The Cleavage of the Urokinase Receptor Regulates Its Multiple Functions
2002; Elsevier BV; Volume: 277; Issue: 49 Linguagem: Inglês
10.1074/jbc.m207494200
ISSN1083-351X
AutoresNunzia Montuori, Maria Vincenza Carriero, Salvatore Salzano, Guido Rossi, Pia Ragno,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoThe urokinase-type plasminogen activator (uPA) is able to cleave its cell surface receptor (uPAR) anchored to the cell membrane through a glycophosphatidylinositol tail. The cleavage leads to the formation of cell surface truncated forms, devoid of the N-terminal domain 1 (D1) and unmasks or disrupts, depending on the cleavage site, a sequence in the D1-D2 linker region (residues 88–92), which in the soluble form is a potent chemoattractant for monocyte-like cells. To investigate the possible role(s) of the cleaved forms of cell surface glycophosphatidylinositol-anchored uPAR, uPAR-negative human embrional kidney 293 cells were transfected with the cDNA of intact uPAR (uPAR-293) or with cDNAs corresponding to the truncated forms of uPAR exposing (D2D3-293) or lacking (D2D3wc-293) the peptide 88–92 (P88–92). Cell adhesion assays and co-immunoprecipitation experiments indicated that the removal of D1, independently of the presence of P88–92, abolished the lateral interaction of uPAR with integrins and its capability to regulate integrin adhesive functions. The expression of intact uPAR induced also a moderate increase in 293 cell proliferation, which was accompanied by the activation of ERK. Also this effect was abolished by D1 removal, independently of the presence of P88–92. The expression of intact and truncated uPARs regulated cell directional migration toward uPA, the specific uPAR ligand, and toward fMLP, a bacterial chemotactic peptide. In fact, the uPA-dependent cell migration required the expression of intact uPAR, including D1, whereas the fMLP-dependent cell migration required the expression of a P88–92 containing uPAR and was independent of the presence of D1. Together these observations indicate that uPA-mediated uPAR cleavage and D1 removal, occurring on the cell surface of several cell types, can play a fundamental role in the regulation of multiple uPAR functions. The urokinase-type plasminogen activator (uPA) is able to cleave its cell surface receptor (uPAR) anchored to the cell membrane through a glycophosphatidylinositol tail. The cleavage leads to the formation of cell surface truncated forms, devoid of the N-terminal domain 1 (D1) and unmasks or disrupts, depending on the cleavage site, a sequence in the D1-D2 linker region (residues 88–92), which in the soluble form is a potent chemoattractant for monocyte-like cells. To investigate the possible role(s) of the cleaved forms of cell surface glycophosphatidylinositol-anchored uPAR, uPAR-negative human embrional kidney 293 cells were transfected with the cDNA of intact uPAR (uPAR-293) or with cDNAs corresponding to the truncated forms of uPAR exposing (D2D3-293) or lacking (D2D3wc-293) the peptide 88–92 (P88–92). Cell adhesion assays and co-immunoprecipitation experiments indicated that the removal of D1, independently of the presence of P88–92, abolished the lateral interaction of uPAR with integrins and its capability to regulate integrin adhesive functions. The expression of intact uPAR induced also a moderate increase in 293 cell proliferation, which was accompanied by the activation of ERK. Also this effect was abolished by D1 removal, independently of the presence of P88–92. The expression of intact and truncated uPARs regulated cell directional migration toward uPA, the specific uPAR ligand, and toward fMLP, a bacterial chemotactic peptide. In fact, the uPA-dependent cell migration required the