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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 (Kd = 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.
*
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.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 2000 by the American Society for Biochemistry and Molecular Biology.
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