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First published online 1 August 2006
doi: 10.1242/jcs.03067

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Activation of urokinase receptor by a novel interaction between the connecting peptide region of urokinase and vß5 integrin

Paola Franco^1,*, Immacolata Vocca^1,*, Maria V. Carriero², Daniela Alfano¹, Letizia Cito¹, Immacolata Longanesi-Cattani², Paolo Grieco³, Liliana Ossowski⁴ and Maria P. Stoppelli^1,

¹ Institute of Genetics and Biophysics `Adriano Buzzati-Traverso', National Research Council, Via P. Castellino 111, 80131 Naples, Italy
² Department of Experimental Oncology, National Cancer Institute of Naples, Via M. Semmola, Naples, Italy
³ Department of Pharmaceutical and Toxicological Chemistry, University Federico II, Naples, Italy
⁴ Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA

View larger version (26K): [in a new window]   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.

View larger version (20K): [in a new window]   Fig. 2. High-affinity specific binding of GFa and CPp to HEK-293 cells. (A) 125I-GFa (150,000 cpm/sample) was incubated with HEK-293 cells with increasing concentrations of unlabeled effectors for 3 hours at 4°C. After extensive washing the cell-surface-associated radioactivity was determined. The 125I-GFa-specific binding to cells in the absence of competitor was 1300 cpm. Results are presented as the mean of specific binding ± s.d. in percent. Experiments were carried out in duplicate and data shown here are representative of three independent experiments. (B) 125I-CPp (150,000 cpm/sample) was incubated with HEK-293 or HEK-293/uPAR-12 cells with increasing concentrations of the indicated unlabeled effectors for 3 hours at 4°C as described above. The 125I-CPp-specific binding to cells in the absence of competitor was 2270 cpm. Results are presented as the mean of specific binding ± s.d. in percent. Experiments were carried out in duplicate and data shown are representative of three independent experiments. (C) 50 nM uPA 1-158 was incubated with 5 µl of conditioned medium from LB6 cells expressing soluble uPAR (suPAR) for 1 hour at 37°C with or without the indicated concentrations of CPp or GFDp. Samples were crosslinked with 1 mM DSS for 15 minutes on ice and loaded onto a 12.5% SDS-PAGE. Arrows indicate the suPAR and the complex between suPAR and uPA 1-158 (suPAR/uPA 1-158).

View larger version (27K): [in a new window]   Fig. 3. Specific binding of GFa and CPp to vß5 integrin. (A) 125I-CPp was incubated with aliquots of HEK-293 cells (as described in the legend to Fig. 2B) that had been preincubated with the indicated antibody dilution for 1 hour at 4°C. When indicated, 125I-CPp was preincubated either with purified vß5 integrin (250 or 500 ng) or 1ß1 integrin (250 ng) in a total volume of 0.2 ml for 1 hour prior to radioreceptor-binding assay. The white bar (furthest right) refers to the radioactivity specifically bound to HEK-293-v cells, which had been exposed to 125I-CPp for 3 hours at 4°C. Non-specific binding was assessed by including additional duplicate samples containing unlabeled 10 nM CPp. Specific binding to HEK-293 cells was taken as 100% and the extent of inhibition by anti-integrin antibodies or by vß5 integrin or 1ß1 integrin was calculated relative to this value. Values represent the mean ± s.d. of two independent experiments performed in duplicate. *P<0.005; **P<0.0006. (B) 24-well cell culture dishes were coated with 1 mg/ml BSA or 25 µg/ml vitronectin or 100 pM GFa overnight at 4°C and extensively washed with 1x PBS. For each sample, 105 HEK-293 cells were preincubated with the indicated dilution of antibodies or non-treated (none) in a final volume of 0.3 ml for 1 hour and allowed to adhere in an 1-hour-assay at 37°C. The number of adherent cells was counted and is given as the percentage of the total cell population, representing the average of three different experiments performed in duplicate. *P<0.0001 compared with GFa. (C) 6% SDS-PAGE followed by western blotting with anti-v integrin polyclonal antibody. 15 µl of protein A-anti-uPA antibody Sepharose with (+ GFa) or without (- GFa) 5 µg of GFa was incubated for 2 hours at 25°C in a total volume of 60 µl, washed and further incubated with 500 ng of vß5 integrin. Sepharose-bound proteins were isolated by centrifugation and the supernatants (Sup) and the eluates (El) were separated by 7.5% SDS-PAGE. 100 ng of purified vß5 integrin protein was loaded as a control.

View larger version (39K): [in a new window]   Fig. 4. Cytoskeletal rearrangements in migrating HEK-293/uPAR-25 cells. (A,B) HEK-293/uPAR-25 cells were seeded onto a glass slide and allowed to migrate towards 1 nM uPA 1-158 or diluents (Control) for 4 hours in a Dunn chamber (Allen et al., 1998). At the end of the incubation, cells were stained with Rhodamine-phalloidin, observed under an inverted fluorescence microscope and analysed as described in Materials and Methods. Original magnification, x400.

View larger version (29K): [in a new window]   Fig. 5. Cytoskeletal rearrangements in HEK-293/uPAR-25 cells exposed to GFDp and/or CPp. Cells were collected by a mild tryspinization, incubated in suspension with diluents or His-uPA or GFDp or CPp at the indicated concentrations for 1 hour at 23°C. In the combinations, a molar ratio of 1:1 CPp:GFDp was employed. When specified, cells were incubated with diluents (none), or preloaded with 50 µg/ml RGD peptide, 5 µg/ml anti-uPAR 399 polyclonal or with the specific anti-integrin antibodies at a dilution of 1:30 for 1 hour. F-actin was detected with Rhodamine-phalloidin; uPAR was detected with anti-uPAR 399 polyclonal antibody followed by a secondary FITC-conjugated anti-rabbit antibody, as specified in Materials and Methods. (A) A representative confocal image of GFDp-treated HEK-293/uPAR-25 cells double-stained with Rhodamine-phalloidin and anti-uPAR antibodies. Original magnification, x630. (B,C) Values reported on the y-axis correspond to the net percentage of cells exhibiting F-actin-enriched protrusions upon exposure to the indicated effectors. Data represent the mean of three independent experiments ± s.d. (error bars), performed in triplicate and evaluated by two independent observers. *P<0.005; **P<0.0001.

View larger version (20K): [in a new window]   Fig. 6. Combined effects of individual uPA domains leading to enhancement of cell migration and association of uPAR with vß5 integrin. (A,B) HEK-293/uPAR-25 cells were allowed to migrate towards (A) increasing concentrations of the indicated effectors for 3 hours or (B) 0.1 nM of the effectors for 15, 30, 60 and 120 minutes (B). In the combinations, a molar ratio of 1:1 was employed. When indicated, CPp or uPA 1-158 were preincubated with 100 ng of purified vß5 integrin in 0.2 ml of 0.1% BSA in DMEM for 1 hour at 37°C. In all cases, cells were assayed for directional migration in Boyden chambers as specified in the legend to Fig. 1. The results are expressed as mean ± s.d. of three independent experiments performed in triplicate. (C) Lysates (400 µg/sample) of HEK-293/uPAR-25 cells exposed for 60 minutes to the indicated peptides or diluents (NT), were immunoprecipitated with 5 µg/ml VNR147 anti-v integrin monoclonal antibody. 1 µg of anti-v integrin antibody was loaded as a control. The resulting proteins were analysed by western blotting using R4 anti-uPAR or polyclonal anti-v integrin antibodies.

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