A Composite Role of Vitronectin and Urokinase in the Modulation of Cell Morphology upon Expression of the Urokinase Receptor
2008; Elsevier BV; Volume: 283; Issue: 22 Linguagem: Inglês
10.1074/jbc.c700214200
ISSN1083-351X
AutoresThore Hillig, Lars H. Engelholm, Signe Ingvarsen, Daniel H. Madsen, Henrik Gårdsvoll, Jørgen K. Larsen, Michael Ploug, Keld Danø, Lars Kjøller, Niels Behrendt,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoThe urokinase receptor, urokinase receptor (uPAR), is a glycosylphosphatidylinositol-anchored membrane protein engaged in pericellular proteolysis and cellular adhesion, migration, and modulation of cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin, and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear whether the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements or whether lateral interactions between uPAR and integrins can induce the same response. In this report, we show that both of these triggering mechanisms can be operative and that uPAR-dependent modulation of cell morphology can indeed occur independently of a direct vitronectin binding. Expression of wild-type uPAR on HEK293 cells led to pronounced vitronectin adhesion and cytoskeletal rearrangements, whereas a mutant uPAR, uPARW32A with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPARW32A with the protease ligand, pro-uPA, or its receptor-binding domain, the ability to induce cytoskeletal rearrangements was restored, although this did not rescue the uPAR-vitronectin binding and adhesion capability. On the other hand, using other uPAR variants, we could show that uPAR-vitronectin adhesion is indeed capable and sufficient to induce the same morphological rearrangements. This was shown with cells expressing a different single-site mutant, uPARY57A, in the presence of a synthetic uPAR-binding peptide, as well as with wild-type uPAR, which underwent cytoskeletal rearrangements even when cultivated in uPA-deficient serum. Blocking of integrins with an Arg-Gly-Asp-containing peptide counteracted the matrix contacts necessary to initiate the uPAR-dependent cytoskeletal rearrangements, whereas inactivation of the Rac signaling pathway in all cases suppressed the occurrence of the same events. The urokinase receptor, urokinase receptor (uPAR), is a glycosylphosphatidylinositol-anchored membrane protein engaged in pericellular proteolysis and cellular adhesion, migration, and modulation of cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin, and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear whether the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements or whether lateral interactions between uPAR and integrins can induce the same response. In this report, we show that both of these triggering mechanisms can be operative and that uPAR-dependent modulation of cell morphology can indeed occur independently of a direct vitronectin binding. Expression of wild-type uPAR on HEK293 cells led to pronounced vitronectin adhesion and cytoskeletal rearrangements, whereas a mutant uPAR, uPARW32A with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPARW32A with the protease ligand, pro-uPA, or its receptor-binding domain, the ability to induce cytoskeletal rearrangements was restored, although this did not rescue the uPAR-vitronectin binding and adhesion capability. On the other hand, using other uPAR variants, we could show that uPAR-vitronectin adhesion is indeed capable and sufficient to induce the same morphological rearrangements. This was shown with cells expressing a different single-site mutant, uPARY57A, in the presence of a synthetic uPAR-binding peptide, as well as with wild-type uPAR, which underwent cytoskeletal rearrangements even when cultivated in uPA-deficient serum. Blocking of integrins with an Arg-Gly-Asp-containing peptide counteracted the matrix contacts necessary to initiate the uPAR-dependent cytoskeletal rearrangements, whereas inactivation of the Rac signaling pathway in all cases suppressed the occurrence of the same events. The urokinase