Mapping of the Vitronectin-binding Site on the Urokinase Receptor
2007; Elsevier BV; Volume: 282; Issue: 18 Linguagem: Inglês
10.1074/jbc.m610184200
ISSN1083-351X
AutoresHenrik Gårdsvoll, Michael Ploug,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoThe urokinase-type plasminogen activator receptor (uPAR) has been implicated as a modulator of several biochemical processes that are active during tumor invasion and metastasis, e.g. extracellular proteolysis, cell adhesion, and cell motility. The structural basis for the high affinity interaction between the urokinase-type plasminogen activator (uPA) and uPAR, which focuses cell surface-associated plasminogen activation in vivo, is now thoroughly characterized by site-directed mutagenesis studies and x-ray crystallography. In contrast, the structural basis for the interaction between uPAR and the extracellular matrix protein vitronectin, which is involved in the regulation of cell adhesion and motility, remains to be clarified. In this study, we have identified the functional epitope on uPAR that is responsible for its interaction with the full-length, extended form of vitronectin by using a comprehensive alanine-scanning library of purified single-site uPAR mutants (244 positions tested). Interestingly, the five residues identified as "hot spots" for vitronectin binding form a contiguous epitope consisting of two exposed loops connecting the central fourstranded β-sheet in uPAR domain I (Trp32, Arg58, and Ile63) as well as a proximal region of the flexible linker peptide connecting uPAR domains I and II (Arg91 and Tyr92). This binding topology provides the molecular basis for the observation that uPAR can form a ternary complex with uPA and vitronectin. Furthermore, it raises the intriguing possibility that the canonical receptor and inhibitor for uPA (uPAR and PAI-1) may have reached a convergent solution for binding to the somatomedin B domain of vitronectin. The urokinase-type plasminogen activator receptor (uPAR) has been implicated as a modulator of several biochemical processes that are active during tumor invasion and metastasis, e.g. extracellular proteolysis, cell adhesion, and cell motility. The structural basis for the high affinity interaction between the urokinase-type plasminogen activator (uPA) and uPAR, which focuses cell surface-associated plasminogen activation in vivo, is now thoroughly characterized by site-directed mutagenesis studies and x-ray crystallography. In contrast, the structural basis for the interaction between uPAR and the extracellular matrix protein vitronectin, which is involved in the regulation of cell adhesion and motility, remains to be clarified. In this study, we have identified the functional epitope on uPAR that is responsible for its interaction with the full-length, extended form of vitronectin by using a comprehensive alanine-scanning library of purified single-site uPAR mutants (244 positions tested). Interestingly, the five residues identified as "hot spots" for vitronectin binding form a contiguous epitope consisting of two exposed loops connecting the central fourstranded β-sheet in uPAR domain I (Trp32, Arg58, and Ile63) as well as a proximal region of the flexible linker peptide connecting uPAR domains I and II (Arg91 and Tyr92). This binding topology provides the molecular basis for the observation that uPAR can form a ternary complex with uPA and vitronectin. Furthermore, it raises the intriguing possibility that the canonical receptor and inhibitor for uPA (uPAR and PAI-1) may have reached a convergent solution for binding to the somatomedin B domain of vitronectin. The urokinase-type plasminogen activator receptor (uPAR 2The abbreviations used are: uPAR, uPA receptor; uPA, urokinase-type plasminogen activator; ATF, amino-terminal fragment of uPA; PAI-1, plasminogen activator inhibitor 1; SMB, somatomedin-B like domain of vitronectin; Vn, vitronectin; WT, wild type; RU, resonance units; GFD, growth factor-like domain of uPA. 2The abbreviations used are: uPAR, uPA receptor; uPA, urokinase-type plasminogen activator; ATF, amino-terminal fragment of uPA; PAI-1, plasminogen activator inhibitor 1; SMB, somatomedin-B