expression of intact uPAR, including D1, whereas the fMLP-dependent cell migration required the expression of a P88–92 containing uPAR and was independent of the presence of D1. Together these observations indicate that uPA-mediated uPAR cleavage and D1 removal, occurring on the cell surface of several cell types, can play a fundamental role in the regulation of multiple uPAR functions. Cell migration is the result of a complex balance between localized proteolysis, dynamic cell extracellular matrix interactions, and cytoskeletal reorganization. The receptor for the urokinase-type plasminogen activator (uPAR) 1The abbreviations used are: uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; suPAR, soluble uPAR; VN, vitronectin; CG, collagen; FN, fibronectin; LM, laminin; GPI, glycophosphatidylinositol; fMLP, N-formyl-Met-Leu-Phe; FPR, N-formyl peptide receptor; Pipes, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline; ATF, N-terminal fragment; ERK, extracellular signal-regulated kinase appears to be a key molecule in the coordination of these different events. uPAR, in fact, promotes cell-associated proteolysis by binding its specific ligand, the serine-protease urokinase (uPA), which converts locally the widely distributed zymogen plasminogen into active plasmin, a broad spectrum protease that degrades extracellular matrix proteins either directly or by activating other proteases (1Irigoyen J.P. Munoz-Canoves P. Montero L. Koziczak M. Nagamine Y. Cell Mol. Life Sci. 1999; 56: 104-132Google Scholar, 2Stoppelli M.P. Heino J. Kahari V.M. Cell Invasion. Londes Bioscience, 2001: 128-141Google Scholar). Furthermore, uPAR interacts with cell adhesion specialized molecules, such as integrins of the β1, β2, and β3 families (3Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Google Scholar, 4Xue W. Kindzelskii A.L. Todd III, R.F. Petty H.R. J. Immunol. 1994; 152: 4630-4640Google Scholar, 5Xue W. Mizukami I. Todd III, R.F. Petty H.R. Cancer Res. 1997; 57: 1682-1689Google Scholar), thus regulating their functions, and binds vitronectin (VN), a component abundant in tumor-associated extracellular matrices (6Wei Y. Waltz D.A. Rao N. Drummond R.J. Rosenberg S. Chapman H.A. J. Biol. Chem. 1994; 269: 32380-32388Google Scholar). The interactions with integrins and VN are positively regulated by uPA (6Wei Y. Waltz D.A. Rao N. Drummond R.J. Rosenberg S. Chapman H.A. J. Biol. Chem. 1994; 269: 32380-32388Google Scholar,7Wei Y. Eble J.A. Wang Z. Kreidberg J.A. Chapman H.A. Mol. Biol. Cell. 2001; 12: 2975-2986Google Scholar). Both uPA and VN can induce uPAR-mediated cytoskeletal reorganization and cell migration (8Fibbi G. Ziche M. Morbidelli L. Magnelli L. Del Rosso M. Exp. Cell Res. 1988; 179: 385-395Google Scholar, 9Busso N. Masur S.K. Lazega D. Waxman S. Ossowski L. J. Cell Biol. 1994; 126: 259-270Google Scholar, 10Yebra M. Parry G.C. Stromblad S. Mackman N. Rosenberg S. Mueller B.M. Cheresh D.A. J. Biol. Chem. 1996; 271: 29393-29399Google Scholar, 11Carriero M.V. Del Vecchio S. Capozzoli M. Franco P. Fontana L. Zannetti A. Botti G. D'Aiuto G. Salvatore M. Stoppelli M.P. Cancer Res. 1999; 59: 5307-5314Google Scholar, 12Degryse B. Orlando S. Resnati M. Rabbani S.A. Blasi F. Oncogene. 2001; 20: 2032-2043Google Scholar). uPAR is synthesized as a single polypeptide chain of 313 amino acid residues, preceded by a 21-residue signal peptide. Post-translational events lead to the cleavage of the last 30 C-terminal residues and to the attachment of a glycophosphatidylinositol (GPI) tail to Gly283 (13Dano K. Behrendt N. Brunner N. Ellis V. Ploug M. Pyke C. Fibrinolysis. 