Characterization of low density lipoprotein receptor ligand interactions by fluorescence resonance energy transfer.
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.
Yamamoto
Taichi
T
Lipid Biology in Health and Disease Research Group, Children's Hospital Oakland Research Institute, CA 94609, USA.
Lamoureux
Johanne
J
Ryan
Robert O
RO
D053318:Apolipoprotein E3; D001057:Apolipoproteins E / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009282:Naphthalenesulfonates / Q000737:chemistry; D011973:Receptors, LDL / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry / Q000172:deficiency
D053318:Apolipoprotein E3; D001057:Apolipoproteins E; D008024:Ligands; D009282:Naphthalenesulfonates; D011973:Receptors, LDL; C006421:1,5-I-AEDANS; D014364:Tryptophan; D004492:Edetic Acid