Identification of the Minimal Functional Unit in the Low Density Lipoprotein Receptor-related Protein for Binding the Receptor-associated Protein (RAP)

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 ∼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 anEscherichia 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 CysIV and CysV. 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.

[1]  Deborah Fass,et al.  Molecular basis of familial hypercholesterolaemia from structure of LDL receptor module , 1997, Nature.

[2]  B. Hyman,et al.  Expression of the Very Low‐Density Lipoprotein Receptor (VLDL‐r), an Apolipoprotein‐E Receptor, in the Central Nervous System and in Alzheimer's Disease , 1996, Journal of neuropathology and experimental neurology.

[3]  A. Schwartz,et al.  Identification of Domains on the 39‐kDa Protein That Inhibit the Binding of Ligands to the Low Density Lipoprotein Receptor–Related Protein , 1993, Annals of the New York Academy of Sciences.

[4]  D. Strickland,et al.  The 39-kDa Receptor-associated Protein Modulates Lipoprotein Catabolism by Binding to LDL Receptors (*) , 1995, The Journal of Biological Chemistry.

[5]  Peter Roepstorff,et al.  Improved resolution and very high sensitivity in MALDI TOF of matrix surfaces made by fast evaporation , 1994 .

[6]  M. Peitsch,et al.  Cloning of the TMPRSS2 gene, which encodes a novel serine protease with transmembrane, LDLRA, and SRCR domains and maps to 21q22.3. , 1997, Genomics.

[7]  D. Decamp,et al.  Three complement-type repeats of the low-density lipoprotein receptor-related protein define a common binding site for RAP, PAI-1, and lactoferrin. , 1998, Blood.

[8]  K. Goto,et al.  Human Apolipoprotein E Receptor 2 , 1996, The Journal of Biological Chemistry.

[9]  N. Daly,et al.  Three-dimensional structure of the second cysteine-rich repeat from the human low-density lipoprotein receptor. , 1995, Biochemistry.

[10]  P. Bates,et al.  Conversion of a human low-density lipoprotein receptor ligandbinding repeat to a virus receptor: identification of residues important for ligand specificity. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[11]  L. Ellgaard,et al.  Dissection of the domain architecture of the alpha2macroglobulin-receptor-associated protein. , 1997, European journal of biochemistry.

[12]  S. Moestrup,et al.  Nested sets of protein fragments and their use in epitope mapping: characterization of the epitope for the S4D5 monoclonal antibody binding to receptor associated protein. , 1995, Journal of immunological methods.

[13]  S. Moestrup,et al.  The carboxy‐terminal domain of the receptor‐associated protein binds to the Vps10p domain of sortilin , 1998, FEBS letters.

[14]  N. Tommerup,et al.  Molecular Characterization of a Novel Human Hybrid-type Receptor That Binds the α2-Macroglobulin Receptor-associated Protein* , 1996, The Journal of Biological Chemistry.

[15]  G. Bu,et al.  Receptor-associated Protein Is a Folding Chaperone for Low Density Lipoprotein Receptor-related Protein* , 1996, The Journal of Biological Chemistry.

[16]  M. Jones,et al.  Characterization and tissue-specific expression of the human 'very low density lipoprotein (VLDL) receptor' mRNA. , 1994, Human molecular genetics.

[17]  S. Moestrup,et al.  Alpha 2-macroglobulin-proteinase complexes, plasminogen activator inhibitor type-1-plasminogen activator complexes, and receptor-associated protein bind to a region of the alpha 2-macroglobulin receptor containing a cluster of eight complement-type repeats. , 1993, The Journal of biological chemistry.

[18]  S. Moestrup,et al.  The human alpha 2-macroglobulin receptor contains high affinity calcium binding sites important for receptor conformation and ligand recognition. , 1990, Journal of Biological Chemistry.

[19]  N. Daly,et al.  Disulfide bridges of a cysteine-rich repeat of the LDL receptor ligand-binding domain. , 1995, Biochemistry.

[20]  P. Gettins,et al.  NMR Solution Structure of Complement-like Repeat CR8 from the Low Density Lipoprotein Receptor-related Protein* , 1999, The Journal of Biological Chemistry.

[21]  T. Springer,et al.  An extracellular beta-propeller module predicted in lipoprotein and scavenger receptors, tyrosine kinases, epidermal growth factor precursor, and extracellular matrix components. , 1998, Journal of molecular biology.

[22]  S. Moestrup,et al.  Analysis of ligand recognition by the purified alpha 2-macroglobulin receptor (low density lipoprotein receptor-related protein). Evidence that high affinity of alpha 2-macroglobulin-proteinase complex is achieved by binding to adjacent receptors. , 1991, The Journal of biological chemistry.

[23]  G. Bu,et al.  The Domain Structure of Human Receptor-associated Protein , 1998, The Journal of Biological Chemistry.

[24]  H. Vorum,et al.  The receptor‐associated protein (RAP) binds calmodulin and is phosphorylated by calmodulin‐dependent kinase II. , 1996, The EMBO journal.