1994; 8: 189-203Google Scholar). The mature protein has a three-domain structure: D1 is the N-terminal domain, D2 connects D1 to D3, and D3 is the C-terminal domain that anchors the molecule to the cell membrane through the GPI tail (13Dano K. Behrendt N. Brunner N. Ellis V. Ploug M. Pyke C. Fibrinolysis. 1994; 8: 189-203Google Scholar). However, despite the lack of a transducing cytoplasmic tail, the receptor is able to activate cell signaling pathways, probably by interacting with other cell surface molecules (14Ossowski L. Aguirre-Ghiso J.A. Curr. Opin. Cell Biol. 2000; 12: 613-620Google Scholar). uPA binds the D1 of the receptor and retains its proteolytic activity after the binding; the activity can be inhibited by two specific inhibitors, type 1 (PAI-1) and type 2 (PAI-2) (1Irigoyen J.P. Munoz-Canoves P. Montero L. Koziczak M. Nagamine Y. Cell Mol. Life Sci. 1999; 56: 104-132Google Scholar, 2Stoppelli M.P. Heino J. Kahari V.M. Cell Invasion. Londes Bioscience, 2001: 128-141Google Scholar). PAI-1 also appears to be involved in the regulation of uPAR-mediated cell migration, because it promotes the internalization of the uPA-uPAR complex and competes with uPAR for the binding to VN (15Deng G. Curriden S.A. Wang S. Rosenberg S. Loskutoff D.J. J. Cell Biol. 1996; 134: 1563-1571Google Scholar). The VN-binding domain of uPAR is controversial (6Wei Y. Waltz D.A. Rao N. Drummond R.J. Rosenberg S. Chapman H.A. J. Biol. Chem. 1994; 269: 32380-32388Google Scholar, 16Hoyer-Hansen G. Behrendt N. Ploug M. Dano K. Preissner K.T. FEBS Lett. 1997; 420: 79-85Google Scholar); the intact uPAR molecule is however required for an efficient binding to both vitronectin (16Hoyer-Hansen G. Behrendt N. Ploug M. Dano K. Preissner K.T. FEBS Lett. 1997; 420: 79-85Google Scholar, 17Sidenius N. Blasi F. FEBS Lett. 2000; 470: 40-46Google Scholar) and uPA (18Ploug M. Ellis V. Dano K. Biochemistry. 1994; 33: 8991-8997Google Scholar), thus suggesting the cooperation of different domains (19Behrendt N. Ronne E. Dano K. J. Biol. Chem. 1996; 271: 22885-22894Google Scholar). The region of uPAR involved in the interaction with integrins has not yet been identified. uPAR can be released by several cell lines, and a soluble form of the receptor (suPAR) has been detected in human plasma and urine (20Ronne E. Pappot H. Grondahl-Hansen J. Hoyer-Hansen G. Plesner T. Hansen N.E. Dano K. Br. J. Haematol. 1995; 89: 576-581Google Scholar, 21Sier C.F. Sidenius N. Mariani A. Aletti G. Agape V. Ferrari A. Casetta G. Stephens R.W. Brunner N. Blasi F. Lab. Invest. 1999; 79: 717-722Google Scholar). The soluble form of the receptor, deprived of the D1 domain by cleavage with chymotrypsin, is a potent chemoattractant for monocyte-like cells (22Resnati M. Guttinger M. Valcamonica S. Sidenius N. Blasi F. Fazioli F. EMBO J. 1996; 15: 1572-1582Google Scholar). In fact, chymotrypsin cleaves uPAR between Tyr87 and Ser88, thus unmasking a region with chemotactic properties, corresponding to the 88–92 residues of uPAR (P88–92) (23Fazioli F. Resnati M. Sidenius N. Higashimoto Y. Appella E. Blasi F. EMBO J. 1997; 16: 7279-7286Google Scholar). It has been recently reported that P88–92-induced chemotaxis is mediated by the low affinity receptor for the N-formyl-Met-Leu-Phe (fMLP), a peptide of bacterial origin (24Resnati M. Pallavicini I. Wang J.M. Oppenheim J. Serhan C.N. Romano M. Blasi F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1359-1364Google Scholar). Cell surface uPAR also can be cleaved in vitro within the D1-D2 connecting region, by several proteolytic enzymes, such as chymotrypsin, plasmin, and uPA itself (25Hoyer-Hansen G. Ronne E. Solberg H. Behrendt N. Ploug M. Lund L.R. Ellis V. Dano K. J. Biol. Chem. 1992; 267: 18224-18229Google Scholar, 26Ragno P. Montuori N. Covelli B. Hoyer-Hansen G. Rossi G. Cancer Res. 1998; 58: 1315-1319Google