Recombinant toxins that bind to the urokinase receptor are cytotoxic without requiring binding to the α2-macroglobulin receptor

Abstract

The α2-macroglobulin receptor (α2MR) 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 α2MR, 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 α2MR. 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 α2MR from other proteins, or by incubation with phorbol myristate acetate, which is known to decrease the number of α2MRs in U937 cells or by antibodies to α2MR. Therefore, these recombinant toxins appear to internalize via uPAR without association with the α2MR.