The epidermal growth factor homology domain of the LDL receptor drives lipoprotein release through an allosteric mechanism involving H190, H562, and H586

Abstract

The low density lipoprotein (LDL) receptor (LDLR) mediates efficient endocytosis of VLDL, VLDL remnants, and LDL. As part of the endocytic process, the LDLR releases lipoproteins in endosomes. The release process correlates with an acid-dependent conformational change in the receptor from an extended, "open" state to a compact, "closed" state. The closed state has an intramolecular contact involving H190, H562, and H586. The current model for lipoprotein release holds that protonation of these histidines drives the conformational change that is associated with release. We tested the roles of H190, H562, and H586 on LDLR conformation and on lipoprotein binding, uptake, and release using variants in which the three histidines were replaced with alanine (AAA variant) or in which the histidines were replaced with charged residues that can form ionic contacts at neutral pH (DRK variant). Contrary to expectation, both the AAA and the DRK variants exhibited normal acid-dependent transitions from open to closed conformations. Despite this similarity, both the AAA and DRK mutations modulated lipoprotein release, indicating that H190, H562, and H586 act subsequent to the conformational transition. These observations also suggest that the intramolecular contact does not drive release through a competitive mechanism. In support of this possibility, mutagenesis experiments showed that beta-VLDL binding was inhibited by mutations at D203 and E208, which are exposed in the closed conformation of the LDLR. We propose that H190, H562, and H586 are part of an allosteric mechanism that drives lipoprotein release.

FIGURE 1.

Mutations at H190, H562, and H586 have little effect on LDLR ectodomain conformation. H190, H562, and H586 come together at the interface between the β-propeller region of the EGF homology domain and LA repeats 4 and 5. The left panel of A shows the orientation of the three histidines in the closed conformation of the LDLR. The right panel of A shows a model of the interface with the H190D, H562R, and H586K mutations. These mutations were designed to form two ionic contacts: one between K586 and D149, and one between R562 and D190. In panel B, gel filtration was used to determine the hydrodynamic (Stokes) radius of ectodomains from normal (WT), LDLR-AAA (AAA), and LDLR-DRK (DRK) receptors as a function of pH.

FIGURE 2.

Fibroblasts expressing normal (WT), LDLR-AAA (AAA), or LDLR-DRK (DRK) have similar total and surface receptor expression. A, cell lysates from LDLR^-/- fibroblasts that were infected with vector (Vector), normal LDLR (WT), LDLR-AAA (AAA), or LDLR-DRK (DRK) retroviruses or from normal human fibroblasts (NHF) were electrophoresed on 5–17% SDS-PAGE gels, transferred to nylon membranes and immunoblotted for the LDLR. CD44, a transmembrane protein expressed on the cell surface, was used as a loading control. B, the surface expression of LDLR was determined by flow cytometry using the C7 monoclonal antibody to the LDLR. Fluorescence values are presented as a fraction of the WT value.

FIGURE 3.

The effect of the AAA and DRK mutations on lipoprotein binding. Saturation binding of ¹²⁵I-LDL (A) and ¹²⁵I-β-VLDL (B) was performed using LDLR^-/- fibroblasts that were infected with normal LDLR (WT), LDLR-AAA (AAA), or LDLR-DRK (DRK) retroviruses. Experiments were performed in triplicate and data are presented as means ± S.D.

FIGURE 4.

The AAA mutation hinders acid-dependent lipoprotein release, while the DRK mutation potentiates β-VLDL release in vitro. Release of prebound ¹²⁵I-LDL (A and C) or ¹²⁵I-β-VLDL (B and D) from fibroblasts expressing normal LDLR (WT), LDLR-AAA (AAA), or LDLR-DRK (DRK) was measured after 30 min at 4 °C (A and B) or 37°C(C and D) in medium buffered at pH 5.5, 6.0, 6.5, 7.0, or 7.5. In the 37 °C trials, 0.45 m sucrose was included in the medium to prevent clathrin-coated pit internalization. All experiments were performed in triplicate and are reported as the mean of the fraction of cell-associated lipoprotein remaining ± S.D.

FIGURE 5.

