Fig. 1. GFD-independence of uPA chemotactic ability. (A) Schematic representation of the human urokinase structure, showing the N-terminal growth-factor-like domain (GFD, residues 1-49), the kringle domain (residues 50-131), the CP region (residues 132-158) and the catalytic domain (residues 159-411). Histidine-tagged wild-type human uPA (His-uPA), the histidine-tagged variant lacking amino acid residues 9-45 (GFa) and the untagged N-terminal 158 aminoacids (uPA 1-158) were obtained as secreted products in the P. pastoris expression system. The two peptides (CPp, residues 135-158 and GFDp, residues 12-32) employed in this analysis are also indicated. (B) Radioreceptor competition assay of ¹²⁵I-His-uPA (10⁵ cpm/sample) and the indicated nanomolar concentrations of unlabeled His-uPA or GFa to U937 cells. The results are the mean of two independent experiments performed in duplicate, error bars indicate ±s.d. (C) 50 µg of membrane extracts of U937, HEK-293, HEK-293/uPAR-12 or 2.5 µg of HEK-293/uPAR-25 were resolved on a 10% SDS-PAGE followed by western blotting with R2 anti-uPAR monoclonal antibody. (D) HEK-293, HEK-293/uPAR-12 and HEK-293/uPAR-25 cell lines were subjected to a directional migration assay in Boyden chambers. 10⁵ cells/sample were allowed to migrate for 3 hours towards 0.1 nM His-uPA or GFa or diluents. Migration in the absence of effectors or random migration was taken as 100%. Data are presented as the mean ± s.d. of three separate experiments performed in duplicate. Statistical analysis was with Student's t-test. ^*P<0.01; ^**P<0.0001 when compared with the untreated relative control cells (none). (E) HEK-293/uPAR-25 cells were assayed for directional migration towards the indicated effectors over the specified concentration range as specified above. In the combinations, a molar ratio of 1:1 was employed. The results are expressed as mean ± s.d. of three independent experiments performed in triplicate.