Structural requirements for PCSK9-mediated degradation of the low-density lipoprotein receptor

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that controls plasma LDL cholesterol levels by posttranslational regulation of the LDL receptor (LDLR). Previously, we showed that PCSK9 binds specifically to an EGF-like repeat (EGF-A) in LDLR and reroutes the receptor from endosomes to lysosomes rather than to the cell surface. Here, we defined the regions in LDLR and PCSK9 that are required for receptor degradation and examined the relationship between PCSK9 binding and LDLR conformation. Addition of PCSK9 to cultured hepatocytes promoted degradation of WT LDLR and of receptors lacking up to four ligand binding domains, EGF-B or the clustered O-linked sugar region. In contrast, LDLRs lacking the entire ligand binding domain or the beta-propeller domain failed to be degraded, although they bound and internalized PCSK9. Using gel filtration chromatography, we assessed the effects of PCSK9 binding on an acid-dependent conformational change that happens in the extracellular domain of the LDLR. Although PCSK9 prevented the reduction in hydrodynamic radius of the receptor that occurs at a reduced pH, the effect was not sufficient for LDLR degradation. A truncated version of PCSK9 containing the prodomain and the catalytic domain, but not the C-terminal domain, bound the receptor but did not stimulate LDLR degradation. Thus, domains in both the LDLR and PCSK9 that are not required for binding (or internalization) are essential for PCSK9-mediated degradation of the LDLR.

Fig. 1.

PCSK9 interferes with an acid-dependent conformational change in the LDLR. Purified extracellular domain of LDLR (60 μg) was incubated in the presence or absence of recombinant PCSK9 (160 μg) for 2 h at room temperature. The samples were centrifuged, and the supernatants were loaded onto Superose 12 10/300 GL columns equilibrated with Buffer A. The elution patterns of LDLR and PCSK9 were determined by SDS/PAGE and immunoblotting of the collected fractions. The LDLR and PCSK9 were detected using monoclonal antibodies (HL-1 and 15A6, respectively). The elution positions of the following protein standards are shown: γ-globulin (158 kDa) and ovalbumin (44 kDa).

Fig. 2.

Structural requirements in the ligand binding domain of the LDLR for PCSK9-mediated LDLR degradation. Hepa1c1c7 cells expressing HA-tagged WT or mutant LDLRs were incubated with 2.0 μg/ml purified PCSK9 (D374Y) for 4 h. Cells were lysed in 150 μl of lysis buffer, and analyzed by SDS/PAGE (8%) and immunoblotting as described in Methods. LDLR was detected with an anti-HA polyclonal antibody (A and B) or with a monoclonal antibody, HL-1 (C). Calnexin was detected by a polyclonal antibody. Similar results were obtained in two independent experiments.

Fig. 3.

PCSK9 binding and internalization of LDLR:ΔR1-R7. (A and B). Experiments were performed as described in the legend to Fig. 2 except that antibody 3143 was used in A to detect LDLR. (C) Hepa1c1c7 cells expressing WT or mutant LDLR were treated with 30 μM monensin for 4 h. The cells were biotinylated exactly as described. Biotinylated proteins from the cell surface (pellet) and proteins from the whole cell lysate were analyzed by SDS/PAGE (8%) and immunoblotting. LDLR was detected using a polyclonal anti-HA antibody. Transferrin receptor (TfR) and Na⁺/K⁺-ATPase(α1) were detected with monoclonal antibodies and calnexin was detected with a polyclonal antibody.

Fig. 4.

Structural requirements within the EGF precursor homology domain of the LDLR for PCSK9-mediated degradation. (A–C) The experiments were performed as described in the legend to Fig. 2. (D) The experiments were performed exactly as described in the legend to Fig. 3C. Similar results were obtained in two additional independent experiments.

Fig. 5.

Interaction of the extracellular domain of LDLR with WT and mutant PCSK9. (A) Coimmunoprecipitation of the extracellular domain of LDLR and PCSK9. A total of 1 μg of full-length (amino acids 1–692) (F), N-terminal (amino acids 1–454) (N), or C-terminal (amino acids 425–692) (C) PCSK9 was incubated overnight at 4°C with the extracellular domain of LDLR (1 μg) in the presence of an anti-LDLR polyclonal antibody (4548) and protein A agarose. After centrifugation, proteins were eluted from the pellets and subjected to electrophoresis on a 4–12% NuPage Bis-Tris gradient gel. (B) Degradation of LDLR by PCSK9 in Huh-7 cells. Cells were incubated in 1 ml of DMEM containing 5% (vol/vol) newborn calf lipoprotein-poor serum, 10 μg/ml cholesterol, 1 μg/ml 25-HC, and 5.0 μg/ml purified PCSK9 for 4 h. Cell lysates were subjected to immunoblotting using a monoclonal antibody to LDLR. PCSK9 was detected using monoclonal antibodies (15A6 or 13D3). Calnexin was detected by a polyclonal antibody.

Fig. 6.

Degradation of ¹²⁵I-labeled PCSK9 in HepG2 cells. HepG2 cells were incubated in 2 ml of medium A plus 2.5 μg/ml ¹²⁵I-labeled PCSK9 (600 cpm/ng) for 1 h. After washing, cells were incubated in medium A at 37°C. The amounts of ¹²⁵I-labeled TCA-soluble and TCA-insoluble material were determined in the cells and medium at the indicated time intervals. (B) Cells were incubated with labeled PCSK9 (1 μg) for 3 h at 4°C and then 40 min at 37°C before adding stripping buffer. After the indicated times, 10% of the protein in the cell lysate and medium was subjected to immunoblotting as described. This experiment was repeated twice and the results were similar.