receptor (uPAR) 3The abbreviations used are: uPA, urokinase plasminogen activator; uPAR, uPA receptor; PBS, phosphate-buffered saline; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; ATF, amino-terminal fragment of uPA; GFD, growth factor domain of uPA; WT, wild type. 3The abbreviations used are: uPA, urokinase plasminogen activator; uPAR, uPA receptor; PBS, phosphate-buffered saline; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; ATF, amino-terminal fragment of uPA; GFD, growth factor domain of uPA; WT, wild type. is a glycosylphosphatidyl-inositol-anchored membrane protein that binds the plasminogen activator, uPA and the extracellular matrix protein, vitronectin (for review, see Ref. 1Behrendt N. Biol. Chem. 2004; 385: 103-136Crossref PubMed Scopus (86) Google Scholar). uPAR plays several important roles in the interplay of cells with their surrounding matrix, including the governing of plasmin-dependent fibrinolysis and matrix degradation (2Bugge T.H. Flick M.J. Danton M.J. Daugherty C.C. Rømer J. Danø K. Carmeliet P. Collen D. Degen J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5899-5904Crossref PubMed Scopus (231) Google Scholar), the direct adhesion on vitronectin-containing matrices (3Wei Y. Waltz D.A. Rao N. Drummond R.J. Rosenberg S. Chapman H.A. J. Biol. Chem. 1994; 269: 32380-32388Abstract Full Text PDF PubMed Google Scholar), and the modulation of morphology and adhesive properties of cells toward both vitronectin and other matrix proteins (4Kjøller L. Hall A. J. Cell Biol. 2001; 152: 1145-1157Crossref PubMed Scopus (174) Google Scholar, 5Kjøller L. Biol. Chem. 2002; 383: 5-19Crossref PubMed Scopus (102) Google Scholar, 6Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Crossref PubMed Scopus (696) Google Scholar). Being expressed on tumor-associated stromal cells (7Nielsen B.S. Rank F. Illemann M. Lund L.R. Danø K. Int. J. Cancer. 2007; 120: 2086-2095Crossref PubMed Scopus (87) Google Scholar, 8Pyke C. Ralfkiaer E. Rønne E. Høyer-Hansen G. Kirkeby L. Danø K. Histopathology (Oxf.). 1994; 24: 131-138Crossref PubMed Scopus (108) Google Scholar) and, in some cancers, on the tumor cells themselves (9Rømer J. Pyke C. Lund L.R. Ralfkiaer E. Danø K. J. Investig. Dermatol. 2001; 116: 353-358Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), uPAR is assumed to be important for cancer invasion, both through the localization of matrix degrading processes (10Danø K. Behrendt N. Høyer-Hansen G. Johnsen M. Lund L.R. Ploug M. Rømer J. Thromb. Haemostasis. 2005; 93: 676-681Crossref PubMed Scopus (386) Google Scholar) and through the modulation of cell adhesion and migration (11Blasi F. Carmeliet P. Nat. Rev. Mol. Cell. Biol. 2002; 3: 932-943Crossref PubMed Scopus (1064) Google Scholar). Therefore, a considerable research effort is devoted to the unraveling of the molecular and cellular interactions of uPAR. Recent studies on the three-dimensional structure of uPAR (12Llinas P. Le Du M.H. Gårdsvoll H. Danø K. Ploug M. Gilquin B. Stura E.A. Menez A. EMBO J. 2005; 24: 1655-1663Crossref PubMed Scopus (203) Google Scholar, 13Huai Q. Mazar A.P. Kuo A. Parry G.C. Shaw D.E. Callahan J. Li Y. Yuan C. Bian C. Chen L. Furie B. Furie B.C. Cines D.B. Huang M. Science. 2006; 311: 656-659Crossref PubMed Scopus (258) Google Scholar), combined with site-directed mutagenesis (14Gårdsvoll H. Danø K. Ploug M. J. Biol. Chem. 1999; 274: 37995-38003Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 15Gårdsvoll H. Ploug M. J. Biol. Chem. 2007; 282: 13561-13572Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 16Madsen C.D. Ferraris G.M. Andolfo A. Cunningham O. Sidenius N. J. Cell Biol. 2007; 177: 927-939Crossref PubMed Scopus (198) Google Scholar, 17Gårdsvoll H. Gilquin B. Le Du M.H. Menez A. Jorgensen T.J. Ploug M. J. Biol. Chem. 2006; 281: 19260-19272Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), have led to a detailed understanding of the structural basis for the binding of uPA and vitronectin to uPAR. In addition to these binding reactions, however, an increasing number of reports point to downstream effects of uPAR interactions that appear to include additional molecular partners, such as β1, β2, and β3 integrins (6Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Crossref PubMed Scopus (696) Google Scholar, 18Xue W. Kindzelskii A.L. Todd III, R.F. Petty H.R. J. 