Scholar). Cleavage leads to the release of the D1 domain, thus leaving on the cell surface a two domains D2D3-uPAR. Cleaved forms of uPAR, lacking the uPA-binding domain, are widely expressed both in vivo and in vitro, e.g. on the surface of different cell lines (25Hoyer-Hansen G. Ronne E. Solberg H. Behrendt N. Ploug M. Lund L.R. Ellis V. Dano K. J. Biol. Chem. 1992; 267: 18224-18229Google Scholar, 26Ragno P. Montuori N. Covelli B. Hoyer-Hansen G. Rossi G. Cancer Res. 1998; 58: 1315-1319Google Scholar, 27Hoyer-Hansen G. Solberg H. Ronne E. Behrendt N. Dano K. Preissner K.T. Rosenblatt S. Kost C. Wegerhoff J. Mosher D. Biology of Vitronectin and Their Receptors. Elsevier Science Publishers B.V., Amsterdam1993Google Scholar, 28Mazzieri R. Hoyer-Hansen G. Ronne E. Lober D. Vagnarelli P. Raimondi E. De Carli L. Dano K. Mignatti P. Fibrinolysis. 1994; 8: 344-352Google Scholar, 29Solberg H. Romer J. Brunner N. Holm A. Sidenius N. Dano K. Hoyer-Hansen G. Int. J. Cancer. 1994; 58: 877-881Google Scholar), in normal and neoplastic thyroid tissues (26Ragno P. Montuori N. Covelli B. Hoyer-Hansen G. Rossi G. Cancer Res. 1998; 58: 1315-1319Google Scholar), in blast cells of patients with acute leukemia (30Mustjoki S. Sidenius N. Sier C.F. Blasi F. Elonen E. Alitalo R. Vaheri A. Cancer Res. 2000; 60: 7126-7132Google Scholar), in cystic fluids from benign and malignant ovarian tumors (31Wahlberg K. Hoyer-Hansen G. Casslen B. Cancer Res. 1998; 58: 3294-3298Google Scholar), in human foreskin microvascular endothelial cells (32Koolwijk P. Sidenius N. Peters E. Sier C.F. Hanemaaijer R. Blasi F. van Hinsbergh V.W. Blood. 2001; 97: 3123-3131Google Scholar), and in human xenograft tumors implanted in mice (29Solberg H. Romer J. Brunner N. Holm A. Sidenius N. Dano K. Hoyer-Hansen G. Int. J. Cancer. 1994; 58: 877-881Google Scholar, 33Holst-Hansen C. Hamers M.J. Johannessen B.E. Brunner N. Stephens R.W. Br. J. Cancer. 1999; 81: 203-211Google Scholar). In monocyte-like cells, N-terminal sequencing of the cleaved forms and uPAR cleavage inhibition by uPA-inactivating antibodies showed that uPA could be physiologically responsible for D1 removal by cleaving the receptor at two specific sites, between Arg83and Ala84 and between Arg89 and Ser90, in the D1-D2 linker region (34Hoyer-Hansen G. Ploug M. Behrendt N. Ronne E. Dano K. Eur. J. Biochem. 1997; 243: 21-26Google Scholar). Thus, uPA-mediated cleavage can leave on the cell surface a truncated receptor exposing at the N terminus an intact P88–92 (the sequence with chemotactic properties) or a truncated receptor where this specific sequence has been partially removed. It has been recently reported that uPA could be involved in the cell surface uPAR cleavage also in vivo (35Zhou H.M. Nichols A. Meda P. Vassalli J.D. EMBO J. 2000; 19: 4817-4826Google Scholar) and that the enzyme cleaves with high efficiency only the GPI-anchored receptor and not the soluble form (36Hoyer-Hansen G. Pessara U. Holm A. Pass J. Weidle U. Dano K. Behrendt N. Biochem. J. 2001; 358: 673-679Google Scholar). Because cleaved uPAR is unable to bind uPA and VN, uPA-mediated cleavage acts as a negative feedback regulatory mechanism of the cell surface-associated plasminogen-activation and of cell adhesion to VN. We now investigate whether the removal of D1 from the cell surface uPAR can interfere also with other known functions of intact uPAR, such as cell adhesion, migration, and proliferation. Moreover, we investigate whether the presence, the exposure, or the deletion of P88–92, whose unmasking appears to be necessary for the chemotactic activity of soluble uPAR, can play a role in the functions of the cell-anchored receptor. uPAR cDNA was kindly provided by Dr. M. P. Stoppelli (Istituto