Abstract
The current paper describes a solid phase ligand binding assay for the low density lipoprotein (LDL) receptor that takes advantage of the domain structure of the protein. An antibody directed against one domain, e.g. the cytoplasmic tail, is adsorbed to a microtiter well. A detergent solution containing the LDL receptor is added, and the receptor is allowed to bind to the antibody. The wells are then washed, and one of the following radioiodinated ligands is added: 125I-LDL or an 125I-labeled monoclonal antibody directed against a different domain than the antibody adsorbed to the well. Under these conditions, the human LDL receptor shows high affinity for 125I-LDL and for 125I-IgG-HL1, a monoclonal antipeptide antibody directed against a 10-amino-acid "linker" between repeats 4 and 5 in the ligand binding domain. The binding affinity is the same at 4 degrees C and 37 degrees C. The binding of 125I-LDL and 125I-IgG-HL1 occurs with 1:1 molar stoichiometry, suggesting that the human LDL receptor binds 1 mol of LDL per mol of receptor. The acid-dependent dissociation of 125I-LDL and 125I-labeled monoclonal antibody from LDL receptors that is observed in intact cells was also shown to occur in the solid phase binding assay. We used the solid phase assay to demonstrate the secretion of LDL receptors from monkey cells that have been transfected with a cDNA encoding a truncated form of the human receptor that lacks the membrane-spanning domain. This assay may be useful in measuring the relative amounts of the intact LDL receptor in tissue extracts and the secreted receptor in transfected cells.
Highlights
The current paper describes a solid phase ligand ing into the cytoplasm
Theacid-dependent dissocia- can be used to study low density lipoprotein (LDL) binding activity of receptors in tion of lZ6I-LDLand '261-labeled monoclonalantibody from LDL receptors that isobserved in intact cells was shown to occur in the solid phase binding assay
We used the solid phase assay to demonstrate the secretion of LDL receptors frommonkey cells that have been transfected with a cDNA encoding a truncated form of the human receptor that lacks themembranespanning domain
Summary
The wells were washed four times with 0.25 ml of ice-cold BSA. Other protein determinations weremade by the method of buffer A containing 0.5% BSA (buffer B), and solutions containing Lowry [18]using BSA as a standard. LDL receptors (2-20 ngof purified receptor or 100-125 pg ofpartially purified proteins) were added in a volume of 0.1 ml.
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