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Such interactions are likely to be involved in signaling effects that ultimately govern cell morphology and migration as well as proliferation (6Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Crossref PubMed Scopus (696) Google Scholar, 24Nguyen D.H. Hussaini I.M. Gonias S.L. J. Biol. Chem. 1998; 273: 8502-8507Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 25Aguirre Ghiso J.A. Kovalski K. Ossowski L. J. Cell Biol. 1999; 147: 89-104Crossref PubMed Scopus (460) Google Scholar), but their molecular basis is still much less well defined than that of the uPA and vitronectin binding reactions. Therefore, although direct interactions between uPAR and integrins have indeed been demonstrated (20Pluskota E. Soloviev D.A. Plow E.F. Blood. 2003; 101: 1582-1590Crossref PubMed Scopus (99) Google Scholar, 26Wei Y. Eble J.A. Wang Z. Kreidberg J.A. Chapman H.A. Mol. Biol. Cell. 2001; 12: 2975-2986Crossref PubMed Scopus (223) Google Scholar, 27Carriero M.V. Franco P. Gargiulo L. Vocca I. Cito L. Fontana L. Iaccarino C. Del P.G. Guardiola J. Stoppelli M.P. Biol. Chem. 2002; 383: 107-113Crossref PubMed Scopus (12) Google Scholar), an unresolved question is whether the signaling reactions do actually depend on a defined molecular interplay between these membrane proteins and uPAR or whether they are secondary phenomena brought about, e.g. by uPAR-mediated matrix adhesion as proposed recently (16Madsen C.D. Ferraris G.M. Andolfo A. Cunningham O. Sidenius N. J. Cell Biol. 2007; 177: 927-939Crossref PubMed Scopus (198) Google Scholar). In this report, we address an important aspect of this question by demonstrating a striking uPAR-dependent effect on the cytoskeleton that is independent of the uPAR-mediated cell adhesion. Protein Reagents—The following recombinant proteins and synthetic reagents were produced as described previously: recombinant human full-length pro-uPA and its isolated amino-terminal fragment (ATF; residues 1–143) (15Gårdsvoll H. Ploug M. J. Biol. Chem. 2007; 282: 13561-13572Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), the uPA growth factor domain (GFD; residues 1–48) (17Gårdsvoll H. Gilquin B. Le Du M.H. Menez A. Jorgensen T.J. Ploug M. J. Biol. Chem. 2006; 281: 19260-19272Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and the synthetic peptide antagonist, AE120 (28Ploug M. Ostergaard S. Gårdsvoll H. Kovalski K. Holst-Hansen C. Holm A. Ossowski L. Danø K. Biochemistry. 2001; 40: 12157-12168Crossref PubMed Scopus (165) Google Scholar). The cyclic peptides, cyclo(Arg-Gly-Asp-d-Phe-Val) and cyclo(Arg-Ala-Asp-d-Phe-Val), were purchased from Peptides International, Louisville, KT. The following reagents were purchased from the commercial sources indicated: human vitronectin (BD Biosciences) and FITC-phalloidin (Invitrogen). Transfection of HEK Cells with cDNAs Encoding uPAR Wild-type and Single-site Mutants or Dominant-negative Rac Construct—cDNA constructs encoding the human uPARWT full-length protein (signal peptide of 22 amino acid residues followed by residues 1–313; numbering referring to Ref. 29Behrendt N. Ploug M. Patthy L. Houen G. Blasi F. Danø K. J. Biol. Chem. 1991; 266: 7842-7847Abstract Full Text PDF PubMed Google Scholar) and uPAR proteins with single site substitutions at position 32 (uPARW32A) and 57 (uPARY57A) were obtained by ligating the corresponding DNA constructs encoding soluble, truncated uPARs (14Gårdsvoll H. Danø K. Ploug M. J. Biol. Chem. 1999; 274: 37995-38003Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) into the full-length uPAR expression plasmid, pRc/CMV-uPAR (4Kjøller L. Hall A. J. Cell Biol. 2001; 152: 1145-1157Crossref PubMed Scopus (174) Google Scholar), using appropriate