Internazionale di Genetica e Biofisica, CNR, Naples, Italy). Mouse anti-uPAR monoclonal antibodies R4 and R2 were kindly provided by Dr. G. Hoyer-Hansen (Finsen Laboratory, Copenhagen, Denmark), whereas the rabbit polyclonal anti-uPAR 399 antibody was from American Diagnostica (Greenwich, CT). Rabbit polyclonal antibodies against αv and β1integrins were kindly provided by Dr. G. Tarone (University of Turin, Turin, Italy); the rabbit polyclonal antibodies against α3 and α5 integrins were from CHEMICON (Temecula, CA), and those against caveolin were from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG were from Bio-Rad; fluorescein isothiocyanate-labeled goat anti-rabbit IgG were from Jackson Lab (West Grove, PA). An ECL detection kit and the protein A-Sepharose were fromAmersham Biosciences. Polyvinylidene fluoride filters were from Millipore (Windsor, MA), and the PCR kit was from Perkin-Elmer (Branchburg, NJ). Collagen, laminin, fibronectin, and vitronectin were from Collaborative Research (Bedford, MA). The pertussis toxin was obtained by Calbiochem (Darmstadt, Germany), and the chemotaxis polyvinylpyrrolidone-free filters were from Corning; TRIzol reagent and Superscript II (Moloney murine leukemia virus reverse transcriptase) were Invitrogen. Human embrional kidney 293 (HEK-293) cells and transfected cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum. Immunoprecipitation was performed as previously described (3Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Google Scholar). The cells (5 × 106/sample) were washed twice with microtubule stabilization buffer (0.1m Pipes, pH 6.9, 2 m glycerol, 1 mmEGTA, 1 mm magnesium acetate) and then extracted in 0.2% Triton X-100 with additional protease inhibitors. The insoluble residue, enriched in cytoskeleton-associated proteins, was solubilized in RIPA buffer (150 mm NaCl, 50 mm Tris-HCl, pH 7.5, 1% deoxycholate, 0.1% SDS, 1% Triton-X-100, and protease inhibitors) and preincubated with nonimmune serum and 10% protein A-Sepharose for 2 h at 4 °C. After centrifugation, the supernatants were incubated with 2 μl of antisera to β1, α3, α5, and αv integrins, with 2 μl of antiserum to caveolin, or with 2 μl of nonimmune serum for 2 h at 4 °C. After 30 min of incubation with 10% protein A-Sepharose at room temperature, the immunoprecipitates were washed, subjected to 9% SDS-PAGE, and analyzed by Western blot using R2 monoclonal anti-uPAR antibody at a concentration of 1 μg/ml. The cells were lysed in 1% Triton X-100 with PBS in the presence of protease inhibitors (Sigma); the protein content was measured by a colorimetric assay (Bio-Rad). 0.5–1 μg of protein of transfected cell lysates was electrophoresed with 9% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% nonfat dry milk and probed with 1 μg/ml of R2 or R4 anti-uPAR monoclonal antibodies directed to uPAR domains 2 and 3 and therefore able to detect both intact and truncated uPARs. Finally, washed filters were incubated with horseradish peroxidase-conjugated anti-mouse antibody and detected by ECL. The cells were washed and harvested by PBS-EDTA 10 mm. After three washes in Ca2+,Mg2+-containing PBS, 5 × 105 cells were incubated with 1 μg/ml of 399 anti-uPAR polyclonal antibody for 1 h at 4 °C. Purified rabbit immunoglobulins were used as a negative control. The cells were then washed in PBS with 0.1% BSA and incubated with a fluorescein isothiocyanate-labeled goat anti-rabbit IgG for 30 min at 4 °C. Finally, the cells were washed and analyzed by flow cytometry using a FACScan (Becton Dickinson, Mountain View, CA). 