Recombinant toxins that bind to the urokinase receptor are cytotoxic without requiring binding to the alpha(2)-macroglobulin receptor.
The alpha(2-)macroglobulin receptor (alpha(2)MR) has been reported to mediate the internalization of the urokinase plasminogen activator receptor (uPAR) via ligand binding to both receptors. To target malignant uPAR-expressing cells and to determine whether uPAR can internalize without ligand binding to alpha(2)MR, we engineered two recombinant toxins, ATF-PE38 and ATF-PE38KDEL. Each consists of the amino-terminal fragment (ATF) of human urokinase and a truncated form of Pseudomonas exotoxin (PE) devoid of domain Ia, which binds alpha(2)MR. ATF-PE38 and ATF-PE38KDEL were cytotoxic toward malignant uPAR-bearing cells, with IC(50) values as low as 0.02 ng/ml (0.3 pM). Cytotoxicity could be blocked using either recombinant urokinase or free ATF, indicating that the cytotoxicity of the recombinant toxins was specific. Radiolabeled ATF-PE38 had high affinity for uPAR (K(d) = 0.4-8 nM) on a variety of different malignant cell types and internalized at a rate similar to that of ATF. The cytotoxicity was not diminished by receptor-associated protein, which binds and shields the alpha(2)MR from other proteins, or by incubation with phorbol myristate acetate, which is known to decrease the number of alpha(2)MRs in U937 cells or by antibodies to alpha(2)MR. Therefore, these recombinant toxins appear to internalize via uPAR without association with the alpha(2)MR.
Rajagopal
V
V
Laboratory of Molecular Biology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Kreitman
R J
RJ
D036002:ADP Ribose Transferases; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D001692:Biological Transport; D004355:Drug Stability; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008213:Lymphocyte Activation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011550:Pseudomonas aeruginosa; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018675:Toxicity Tests; D014407:Tumor Cells, Cultured / Q000187:drug effects; D020298:U937 Cells; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000970:Antineoplastic Agents; D001427:Bacterial Toxins; D005098:Exotoxins; D000922:Immunotoxins; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D013755:Tetradecanoylphorbol Acetate; D014568:Urokinase-Type Plasminogen Activator; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C524898:PLAUR protein, human