restriction sites. HEK cells, (HEK293, purchased from ECACC) were transfected with the resulting uPAR expression vectors or with vector alone (mock-transfected) by electroporation followed by cloning by limiting dilution and characterization of transfectant clones by flow cytometry. Details for these procedures can be found in the supplemental materials. For transfection with a dominant-negative Rac construct, the plasmid pRK5-myc-Rac1-T17N (N17Rac) (4Kjøller L. Hall A. J. Cell Biol. 2001; 152: 1145-1157Crossref PubMed Scopus (174) Google Scholar, 30Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (802) Google Scholar) was used for electroporation. After transformation, the cells were immediately seeded on coverslips coated with vitronectin in the presence or absence of pro-uPA. Further details are described in the supplemental materials. Adhesion Assays—Vitronectin adhesion assays with transfected cells were done in vitronectin-coated 96-well culture plates, using triplicate samples of 1 × 105 cells in PBS with 5 mm EDTA and 0.5% bovine serum albumin. After incubation and washing, adherent cells were quantified by thiazolyl blue tetrazolium bromide assay (31Liu S. Bugge T.H. Leppla S.H. J. Biol. Chem. 2001; 276: 17976-17984Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The complete procedure is available in the supplemental materials. Analyses of Cell Morphology and Cytoskeletal Rearrangements—Glass coverslips were placed in a 24-well culture plate and coated with 50 μl of a 5 μg/ml solution of vitronectin in PBS/calcium/magnesium (PBS with 0.9 mm CaCl2 and 0.5 mm MgCl2) followed by blocking with 2 mg/ml bovine serum albumin in PBS/calcium/magnesium. After washing the wells, transfected cells were seeded on the coverslips and grown for 2 days at 37 °C in 1 ml of serum-containing growth medium (see above), supplemented with 400 μg/ml Geneticin. In some experiments, this was followed by the addition of pro-uPA or other reagents, as indicated followed by culture for another 2 days with the cells still being subconfluent. In some experiments, cells were cultured for different time periods, as indicated. For examination of lamellipodia and cytoskeletal rearrangements, cells were fixed and permeabilized followed by staining with FITC-labeled phalloidin (see supplemental materials for details). The cells were then examined by fluorescence microscopy, using a Leica DM4000B fluorescence microscope with a Leica DFC-480 camera. Scoring of lamellipodia-positive cells (16Madsen C.D. Ferraris G.M. Andolfo A. Cunningham O. Sidenius N. J. Cell Biol. 2007; 177: 927-939Crossref PubMed Scopus (198) Google Scholar) was done blindly in randomly selected fields from fluorescence microscopy of the FITC-phalloidin treated samples. For each cell type and each of the experimental conditions tested, five microscope fields were examined by four skilled researchers who were unaware of the sample identity. Each investigator assigned the number 0 (lamellipodia-negative), 0.5 (ambiguous), or 1 (lamellipodia-positive) to each field examined. The final score of each sample was defined as the cumulative number (0–20) obtained from all investigators for all fields from the sample in question after decoding the fields and samples. In specific experiments with uPA-free conditions, FCS was excluded from the cell culture medium and substituted with sterile serum from a uPA-deficient mouse (a kind gift from Dr. Leif R. Lund, the Finsen Laboratory). For studies with cells transfected with the dominant-negative Rac construct, quantification of lamellipodia-positive cells was done by counting of single cells; see supplemental materials. Adhesion Properties and Morphology of HEK Cells Transfected with Mutated uPARs—Based on the biochemical mapping of the vitronectin binding determinant of soluble uPAR (15Gårdsvoll H. Ploug M. J. Biol. Chem. 2007; 282: 13561-13572Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), we set out to study the importance of this binding reaction with cell-associated uPAR. Human embryonic