Abstract
The low density lipoprotein receptor (LDLR) is the prototype of a family of cell surface receptors involved in a wide range of biological processes. A soluble low density lipoprotein receptor (sLDLR) and a tryptophan (Trp)-deficient variant human apolipoprotein E3 (apoE3) N-terminal domain (NT) were used in binding studies. The sole cysteine in apoE3-NT was covalently modified with an extrinsic fluorescence probe, N-(iodoacetyl)-N'-(5-sulfo-1-napthyl)ethylenediamine (AEDANS), and the protein was complexed with lipid. Incubation of sLDLR with AEDANS-Trp-null apoE3-NT dimyristoylphosphatidylcholine (DMPC) disks, but not lipid-free AEDANS-apoE, induced an enhancement in AEDANS fluorescence emission intensity (excitation, 280 nm) consistent with intermolecular energy transfer from excited Trp in sLDLR to receptor-bound apoE. Ligand binding to sLDLR required calcium and was saturable. In competition binding assays, unlabeled apoE3-NT DMPC inhibited AEDANS-apoE DMPC binding to sLDLR more effectively than low density lipoprotein. Fluorescence changes in this system reflected pH-dependent ligand binding and release from sLDLR consistent with models derived from the X-ray crystal structure of the receptor at endosomal pH. Intermolecular energy transfer from excited Trp in LDLR family members to fluorescently tagged ligands represents a sensitive and convenient assay for the characterization of the myriad molecular interactions ascribed to this family of receptor.
Highlights
The low density lipoprotein receptor (LDLR) is the prototype of a family of cell surface receptors involved in a wide range of biological processes
To determine whether AEDANS fluorescence emission enhancement observed upon incubation with soluble low density lipoprotein receptor (sLDLR) is attributable to a specific binding interaction between ligand and receptor, AEDANS-Trp-null apoE3NT DMPC was incubated with BSA (Fig. 1C)
The presence of excess albumin relative to AEDANS-apolipoprotein E (apoE) had no effect on AEDANS fluorescence emission intensity, indicating that this unrelated Trp-containing protein is unable to serve as an energy donor to AEDANS-Trp-null apolipoprotein E3 (apoE3)-N-terminal domain (NT) DMPC in this system
Summary
The low density lipoprotein receptor (LDLR) is the prototype of a family of cell surface receptors involved in a wide range of biological processes. In competition binding assays, unlabeled apoE3-NT DMPC inhibited AEDANS-apoE DMPC binding to sLDLR more effectively than low density lipoprotein Fluorescence changes in this system reflected pH-dependent ligand binding and release from sLDLR consistent with models derived from the X-ray crystal structure of the receptor at endosomal pH. Binding is detected by intermolecular fluorescence resonance energy transfer between excited tryptophan (Trp) residues in a soluble low density lipoprotein receptor (sLDLR) and an extrinsic fluorophore covalently bound to a Trp-deficient protein ligand. Using this assay, apolipoprotein E (apoE) interaction with LDLR shows saturability, a requirement for calcium and ligand lipid association as well as competition by unlabeled ligand and pH-dependent ligand release. All members of this ancient receptor family, of which LDLR is the prototype, possess a modular organization minimally composed of a short intracellular domain, a single membrane-spanning sequence, epidermal growth factor (EGF) precursor homology segments, and a series of complement-
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