Identification of domains on the 39-kDa protein that inhibit the binding of ligands to the low density lipoprotein receptor-related protein.
The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2MR) binds and internalizes several plasma proteins including tissue-type plasminogen activator (t-PA) and alpha 2-macroglobulin-protease complexes (alpha 2M*). A 39-kDa protein that copurifies with LRP/alpha 2MR inhibits the binding and uptake of ligands by LRP/alpha 2MR, including t-PA and alpha 2M*. To define domains on the 39-kDa protein which are essential for inhibition of t-PA and alpha 2M* binding to LRP/alpha 2MR, we have generated bacterial expression constructs encoding discrete regions of the 39-kDa protein as fusion proteins with glutathione S-transferase. Inhibition of t-PA and alpha 2M* binding to LRP/alpha 2MR on rat hepatoma MH1C1 cells are shown to require amino acid residues 18-24 and 100-107 on the 39-kDa protein. Inhibition of t-PA but not alpha 2M* binding to LRP/alpha 2MR is also mediated by residues 200-225 and 311-319. The same 39-kDa protein constructs that inhibit alpha 2M* and t-PA binding to MH1C1 cells are able to bind directly to purified LRP/alpha 2MR immobilized on nitrocellulose. Thus, our studies demonstrate several specific regions on the 39-kDa protein which are required for the inhibition of t-PA and alpha 2M* binding to LRP/alpha 2MR.
Warshawsky
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Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D005982:Glutathione Transferase / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008969:Molecular Sequence Data; D051381:Rats; D011971:Receptors, Immunologic / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D000511:alpha-Macroglobulins / Q000378:metabolism*
D017931:DNA Primers; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; D000511:alpha-Macroglobulins; D006493:Heparin; D005982:Glutathione Transferase; D013917:Thrombin; D010959:Tissue Plasminogen Activator