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.
Highlights
Introduction of LDLR Variants intoLDLRϪ/Ϫ Fibroblasts— LDLR variants were cloned into the pMX-IRES-GFP bicistronic retroviral vector [29]
We examined the role of the three histidines on receptor conformation and on lipoprotein binding, uptake, and release using LDLR variants in which the three histidines were replaced with alanine or in which the histidines were replaced with charged residues that can maintain ionic interactions at neutral pH
H190, H562, and H586 have been proposed to constitute a pH sensor that controls receptor conformation, thereby regulating lipoprotein binding and release (24 –26). To test whether these histidines control the conformational state of the LDLR, we used a baculovirus expression system to produce proteins encompassing the LDLR ectodomain of normal LDLR, of an LDLR variant in which H190, H562, and H586 were replaced with alanine, and of an LDLR variant in which the histidines were replaced with charged residues
Summary
Materials—All cell culture reagents were from Invitrogen (Carlsbad, CA). LDLRϪ/Ϫ primary human fibroblasts, human LDL, and rabbit -migrating VLDL (-VLDL) were a gift from Michael Brown and Joseph Goldstein (Department of Molecular Genetics, UT Southwestern Medical Center, Dallas, TX). Cells were incubated with 10 g/ml 125I-LDL or 5 g/ml 125I--VLDL for 1 h at 4 °C in Medium C (bicarbonate-free MEM supplemented with 20 mM HEPES pH 7.5 and 10% lipoprotein-poor serum). Cells were washed with ice-cold Buffer B (TBS ϩ 1% bovine serum albumin) and incubated with Medium D (bicarbonate-free MEM supplemented with 20 mM HEPES, 20 mM maleate, and 10% lipoprotein-poor serum) at pH 5.5, 6.0, 6.5, 7.0, or 7.5 for 30 min at 4 °C. Cells were incubated with either 10 g/ml 125I-LDL or 5 g/ml 125I--VLDL in Medium E for 30 min at 37 °C, washed with warm Medium E, and incubated for 30 min at 37 °C with Medium F (bicarbonate-free MEM supplemented with 0.45 M sucrose, 20 mM HEPES, 20 mM maleate, and 10% lipoprotein-poor serum) at pH 5.5, 6.0, 6.5, 7.0, or 7.5.
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