The AAA mutation slows release. Release of prebound ¹²⁵I-LDL (A and C) or ¹²⁵I-β-VLDL (B and D) from fibroblasts expressing normal LDLR (WT), LDLR-AAA (AAA), or LDLR-DRK (DRK) in response to pH 5.5 medium was determined at 4 °C (A and B) and 37 °C (C and D) over a 16-min time course. 37 °C trials had 0.45 m sucrose present to prevent coated pit internalization. All experiments were performed in triplicate and are reported as the mean of the fraction of cell-associated lipoprotein remaining ± S.D.

FIGURE 6.

The effect of the AAA and DRK mutation on lipoprotein release in cellular assays. The ability of fibroblasts expressing normal LDLR (WT), LDLR-AAA (AAA), or LDLR-DRK (DRK) to internalize (A and C) and accumulate (B and D) LDL (A and B), and β-VLDL (C and D) was determined. Internalization assays determined the ratio of internal/surface ¹²⁵I-LDL (A) or ¹²⁵I-β-VLDL (C) over 15 min at 37 °C. The accumulation assays determined the amount of Alexa546-LDL (B) or DiI-β-VLDL (D) fluorescence associated with each cell type over 4 h. Data are presented as the percent of normal (WT) at 4 h. All experiments were performed in triplicate and are presented as means ± S.D.

FIGURE 7.

The reduction in β-VLDL internalization by the AAA and DRK mutations involves the FDNPVY sequence. A shows immunoblots of cell lysates from LDLR^-/- fibroblasts infected with retroviruses expressing no LDLR (Vector), normal LDLR (WT), LDLR-Y807C (YC), LDLR-Y807C+AAA (AC), or LDLR-Y807C+DRK (DC). B shows the internal/surface ratios of β-VLDL endocytosis at the indicated times at 37 °C. Experiments were performed in triplicate, and the data are presented as means ± S.D.

FIGURE 8.

Sequence comparisons. A compares the amino acid sequence of LA5 from 12 species. E180, D196, D200, D203, D206, and E207 are conserved in all species. E208 is conserved in 8 of the 12 B compares the amino acid sequence of human LA5 with the other six LA repeats of human LDLR. D206 E207 are conserved; E180, D196, D200, and D203 are somewhat conserved; while E187 and E208 are conserved between different repeats. Boxed residues are acidic residues that are conserved with human Residue numbering is for human LA5.

FIGURE 9.

The role of E187, D203, and E208 on lipoprotein binding. A shows the structure of LA5 in ball and stick representation. Carbon is colored dark gray; nitrogen is blue; oxygen is red; and sulfur is yellow. Calcium is shown as a light-gray sphere. E187, D203, and E208 are labeled. B shows immunoblots of cell lysates from LDLR^-/- fibroblasts that were infected with retroviruses expressing no LDLR (Vector), normal LDLR (WT), LDLR-E187K, LDLR-D203K, or LDLR-E208K. The upper portion of B shows the immunoblot for LDLR, while the lower portion shows the immunoblot for CD44, which was used as a loading control. C shows the Scatchard plot of ¹²⁵I-LDL binding to the cell surface of the indicated fibroblasts, while D shows the Scatchard plot of ¹²⁵I-β-VLDL binding.

FIGURE 10.

Model of lipoprotein release by the LDLR. In the depicted models, the LDLR is shown in blue, lipoprotein is in gray, and apolipoprotein is in red. Only the EGF homology domain and LA5 of the LDLR are depicted. In the proposed allosteric model (A), lipoproteins bind to the LDLR via E187, D203, and E208 (apoE) or via E187 (apoB100) (1st and 2nd panels of A). Upon acidification, the EGF homology domain contacts LA5 (3rd panel of A), which drives a conformational change in LA5 that involves H190, H562, and H586. The conformational change in LA5 disrupts the binding sites for apolipoproteins, thereby driving release (4th panel of A). This new model stands in contrast to the previous model (panel B). In the previous model, lipoproteins bound to the same surface as the EGF homology domain (top two panels of B). The previous model also proposed that, as lipoprotein dissociated, the EGF homology domain replaced lipoprotein via an interaction that required protonation of H190, H562, and H586 (bottom two panels of B).