96-well flat bottom microtiter plates (Costar, Cambridge MA) were coated with 2 μg of laminin, vitronectin, fibronectin, collagen, or heat-denatured BSA in PBS as a negative control and incubated overnight at 4 °C. The plates were then rinsed with PBS, incubated 1 h at room temperature with heat-denatured BSA 1% in PBS, and rinsed again. The cells were harvested by 10 mm PBS with EDTA and resuspended in Ca2+,Mg2+-containing PBS. 105 cells were plated in each coated well and incubated 1 h at 37 °C. In RGD competition experiments, the cells were preincubated with 0.5 mg/ml GRGDSP or GRGESP synthetic peptides for 30 min at 4 °C and then loaded in coated wells. Attached cells, after two washes, were fixed with 3% paraformaldehyde in PBS for 10 min and then incubated with 2% methanol for 10 min. The cells were finally stained for 10 min with 0.5% crystal violet in 20% methanol and vigorously washed with water. Stain was eluted by 0.1m sodium citrate in 50% ethanol, pH 4.2, and the absorbance at 540 nm was measured by a spectrophotometer. The cell migration assays were performed in Boyden chambers using 8-μm pore size polyvinylpyrrolidone-free polycarbonate filters coated with 20 μg/ml vitronectin, collagen, fibronectin, or laminin. The transfected cells were harvested by 10 mm EDTA, and then 2 × 105 cells were plated in the upper chamber in serum-free medium. 100 nm fMLP, 1 nm uPA N-terminal fragment (ATF), or serum-free medium was added in the lower chamber. The cells were allowed to migrate for 4 h at 37 °C, 5% CO2. The cells on the lower surface of the filter were then fixed in ethanol, stained with hematoxylin, and counted at 200× magnification (10 random fields/filter). In a separate set of experiments, the cells were preincubated for 1 h at room temperature with 5 μg/ml of 399 anti-uPAR polyclonal antibody or cultured for 18 h at 37 °C with 50 ng/ml pertussis toxin. Total cellular RNA was isolated by lysing cells in TRIzol solution according to the supplier's protocol. RNA was precipitated and quantitated by spectroscopy. 5 μg of total RNA was reversely transcribed with random hexamer primers and 200 units of Moloney murine leukemia virus reverse transcriptase. 1 μl of reversely transcribed DNA was then amplified, using FPR-specific 5′ sense (ATG GAG ACA AAT TCC TCT CTC) and 3′ antisense (CAC CTC TGC AGA AGG TAA AGT) primers, FPRL1-specific 5′ sense (CTT GTG ATC TGG GTG GCT GGA) and 3′ antisense (CAT TGC CTG TAA CTC AGT CTC) primers, or glyceraldehyde-3-phosphate dehydrogenase-specific 5′ sense (TTC ACC ACC ATG GAG AAG GCT) and 3′ antisense (ACA GCC TTG GCA GCA CCA GT) primers, as a control. Semiquantitative PCR was performed for 40 cycles at 68 °C in a thermocycler, and the reaction products were analyzed by electrophoresis in 1% agarose gel containing ethidium bromide, followed by photography under ultraviolet illumination. uPAR cDNA (nucleotide −3/1018) was cloned in theEcoRI site of pcDNA3, and the resulting plasmid was named uPAR-pcDNA3. D2D3-pcDNA3 was generated by PCR amplification of two different regions of uPAR cDNA. First, the region corresponding to the nucleotide −43/65, coding the leader peptide of the protein, was amplified by using the 5′ sense uPAR-4 (AGC TAA GCT TGA AGA CGT GCA GGG ACC CCG C) and the 3′ antisense uPAR-3 (AGC TGA ATT CCC CCA AGA GGC TGG GAC G) primers. The PCR product was digested by HindIII andEcoRI and cloned in pcDNA3 digested with the same enzymes. The resulting plasmid was named L-pcDNA3. Then the region corresponding to nucleotide 315/1008 of uPAR cDNA, coding domains 2 and 3, including the P88–92 peptide, was amplified by using the 5′ sense uPAR-2 (AGC TGA ATT CGG CTG TCA CCT ATT CCC GAA G) and the 3′ antisense uPAR-1 (AGC TTC TAG ATT AGG TCC AGA GGA GAG TGC) primers. The PCR product was digested with EcoRI and XbaI and cloned in L-pcDNA3 digested with the same enzymes. The resulting