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Title
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Identification of the minimal functional unit in the low density lipoprotein receptor-related protein for binding the receptor-associated protein (RAP). A conserved acidic residue in the complement-type repeats is important for recognition of RAP.
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Authors
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O.M.Andersen,
L.L.Christensen,
P.A.Christensen,
E.S.Sørensen,
C.Jacobsen,
S.K.Moestrup,
M.Etzerodt,
H.C.Thogersen.
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Ref.
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J Biol Chem, 2000,
275,
21017-21024.
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PubMed id
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Abstract
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The low density lipoprotein receptor-related protein (LRP), a member of the low
density lipoprotein receptor family, mediates the internalization of a diverse
set of ligands. The ligand binding sites are located in different regions of
clusters consisting of approximately 40 residues, cysteine-rich complement-type
repeats (CRs). The 39-40-kDa receptor-associated protein, a folding
chaperone/escort protein required for efficient transport of functional LRP to
the cell surface, is an antagonist of all identified ligands. To analyze the
multisite inhibition by RAP in ligand binding of LRP, we have used an
Escherichia coli expression system to produce fragments of the entire second
ligand binding cluster of LRP (CR3-10). By ligand affinity chromatography and
surface plasmon resonance analysis, we show that RAP binds to all two-repeat
modules except CR910. CR10 differs from other repeats in cluster II by not
containing a surface-exposed conserved acidic residue between Cys(IV) and
Cys(V). By site-directed mutagenesis and ligand competition analysis, we provide
evidence for a crucial importance of this conserved residue for RAP binding. We
provide experimental evidence showing that two adjacent complement-type repeats,
both containing a conserved acidic residue, represent a minimal unit required
for efficient binding to RAP.
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