kidney (HEK293) cells were transfected with vector alone (mock-transfected), with full-length human wild-type uPAR cDNA (uPARWT), or with uPAR cDNAs with targeted alanine substitutions at positions 32 (uPARW32A) or 57 (uPARY57A). Of these mutant proteins, when analyzed in their soluble form, uPARW32A has impaired vitronectin binding but normal binding of the protease ligand, uPA/pro-uPA (15Gårdsvoll H. Ploug M. J. Biol. Chem. 2007; 282: 13561-13572Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The same was shown to be the case for the corresponding cellular, membrane-bound uPAR mutant in a recent report (16Madsen C.D. Ferraris G.M. Andolfo A. Cunningham O. Sidenius N. J. Cell Biol. 2007; 177: 927-939Crossref PubMed Scopus (198) Google Scholar), published during the course of this work. In contrast, uPARY57A has some reduction in the binding to uPA/pro-uPA (14Gårdsvoll H. Danø K. Ploug M. J. Biol. Chem. 1999; 274: 37995-38003Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) but, when complexed with this ligand, this mutant binds vitronectin normally (15Gårdsvoll H. Ploug M. J. Biol. Chem. 2007; 282: 13561-13572Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Stable transfectant cell lines were isolated and shown to express comparable levels of cell surface uPAR by flow cytometric analysis (supplemental Fig. S1). The cells were then studied with respect to two properties, known from previous studies to be directly or indirectly dependent on the uPAR-vitronectin interaction, i.e. cellular adhesion on a vitronectin matrix (3Wei Y. Waltz D.A. Rao N. Drummond R.J. Rosenberg S. Chapman H.A. J. Biol. Chem. 1994; 269: 32380-32388Abstract Full Text PDF PubMed Google Scholar, 6Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Crossref PubMed Scopus (696) Google Scholar) and induction of cytoskeletal rearrangements (4Kjøller L. Hall A. J. Cell Biol. 2001; 152: 1145-1157Crossref PubMed Scopus (174) Google Scholar). To separate the uPAR-mediated vitronectin adhesion from the integrin-mediated matrix interactions, cell adhesion assays were done as short term experiments in the presence of EDTA (4Kjøller L. Hall A. J. Cell Biol. 2001; 152: 1145-1157Crossref PubMed Scopus (174) Google Scholar) (Fig. 1A). Under these conditions, adhesion was completely uPAR-dependent since efficient adhesion occurred with uPARWT transfectants, whereas no adhesion occurred with the mock-transfected control cells. The two mutant uPARs both failed to induce adhesion capability, in accordance with the notion that neither mutant binds to vitronectin under these conditions. Expression of uPARWT also induced marked changes in cell morphology. The uPARWT cells displayed pronounced protrusions and lamellipodia, which could be observed directly on live cells by phase contrast microscopy (result not shown), as well as by fluorescence microscopy after permeabilization and staining with FITC-phalloidin (Fig. 1B). These membranous structures were accompanied by marked cytoskeletal rearrangements, as also evident on the phalloidin-stained cells. Lamellipodia and cytoskeletal extensions were absent in the mock-transfected cells. This difference between lamellipodia-positive and -negative cell samples was highly striking (Fig. 1B) and was completely unambiguous as evident by the very consistent assignment of cell morphology status after unbiased scoring by four independent researchers (Fig. 1C; see "Experimental Procedures"). When analyzing the two uPAR mutants in the same system, neither of these was capable of inducing morphological change or cytoskeletal rearrangements (Fig. 1, B and C). The Role of pro-uPA in the uPAR-induced Effects—Thus, in accordance with the results of Madsen et al. (16Madsen C.D. Ferraris G.M. Andolfo A. Cunningham O. Sidenius N. J. Cell Biol. 2007; 177: 927-939Crossref PubMed Scopus (198) Google Scholar), transfection with wild-type uPAR led to efficient vitronectin adhesion and changes in cell morphology, whereas non-adhesive uPAR variants failed to induce cytoskeletal rearrangements. To further