plasmid was named D2D3-pcDNA3 and contained a cDNA coding the leader peptide connected to 84–313 residues of uPAR through the insertion of additional Asn and Ser residues. D2D3wc-pcDNA3 was prepared from D2D3-pcDNA3. D2D3-pcDNA3 was subcloned in pGEM 4Z, to substitute the fragment HindIII-NsiI (nucleotide −43/348) with a fragment generated by amplifying the region corresponding to the nucleotide −43/65 of uPAR cDNA with the 5′ sense uPAR-4 (AGC TAA GCT TGA AGA CGT GCA GGG ACC CCG C) and the 3′ antisense uPAR-3 (AGC TGA ATT CCC CCA AGA GGC TGG GAC G) primers digested with the same enzymes. The resulting plasmid was subcloned in pcDNA3 and named D2D3wc-pcDNA3; it contained a cDNA coding the leader peptide connected to 95–313 residues of uPAR through the insertion of an additional Leu residue. 5 × 106 cells, cultured overnight in 100-mm tissue culture dishes, were transfected with 10 μg of uPAR-pcDNA3, D2D3-pcDNA3, D2D3wc-pcDNA3, or control vector pcDNA3 and 60 μl of LipofectAMINE for 5 h at 37 °C (5% CO2). Transfected cells were selected by Geneticin at 1.5 mg/ml; the resulting clones of each transfection were pooled and cultured in the presence of 0.5 mg/ml Geneticin. The cDNAs of two different truncated forms of uPAR were prepared. Both cDNAs coded for a uPAR devoid of D1 and formed by D2 and D3, but they differed for the presence of the sequence corresponding to the peptide 88–92 of the D1-D2 linker region (see Fig.1 for a scheme and "Materials and Methods" for preparation of constructs). The cDNAs of intact uPAR, D2D3-uPAR (containing P88–92), D2D3wc-uPAR (lacking P88–92), and the empty vector were transfected in uPAR-negative 293 cells; the resulting transfected cells were named uPAR-293, D2D3-293, D2D3wc-293, and V-293, respectively. Clones of transfected cells, after selection, were pooled, and expression of the different constructs was analyzed. Western blot of transfected cell lysates with R4 anti-uPAR monoclonal antibody, which is able to detect both intact and truncated forms of uPAR, showed that uPAR-293 cells expressed the 50-kDa intact receptor, whereas D2D3-293 and D2D3wc-293 cells expressed truncated uPARs of the expected 35 kDa (Fig. 2 A). The cDNAs of both uPAR truncated forms were expressed with a similar efficiency as compared with the expression of the intact uPAR cDNA. As expected, control V-293 cells did not express any form of uPAR (Fig.2 A). To investigate whether truncated uPARs were correctly delivered to the cell surface, transfected cell lysates were immunoprecipitated, after cell surface biotinylation, with R4 anti-uPAR monoclonal antibody. Both uPAR truncated forms were labeled by biotin, such as the intact form, thus indicating their cell surface expression (Fig. 2 B). Cell surface expression of uPARs was finally quantitated by flow cytometry with the 399 anti-uPAR polyclonal antibody; mean fluorescence intensity values of D2D3-293 and D2D3wc-293 cells varied of only +10.3% and −13.8%, respectively, as compared with uPAR-293 cells (Fig. 2 C). In conclusion, these results showed that transfected cells expressed almost similar amounts of intact and truncated forms of uPAR on their surface and were therefore suitable for functional assays. Intact uPAR is able to interact with different types of integrins and with caveolin, a protein associated with intracellular signaling pathways and cytoskeletal elements (3Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Google Scholar, 4Xue W. Kindzelskii A.L. Todd III, R.F. Petty H.R. J. Immunol. 