[25]  E R Levy,et al.  Isolation and characterization of LRP6, a novel member of the low density lipoprotein receptor gene family. , 1998, Biochemical and biophysical research communications.

[26]  H. Geuze,et al.  39 kDa receptor‐associated protein is an ER resident protein and molecular chaperone for LDL receptor‐related protein. , 1995, The EMBO journal.

[27]  B. Hyman,et al.  Functional domains of the very low density lipoprotein receptor: molecular analysis of ligand binding and acid-dependent ligand dissociation mechanisms. , 1999, Journal of cell science.

[28]  A. Soutar,et al.  Expression in vitro of alternatively spliced variants of the messenger RNA for human apolipoprotein E receptor-2 identified in human tissues by ribonuclease protection assays. , 1999, European journal of biochemistry.

[29]  M. Metzker,et al.  Cloning of a novel member of the low-density lipoprotein receptor family. , 1998, Gene.

[30]  G. Bu,et al.  Differential Functions of Triplicated Repeats Suggest Two Independent Roles for the Receptor-associated Protein as a Molecular Chaperone* , 1997, The Journal of Biological Chemistry.

[31]  S. Moestrup,et al.  Evidence that the newly cloned low‐density‐lipoprotein receptor related protein (LRP) is the α2‐macroglobulin receptor , 1990, FEBS letters.

[32]  D. Strickland,et al.  Primary structure of alpha 2-macroglobulin receptor-associated protein. Human homologue of a Heymann nephritis antigen. , 1991, The Journal of biological chemistry.

[33]  S. Moestrup,et al.  The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds lipoprotein lipase and beta-migrating very low density lipoprotein associated with the lipase. , 1993, The Journal of biological chemistry.

[34]  A. Schwartz,et al.  The Carboxyl-terminal Domain of Receptor-associated Protein Facilitates Proper Folding and Trafficking of the Very Low Density Lipoprotein Receptor by Interaction with the Three Amino-terminal Ligand-binding Repeats of the Receptor* , 1999, The Journal of Biological Chemistry.

[35]  D. Russell,et al.  Domain map of the LDL receptor: Sequence homology with the epidermal growth factor precursor , 1984, Cell.

[36]  D. Weaver,et al.  Molecular cloning and characterization of LR3, a novel LDL receptor family protein with mitogenic activity. , 1998, Biochemical and biophysical research communications.

[37]  A. V. van Zonneveld,et al.  Molecular Analysis of Ligand Binding to the Second Cluster of Complement-type Repeats of the Low Density Lipoprotein Receptor-related Protein , 1997, The Journal of Biological Chemistry.

[38]  O. Myklebost,et al.  Surface location and high affinity for calcium of a 500‐kd liver membrane protein closely related to the LDL‐receptor suggest a physiological role as lipoprotein receptor. , 1988, The EMBO journal.

[39]  P. Rettenberger,et al.  Ligand Binding Properties of the Very Low Density Lipoprotein Receptor , 1999, The Journal of Biological Chemistry.

[40]  M. Larsson,et al.  Cloning and sequencing of human gp330, a Ca(2+)-binding receptor with potential intracellular signaling properties. , 1996, European journal of biochemistry.

[41]  M. Johnson,et al.  Molecular Cloning of cDNA for Matriptase, a Matrix-degrading Serine Protease with Trypsin-like Activity* , 1999, The Journal of Biological Chemistry.

[42]  P. Kroon,et al.  Expression and disulfide‐bond connectivity of the second ligand‐binding repeat of the human LDL receptor , 1995, FEBS letters.

[43]  S. Ebashi,et al.  Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis. , 1984, Journal of biochemistry.

[44]  H. Pannekoek,et al.  Ligand-receptor interactions of the low density lipoprotein receptor-related protein, a multi-ligand endocytic receptor , 1998 .

[45]  D. Strickland,et al.  The 39-kDa receptor-associated protein regulates ligand binding by the very low density lipoprotein receptor. , 1994, The Journal of biological chemistry.

[46]  M T Hearn,et al.  1,1'-Carbonyldiimidazole-mediated immobilization of enzymes and affinity ligands. , 1987, Methods in enzymology.

[47]  N. Daly,et al.  Three-dimensional structure of a cysteine-rich repeat from the low-density lipoprotein receptor. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[48]  E. Hochuli,et al.  New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. , 1987, Journal of chromatography.

[49]  S. Moestrup,et al.  Purified alpha 2-macroglobulin receptor/LDL receptor-related protein binds urokinase.plasminogen activator inhibitor type-1 complex. Evidence that the alpha 2-macroglobulin receptor mediates cellular degradation of urokinase receptor-bound complexes. , 1992, The Journal of biological chemistry.

[50]  M. Llinás,et al.  Domain Organization of the 39-kDa Receptor-associated Protein* , 1999, The Journal of Biological Chemistry.