pursue this phenomenon, we examined the role of the protease ligand, pro-uPA, which can bind to uPAR simultaneously with the binding of vitronectin to the latter (3Wei Y. Waltz D.A. Rao N. Drummond R.J. Rosenberg S. Chapman H.A. J. Biol. Chem. 1994; 269: 32380-32388Abstract Full Text PDF PubMed Google Scholar). As seen in Fig. 2A, the addition of pro-uPA led to the expected pattern of cell adhesion on vitronectin in all cases. Thus, in accordance with the notion that pro-uPA stimulates the interaction between wild-type uPAR and vitronectin (32Kanse S.M. Kost C. Wilhelm O.G. Andreasen P.A. Preissner K.T. Exp. Cell Res. 1996; 224: 344-353Crossref PubMed Scopus (232) Google Scholar, 33Høyer-Hansen G. Behrendt N. Ploug M. Danø K. Preissner K.T. FEBS Lett. 1997; 420: 79-85Crossref PubMed Scopus (131) Google Scholar), an even stronger adhesion was observed with the uPARWT-expressing cells upon the addition of the protease ligand, as compared with the same cells in the absence of pro-uPA. Furthermore, as expected, the addition of pro-uPA did not lead to adhesion of mock-transfected cells. Finally, a pronounced adhesion capability was induced by the addition of pro-uPA to the uPARY57A cells, whereas adhesion was in all cases negative with the uPARW32A cells. This was in complete agreement with our previous binding studies with isolated uPAR mutant proteins where uPARY57A was shown to bind vitronectin efficiently when saturated with pro-uPA, whereas no affinity for vitronectin could be induced in uPARW32A (15Gårdsvoll H. Ploug M. J. Biol. Chem. 2007; 282: 13561-13572Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Interestingly, however, under these conditions, studies on cell morphology revealed a clear distinction between cellular adhesive properties and the occurrence of cytoskeletal rearrangements. This distinction was evident in the case of the uPARW32A cells, which formed extensive cellular protrusions and lamellipodia in response to pro-uPA (Fig. 2, B and C), although the same uPAR mutant was non-adhesive in the vitronectin adhesion assay as noted above. The other transfectant cell types, however, displayed the same pro-uPA dependence as found in the adhesion assay. Thus, for the uPARY57A cells, lamellipodia were formed only with cells grown in the presence of pro-uPA (Fig. 2, B and C), whereas uPARWT cells were in all cases lamellipodia-positive (Fig. 2C). These cytoskeletal rearrangements were not a result of long term cell culture or high concentrations of pro-uPA because the same outcome was obtained after overnight cell culture with only 10 nm pro-uPA (supplemental Fig. S2A). In conclusion, the uPAR-dependent mechanism for modulation of cell morphology was found to follow the capability for uPAR-mediated vitronectin adhesion in some but not in all cases. The protease ligand could induce uPAR-dependent cytoskeletal rearrangements in uPARW32A transfectants without conferring vitronectin affinity. The Effect of pro-uPA Is Exerted by the Growth Factor Domain and Is Sensitive to Blocking of Integrins—Pro-uPA includes a receptor-binding GFD, a Kringle domain, and a catalytic domain (34Appella E. Robinson E.A. Ullrich S.J. Stoppelli M.P. Corti A. Cassani G. Blasi F. J. Biol. Chem. 1987; 262: 4437-4440Abstract Full Text PDF PubMed Google Scholar), and uPA itself has been shown to have some affinity for vitronectin (35Moser T.L. Enghild J.J. Pizzo S.V. Stack M.S. Biochem. J. 1995; 307: 867-873Crossref PubMed Scopus (46) Google Scholar). Thus, it was important to learn whether the effect of pro-uPA in the phenomena studied in this work required the complete protein or whether only part of the molecule was needed. Therefore, we tested the effect of the isolated receptor-binding units in the same phenomena as studied above. As shown in Fig. 3, A and B, the effects of pro-uPA were in all cases reproduced not only by ATF (including the Kringle and the GFD) but also by the isolated GFD, comprising just 48 amino acid residues. Thus, ATF and GFD both induced a pronounced adhesive capability in