1994; 152: 4630-4640Google Scholar, 5Xue W. Mizukami I. Todd III, R.F. Petty H.R. Cancer Res. 1997; 57: 1682-1689Google Scholar). We tested the ability of uPARs truncated forms to co-immunoprecipitate with β1, αv, α3, and α5 integrins and with caveolin, which, by fluorescence-activated cell sorter analysis, appeared to be expressed to the same extent in the different transfected cells (not shown). Transfected cell lysates were incubated with antibodies directed to β1, αv, α3, α5, and caveolin or with nonimmune serum; immunocomplexes were precipitated with protein A-Sepharose and analyzed by Western blot with the R2 anti-uPAR monoclonal antibody, which, as the R4 antibody, detects both intact and truncated uPARs. The truncated forms of uPAR expressed by D2D3-293 and D2D3wc-293 cells did not co-precipitate with any of the tested antigens, whereas, as expected, the intact form of uPAR expressed by uPAR-293 cells co-precipitated with all of them (Fig.3). Western blot analysis, with the same monoclonal antibody, of cell lysates before immunoprecipitation confirmed a similar expression of intact and truncated uPARs in transfected cells (Fig. 3, upper and lower left panels). The same results (not shown) were obtained in co-immunoprecipitation experiments performed with uPAR-293 cells treated with chymotrypsin, which cleaves uPAR at Tyr87-Ser88, thus leaving on the cell surface a two-domain uPAR carrying P88–92 at the N terminus (23Fazioli F. Resnati M. Sidenius N. Higashimoto Y. Appella E. Blasi F. EMBO J. 1997; 16: 7279-7286Google Scholar). Then removal of D1 abolishes the uPAR capability to interact with different integrins and with caveolin, independently of the presence of the P88–92 containing D1-D2 linker region. Because intact uPAR regulates integrin adhesive functions by interacting with them (3Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Google Scholar), we investigated whether truncated uPAR forms, which do not associate to integrins, lose also the capability to regulate cell adhesion. Adhesion assays were performed by plating transfected cells onto VN-, collagen (CG)-, fibronectin (FN)-, and laminin (LM)-coated wells. Control V-293 cells adhered, to different extents, to all tested substrates (Fig.4, upper panel); their adhesion capability was completely abolished by 2 mm EDTA, thus indicating its total integrin dependence (not shown). In addition, the GRGDSP peptide, inhibitor of RGD-mediated integrin binding, completely abolished V-293 cell adhesion to VN, whereas the related peptide GRGESP was ineffective (Fig. 4, lower panel). The expression of intact uPAR did not affect 293 cell adhesion to VN and caused a 50% reduction of 293 cell adhesion to CG, FN, and LM, as compared with V-293 (Fig. 4, upper panel). 2 mmEDTA also completely abolished uPAR-293 cell adhesion to CG, FN, and LM but not to VN (not shown); the presence of an RGD peptide (Fig. 4,lower panel) did not interfere with uPAR-293 cell adhesion to VN, thus indicating its integrin independence, because uPAR can act as a VN receptor (6Wei Y. Waltz D.A. Rao N. Drummond R.J. Rosenberg S. Chapman H.A. J. Biol. Chem. 1994; 269: 32380-32388Google Scholar). D2D3-293 and D2D3wc-293 cells showed an adhesion behavior to the different substrates very similar to that of V-293 control cells (Fig. 4, upper panel) and was completely abolished by 2 mm EDTA (not shown). The RGD peptide abolished also D2D3-293 and D2D3wc-293 cells adhesion to VN (Fig. 4,lower panel). Adhesion experiments were therefore in agreement with co-precipitation results, because only intact uPAR, which is able to interact with integrins, could regulate their activity; the removal of D1, independently of P88–92, abolishes uPAR interaction with integrins as well as its capability to regulate their functions. Intact uPAR, through lateral interactions with β1 integrins, leads to activation of ERK and cell prolif
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