LRP6 protein regulates low density lipoprotein (LDL) receptor-mediated LDL uptake

Abstract

Genetic variations in LRP6 gene are associated with high serum LDL cholesterol levels. We have previously shown that LDL clearance in peripheral B-lymphocytes of the LRP6(R611C) mutation carriers is significantly impaired. In this study we have examined the role of wild type LRP6 (LRP6(WT)) and LRP6(R611C) in LDL receptor (LDLR)-mediated LDL uptake. LDL binding and uptake were increased when LRP6(WT) was overexpressed and modestly reduced when it was knocked down in LDLR-deficient CHO (ldlA7) cells. These findings implicated LRP6 in LDLR-independent cellular LDL binding and uptake. However, LRP6 knockdown in wild type CHO cells resulted in a much greater decline in LDL binding and uptake compared with CHO-ldlA7 cells, suggesting impaired function of the LDLR. LDLR internalization was severely diminished when LRP6 was knocked down and was restored after LRP6 was reintroduced. Further analysis revealed that LRP6(WT) forms a complex with LDLR, clathrin, and ARH and undergoes a clathrin-mediated internalization after stimulation with LDL. LDLR and LRP6 internalizations as well as LDL uptake were all impaired in CHO-k1 cells expressing LRP6(R611C). These studies identify LRP6 as a critical modulator of receptor-mediated LDL endocytosis and introduce a mechanism by which variation in LRP6 may contribute to high serum LDL levels.

FIGURE 1.

LDLR-independent binding and internalization of LDL by LRP6. CHO-ldlA7 cells were transfected with plasmids encoding HA-tagged LRP6_WT, LRP6_R611C, or with vector alone (CTL). CHO-LdlA7 cells lack LDLR (A). Cells were cultured in 5% human lipoprotein-deficient serum for 24 h followed by the addition of ¹²⁵I-LDL (10 μg/ml) for 2 h at 4 °C. For LDL uptake dil-LDL (10 μg/μml) was added to the medium at 37 °C for 2 h and analyzed by FACS. Cells expressing LRP6_WT had significantly greater LDL binding, and those expressing LRP6_R611C significantly lower LDL binding compared with controls (B). Similarly, cells expressing LRP6_WT had significantly higher LDL uptake compared with vector alone (CTL) and cells expressing LRP6_R611C (C). LRP6-specific shRNA knocked down LRP6 in CHO-ldlA7 cells by >80% (D). LRP6 knockdown by RNA interference modestly reduced LDL binding (E) and uptake (F) compared with GFP shRNA (mean ± S.E.; *, p < 0.05; **,p < 0.01; ***, p < 0.001 by analysis of variance)

FIGURE 2.

Binding and internalization of LDL by LRP6 in the presence of LDLR. CHO-k1 cells were transfected with plasmids encoding HA-tagged LRP6_WT, LRP6_R611C, or with vector (CTL) alone. LDL binding and internalization assays were carried out as described. Cells expressing LRP6_WT had significantly higher LDL binding, and those expressing LRP6_R611C showed significantly lower LDL binding compared with the vector alone (A). LRP6_WT caused higher LDL uptake compared with LRP6_R611C or vector alone (B). LRP6 knockdown by RNA interference significantly reduced LDL binding (C) and uptake (D). The decrement in LDL uptake of CHO-k1 cells was more dramatic compared with CHO-ldlA7 cells, suggesting impairment of the LDLR activity. Interaction between LRP6 and LDLR was further examined by examining the effect of the LPL on LDL uptake. LPL-induced increase in uptake of LDL in CHO-k1 cells expressing LRP6_WT was twice as high compared with vector alone (E) (mean ± S.E.; *, p < 0.05; **, p < 0.01 by analysis of variance).

FIGURE 3.

LRP6 mediates LDLR internalization. LRP6 knockdown by RNA interference significantly impaired LDLR internalization in CHO-k1 cells treated with LDL and recycling inhibitor monensin (A). This effect was rescued with transfection of LRP6_WT (B). Sucrose density gradient centrifugation in CHO-k1 cells shows that LRP6 resides predominantly in the membrane fractions containing clathrin and LDLR compared with caveoline-1 (C). LRP6_WT and LRP6_R611C form complexes with LDLR, clathrin and ARH but not IgG (used as control) M-DRM, detergent-resistant plasma membrane (D). CHO-k1 cells were transfected with plasmids containing either LRP6_WT or LRP6_R611C. Proteins from cell lysates were immunoprecipitated (IP) with either anti-HA or anti-LDLR followed by Western blotting (IB) with either anti-LDLR or anti-HA antibodies, respectively. In addition, after immunoprecipitation with anti-HA, Western blotting was carried out with anti-ARH and anti-clathrin. Immunohistochemical studies in skin fibroblasts of R611C mutation carriers and noncarriers showed colocalization of LDLR and wild type and mutant LRP6 (E). However, LDLR/LRP6 internalization was defective in the skin fibroblasts of R611C mutation carriers. In the upper corner of the right panels higher magnification of the cell surface area, shown by the arrows, are depicted for better visualization).

FIGURE 4.

Clathrin-dependent internalization of LRP6 and its impairment by R611C mutation. LRP6 on the cell surface was biotinylated and were precipitated using neutravidin-agarose. The immunoprecipitated complex was immunoblotted to assess for surface LDLR 30 and 60 min after treatment with LDL. Wild type LRP6 started to internalize 30 min after LDL was added to the medium (A). In contrast, internalization of LRP6_R611C was significantly impaired. Clathrin-specific shRNA knocked down clathrin in MEF cells by more than 90% (B). LRP6 internalization in Cav1^−/− MEFs was comparable to those of the wild type MEFs (C) but was significantly impaired in MEFs after clathrin was knocked down. Immunohistochemical studies in normal human skin fibroblasts showed significant colocalization of LRP6 with clathrin but not with caveolin 1 (D). Clathrin and LRP6 but not Cav-1 internalized in response to LDL stimulation. Immunocoprecipitation (IP) of LRP6, clathrin, and ARH over a time course of 60 min after LDL exposure were carried out. IB, immunoblot. LRP6/clathrin immunocoprecipitation peaked 30 min after stimulation with LDL (E). ARH andLRP6_WT immunocoprecipitated, but their association decreased over time after exposure to LDL (F). LRP6_R611C immunocoprecipitated with ARH, but its association with LRP6 remained unchanged over a time course of 60 min after LDL exposure, suggesting impaired endocytosis.

FIGURE 5.

R611C mutation impairs vesicular LDL uptake. CHO-k1 cells were transfected with vectors containing HA-tagged LRP6_R611C, LRP6_WT, or empty vectors. The total expression of LRP6_R611C and LRP6_WT was not significantly different (A). A pulse-chase study carried out to assess decay of the LRP6_R611C protein showed no change in its expression at specified time interval (B). There was slight reduction in membrane expression levels of LRP6_R611C compared with LRP6_WT (C). Membrane expression levels of LDLR in response to LDL in CHO-k1cells expressing LRP6_R611C and LRP6_WT were compared. LRP6_R611C significantly impaired LDLR internalization in (D). HMGCR, the key enzyme of the LDL synthesis, was expressed at significantly higher levels in cells expressing LRP6_R611C compared with LRP6_WT (E). Immunofluorescent staining of the skin fibroblasts from R611C mutation carriers and noncarriers using antibodies against clathrin and LDLR was carried out (F). LDLR and clathrin internalized after LDL stimulation in the fibroblasts of mutation noncarriers. In contrast, LDLR and, clathrin in the skin fibroblasts of the mutation carriers remained largely on the cell surface after stimulation with LDL.