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J Biol Chem, Vol. 275, Issue 11, 7566-7573, March 17, 2000

Recombinant Toxins That Bind to the Urokinase Receptor Are Cytotoxic without Requiring Binding to the ₂-Macroglobulin Receptor^*

Vivek Rajagopal and Robert J. Kreitman

From the Laboratory of Molecular Biology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892

The _2-macroglobulin receptor (₂MR) 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 ₂MR, 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 ₂MR. ATF-PE38 and ATF-PE38KDEL were cytotoxic toward malignant uPAR-bearing cells, with IC₅₀ 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 ₂MR from other proteins, or by incubation with phorbol myristate acetate, which is known to decrease the number of ₂MRs in U937 cells or by antibodies to ₂MR. Therefore, these recombinant toxins appear to internalize via uPAR without association with the ₂MR.

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^* 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: Laboratory of Molecular Biology, Division of Basic Sciences, NCI, National Institutes of Health, 37/4B27, 37 Convent Dr., MSC 4255, Bethesda, MD 20892. Tel.: 301-496-6947; Fax: 301-480-0843; E-mail: kreitmar@mail.nih.gov.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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