Ligand Binding Properties of the Very Low Density Lipoprotein Receptor

doi:10.1074/jbc.274.13.8973

Ligand Binding Properties of the Very Low Density Lipoprotein Receptor

ABSENCE OF THE THIRD COMPLEMENT-TYPE REPEAT ENCODED BY EXON 4 IS ASSOCIATED WITH REDUCED BINDING OF M_r 40,000 RECEPTOR-ASSOCIATED PROTEIN*

From the ^‡Department of Molecular and Structural Biology, University of Aarhus, DK-8000 Aarhus C, Denmark, the ^§Departments of Cell Biology and Medicine, Baylor College of Medicine, Houston, Texas 77030, and the ^¶Friedrich-Miescher Institute, P.O. Box 2543, CH-4002 Basel, Switzerland

Abstract

The very low density lipoprotein receptor (VLDLR) binds, among other ligands, the M _r 40,000 receptor-associated protein (RAP) and a variety of serine proteinase-serpin complexes, including complexes of the proteinase urokinase-type plasminogen activator (uPA) with the serpins plasminogen activator inhibitor-1 (PAI-1) and protease nexin-1 (PN-1). We have analyzed the binding of RAP, uPA·PAI-1, and uPA·PN-1 to two naturally occurring VLDLR variants, VLDLR-I, containing all eight complement-type repeats, and VLDLR-III, lacking the third complement-type repeat, encoded by exon 4. VLDLR-III displayed ∼4-fold lower binding of RAP than VLDLR-I and ∼10-fold lower binding of the most C-terminal one of the three domains of RAP. In contrast, the binding of uPA·PAI-1 and uPA·PN-1 to the two VLDLR variants was indistinguishable. Surprisingly, uPA·PN-1, but not uPA·PAI-1, competed RAP binding to both VLDLR variants. These observations show that the third complement-type repeat plays a crucial role in maintaining the contact sites needed for optimal recognition of RAP, but does not affect the proteinase-serpin complex contact sites, and that two ligands can show full cross-competition without sharing the same contacts with the receptor. These results elucidate the mechanisms of molecular recognition of ligands by receptors of the low density lipoprotein receptor family.

Footnotes

↵* This work was supported by grants from the Danish Cancer Society, the Danish Medical Research Council, the Novo-Nordisk Foundation, and the Danish Biotechnology Program (to P. A. A.) and by National Institutes of Health Grant HL-16512 (to L. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‖ To whom correspondence should be addressed: Dept. of Molecular and Structural Biology, University of Aarhus, 10C Gustav Wieds Vej, DK-8000 Aarhus C, Denmark. Tel.: 4589425080; Fax: 4586123178; E-mail:pa{at}mbio.aau.dk.
Abbreviations:

LDLR

low density lipoprotein receptor

α₂MR/LRP

α₂-macroglobulin receptor/low density lipoprotein receptor-related protein

VLDLR

very low density lipoprotein receptor

CTR

complement-type repeat

EGF

epidermal growth factor

uPA

urokinase-type plasminogen activator

RAP

M _r40,000 receptor-associated protein

PAI-1

plasminogen activator inhibitor-1

PN-1

protease nexin-1

PCR

polymerase chain reaction

CHO

Chinese hamster ovary

bp

base pairs

TBS

Tris-buffered saline

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid

PAGE

polyacrylamide gel electrophoresis

PVDF

polyvinylidene difluoride

B/F

bound/free
- Received April 23, 1998.
- Revision received January 15, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.