like domain of vitronectin; Vn, vitronectin; WT, wild type; RU, resonance units; GFD, growth factor-like domain of uPA./CD87) is a modular, glycolipid-anchored membrane protein (1Ploug M. Rønne E. Behrendt N. Jensen A.L. Blasi F. Danø K. J. Biol. Chem. 1991; 266: 1926-1933Abstract Full Text PDF PubMed Google Scholar, 2Ploug M. Curr. Pharm. Des. 2003; 9: 1499-1528Crossref PubMed Scopus (150) Google Scholar). Through the specific binding of the urokinase-type plasminogen activator (uPA), uPAR assists in regulating and focalizing cell surface-associated plasminogen activation as demonstrated both in vitro (3Liu S. Bugge T.H. Leppla S.H. J. Biol. Chem. 2001; 276: 17976-17984Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 4Ellis V. Scully M.F. Kakkar V.V. J. Biol. Chem. 1989; 264: 2185-2188Abstract Full Text PDF PubMed Google Scholar) and in vivo (5Bolon I. Zhou H.M. Charron Y. Wohlwend A. Vassalli J.D. Am. J. Pathol. 2004; 164: 2299-2304Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 6Zhou H.M. Nichols A. Meda P. Vassalli J.D. EMBO J. 2000; 19: 4817-4826Crossref PubMed Scopus (90) Google Scholar, 7Liu S. Aaronson H. Mitola D.J. Leppla S.H. Bugge T.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 657-662Crossref PubMed Scopus (113) Google Scholar). The high affinity interaction between uPAR and uPA (KD < 1 nm) has been extensively characterized (2Ploug M. Curr. Pharm. Des. 2003; 9: 1499-1528Crossref PubMed Scopus (150) Google Scholar, 8Gårdsvoll H. Gilquin B. Le Du M.H. Ménez A. Jørgensen T.J. Ploug M. J. Biol. Chem. 2006; 281: 19260-19272Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Although all determinants required for this tight binding are contained within the small growth factor-like domain of uPA (GFD residues 1–48) (9Ploug M. Østergaard S. Hansen L.B. Holm A. Danø K. Biochemistry. 1998; 37: 3612-3622Crossref PubMed Scopus (80) Google Scholar), it critically depends on maintenance of a three-domain, modular structure of uPAR (10Ploug M. Ellis V. Danø K. Biochemistry. 1994; 33: 8991-8997Crossref PubMed Scopus (109) Google Scholar). Accordingly, site-directed mutagenesis and photoaffinity labeling studies have shown that elements located in distinct domains of uPAR are involved in the interactions with both uPA (8Gårdsvoll H. Gilquin B. Le Du M.H. Ménez A. Jørgensen T.J. Ploug M. J. Biol. Chem. 2006; 281: 19260-19272Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 11Gårdsvoll H. Danø K. Ploug M. J. Biol. Chem. 1999; 274: 37995-38003Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 12Li Y. Lawrence D.A. Zhang L. J. Biol. Chem. 2003; 278: 29925-29932Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 13Bdeir K. Kuo A. Mazar A. Sachais B.S. Xiao W. Gawlak S. Harris S. Higazi A.A. Cines D.B. J. Biol. Chem. 2000; 275: 28532-28538Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) and a potent 9-mer peptide antagonist (9Ploug M. Østergaard S. Hansen L.B. Holm A. Danø K. Biochemistry. 1998; 37: 3612-3622Crossref PubMed Scopus (80) Google Scholar, 14Ploug M. Biochemistry. 1998; 37: 16494-16505Crossref PubMed Scopus (55) Google Scholar, 15Ploug M. Østergaard S. Gårdsvoll H. Kovalski K. Holst-Hansen C. Holm A. Ossowski L. Danø K. Biochemistry. 2001; 40: 12157-12168Crossref PubMed Scopus (165) Google Scholar, 16Jørgensen T.J. Gårdsvoll H. Danø K. Roepstorff P. Ploug M. Biochemistry. 2004; 43: 15044-15057Crossref PubMed Scopus (47) Google Scholar). Two crystal structures solved for uPAR in complex with either a linear peptide antagonist (17Llinas P. Le Du M.H. Gårdsvoll H. Danø K. Ploug M. Gilquin B. Stura E.A. Ménez A. EMBO J. 2005; 24: 1655-1663Crossref PubMed Scopus (203) Google Scholar) or the amino-terminal fragment (ATF) of uPA (18Huai 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 (257) Google Scholar, 19Barinka C. Parry G. Callahan J. Shaw D.E. Kuo A. Bdeir K. Cines D.B. Mazar A. Lubkowski J. J. Mol. Biol. 2006; 363: 482-495Crossref PubMed Scopus (108) Google Scholar) have provided the structural basis for the existence of a composite ligand-binding site. The assembly of the three homologous domains in uPAR creates a large and deep central ligand-binding cavity, where