A recombinant pro-urokinase derived mutant missing the growth factor-like domain does not bind to its receptor

Abstract

Pro-urokinase (pro-u-PA) is the single chain, precursor of the urokinase-type plasminogen activator u-PA. Both u-PA and pro-u-PA bind to an extracellular receptor present on the membrane of several cell types of both malignant and normal origin, including endothelial cells. Competition experiments with u-PA fragments and synthetic peptides have suggested that the growth factor-like domain (GFD) of u-PA is involved in receptor binding. To prove the direct involvement of the GFD in receptor binding, we constructed and expressed a mutant u-PA gene missing the GFD. Mutant 9Y45 has u-PA introns C and D fused together, thusdeleting exon IV that codes for the GFD (a.a. 9–45). The product of the mutant gene is a single chain, pro-u-PA derived protein with an apparent Mr of about 43kDa that, upon conversion to the two chain form, acquires full enzymatic activity, suggesting that the molecule has preserved most of its original structure. Mutant 9Y45 is recognised by an anti-human u-PA serum, by a monoclonal antibody directed against the kringle domain but not by a monoclonal raised against the GFD. N-terminal and C-terminal amino acid sequencing of the purified protein confirms that: (a) the GFD is absent; (b) the difference in MW is not due to a truncated protein; (c) the missing a.a. 9–45 are substituted by a novel tyrosine joining the last amino acid in exon III to the first amino acid of exon V. Mutant 9Y45 does not bind to the u-PA receptor as shown by its inability to compete with 125I-labelled DFP-treated u-PA for binding to the u-PA receptor of human U937 cells, even at a concentration 1000-fold higher than that of control pro-u-PA. These data prove that the most important receptor binding determinants of pro-u-PA (and u-PA) reside in the GFD.