the uPARY57A cells but failed to induce any adhesion of the uPARW32A cells (Fig. 3A). In the morphology study, both reagents successfully rescued the lamellipodia phenotype of the uPARW32A as well as the uPARY57A cells, just like the effect exerted by the complete pro-uPA protein (Fig. 3B and supplemental Fig. S2B). In conclusion, the GFD of uPA retains all of the structural features required to elicit these cellular effects.FIGURE 1Vitronectin adhesion and cytoskeletal rearrangements in HEK cells expressing wild-type and mutated uPAR. A, adhesion of uPAR transfected cells on a reconstituted vitronectin matrix. Mock-transfected (Mock) cells or cells expressing uPARWT or uPAR mutant proteins were seeded in vitronectin-coated culture wells in the presence of EDTA and allowed to adhere during a 1-h incubation period at 37 °C. After washing, adherent cells were quantified by thiazolyl blue tetrazolium bromide assay. Each column represents the mean of a triple determination. The standard deviations are indicated. B, morphological changes and cytoskeletal rearrangements. Mock-transfected cells or cells expressing uPARWT or uPAR mutant proteins were cultured for 5 days on vitronectin-coated coverslips. The cells were then fixed and permeabilized followed by FITC-phalloidin staining and examination by fluorescence microscopy. Note the exclusive appearance of lamellipodia and cytoskeletal extensions in the uPARWT-transfected cells. C, quantification of fields with lamellipodia-positive cells. Cells were cultured, stained, and examined by fluorescence microscopy as in B. Each cell type was assigned an arbitrary designation, after which five randomly selected microscope fields for each cell type were scored blindly by four investigators for lamellipodia-positive cells (see "Experimental Procedures"). The cumulative score is represented for each sample.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Dependence on different uPAR ligands and the Rac pathway. A and B, adhesion (A) and lamellipodia-positive cells (B), analyzed as in Fig. 2, A and C, but in the presence of the indicated ligands. Ligands were added at final concentrations of 100 nm (pro-uPA, ATF, and GFD) or 1 μm (synthetic peptide AE120), respectively. Mock, cells transfected with vector alone. C, inactivation of the Rac pathway. Cells were transfected with the dominant-negative Rac expression plasmid, N17Rac, or with vector alone, as indicated, and cultured in the presence of 20 nm pro-uPA. After fixation, permeabilization, and FITC-phalloidin staining, randomly selected microscope fields representing at least 50 cells were scored for the percentage of protrusion-positive cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A non-natural, uPAR-binding peptide, designated AE120, has been developed by phage display technology and combinatorial chemistry and has been shown to interact with uPAR with a high affinity (28Ploug M. Ostergaard S. Gårdsvoll H. Kovalski K. Holst-Hansen C. Holm A. Ossowski L. Danø K. Biochemistry. 2001; 40: 12157-12168Crossref PubMed Scopus (165) Google Scholar). X-ray crystallography of the complex of uPAR and a truncated version of this peptide has shown that it binds directly in the uPA-binding pocket of uPAR (12Llinas P. Le Du M.H. Gårdsvoll H. Danø K. Ploug M. Gilquin B. Stura E.A. Menez A. EMBO J. 2005; 24: 1655-1663Crossref PubMed Scopus (203) Google Scholar), but the conformation of the complex is slightly different from the complex between uPAR and ATF in terms of the interdomain orientation and the flexible regions of the receptor (13Huai Q. Mazar A.P. Kuo A. Parry G.C. Shaw D.E. Callahan J. Li Y. Yuan C. Bian C. Chen L. Furie B. Furie B.C. Cines D.B. Huang M. Science. 2006; 311: 656-659Crossref PubMed Scopus (258) Google Scholar, 36Yuan C. Huang M. CMLS Cell. Mol. Life Sci. 2007; 64: 1033-1037Crossref PubMed Scopus (28) Google Scholar). Therefore, we included AE120 in these studies to learn whether ligation of uPAR with this peptide would mimic t
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