⁸¹⁸>AAA (K) or GFP-LDLR-Y807C+CHN⁸²⁰>AAA (L). Cells were then plated on coverslips, treated with lipoprotein poor media, incubated at 37°C with DiI-β-VLDL for 2 h and visualized for the presence of β-VLDL (red), GFP- (green) and DAPI-stained nuclei (blue). Shown below the fluorescent images is a diagram detailing the mutations and their effect on lipoprotein uptake.

Figure 6

Mutation of the HIC⁸¹⁸ sequence reduces the rate of β-VLDL uptake by the Y807C LDLR by half—LDLR^−/− fibroblasts were infected with bicistronic retroviruses encoding just GFP (vector) or the normal, Y807C, HIC⁸¹⁸>AAA or Y807C+HIC⁸¹⁸>AAA variants of the LDLR, together with GFP. Cells were sorted based upon green fluorescence to >95% homogeneity. (A) A 5 μg weight of total cell lysate from each fibroblast was run on SDS–PAGE gels, transferred to nylon membranes and immunoblotted for the LDLR or for CD44. (B, C) Cells were incubated with 10 μg/ml ¹²⁵I-β-VLDL for 1 h at 4°C, and then shifted to 37°C for 0, 5, 10 or 15 min. Internalized and surface-bound ¹²⁵I-β-VLDL were separated as described in Materials and methods, and the ratio is presented in panel B Individual values for internalized and surface-bound β-VLDL are presented in panel C Each point is the mean of four determinations. Error bars show the standard deviation. The experiment shown is representative of three independent experiments.