aliphatic side chains, provided by uPAR domain I, establish a hydrophobic binding site on one side of the cavity. The high affinity for both uPA and the linear peptide antagonists is achieved by an intimate interaction with this cavity (8Gårdsvoll H. Gilquin B. Le Du M.H. Ménez A. Jørgensen T.J. Ploug M. J. Biol. Chem. 2006; 281: 19260-19272Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 17Llinas P. Le Du M.H. Gårdsvoll H. Danø K. Ploug M. Gilquin B. Stura E.A. Ménez A. EMBO J. 2005; 24: 1655-1663Crossref PubMed Scopus (203) Google Scholar, 18Huai 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 (257) Google Scholar), providing a well defined target site for rational drug design (20Rømer J. Nielsen B.S. Ploug M. Curr. Pharm. Des. 2004; 10: 2359-2376Crossref PubMed Scopus (100) Google Scholar). As opposed to this, the binding sites mediating the interactions between uPAR and its auxiliary binding partners, e.g. vitronectin (21Waltz D.A. Chapman H.A. J. Biol. Chem. 1994; 269: 14746-14750Abstract Full Text PDF PubMed Google Scholar, 22Wei 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 integrins (23Wei 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, 24Wei Y. Czekay R.P. Robillard L. Kugler M.C. Zhang F. Kim K.K. Xiong J.P. Humphries M.J. Chapman H.A. J. Cell Biol. 2005; 168: 501-511Crossref PubMed Scopus (115) Google Scholar), must reside outside this cavity, but the molecular mechanisms underlying these interactions are largely unknown. As these interactions are dependent on or modulated by receptor occupancy with uPA (22Wei 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, 25Sidenius N. Andolfo A. Fesce R. Blasi F. J. Biol. Chem. 2002; 277: 27982-27990Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 26Pluskota E. Soloviev D.A. Plow E.F. Blood. 2003; 101: 1582-1590Crossref PubMed Scopus (99) Google Scholar, 27Sidenius N. Blasi F. FEBS Lett. 2000; 470: 40-46Crossref PubMed Scopus (77) Google Scholar), uPAR may orchestrate the assembly of these ternary complexes on the cell surface and thus assist in the regulation of cell adhesion and migration. In accordance with this proposition, it has been reported that cell lines with low endogenous uPAR expression adhere poorly to vitronectin, but this adhesion is markedly promoted by receptor saturation with exogenously added uPA (21Waltz D.A. Chapman H.A. J. Biol. Chem. 1994; 269: 14746-14750Abstract Full Text PDF PubMed Google Scholar, 27Sidenius N. Blasi F. FEBS Lett. 2000; 470: 40-46Crossref PubMed Scopus (77) Google Scholar). Increased uPAR expression in vitro by transfection or cytokine treatment may, however, uncouple this ligand dependence enabling adhesion to vitronectin by a local high density of unoccupied uPAR (27Sidenius N. Blasi F. FEBS Lett. 2000; 470: 40-46Crossref PubMed Scopus (77) Google Scholar). Partitioning into lipid rafts may generate such a local high density of uPAR and could thus play a regulatory role for uPAR-mediated vitronectin adhesion (28Cunningham O. Andolfo A. Santovito M.L. Iuzzolino L. Blasi F. Sidenius N. EMBO J. 2003; 22: 5994-6003Crossref PubMed Scopus (125) Google Scholar). In contrast to the well characterized uPAR-ATF interaction, the structural elements in the uPAR·uPA complex that are responsible for the interaction with matrix-deposited vitronectin are largely undefined. Two important functional relationships have nonetheless been established for this interaction. First, the binding sites for uPA and vitronectin on uPAR must be completely nonoverlapping because of the ability to form tri-molecular complexes. Second, the integrity of the intact three-domain structure of uPAR is mandatory for this complex formation (27Sidenius N. Blasi F. FEBS Lett. 2000; 470: 40-46Crossref PubMed Scopus (77) Google Scholar, 29Høyer-Hansen G. Behrendt N. Ploug M. Danø K. Preissner K.T. FEBS Lett. 1997; 420: 79-85Crossref PubMed Scopus (131) Google Scholar). So far, only one study (12Li Y. Lawrence D.A. Zhang L. J. Biol. Chem. 2003; 278: 29925-29932Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) has sought to define the functional epitope on uPAR for this interaction. Using homologue-scanning mutagenesis, by which segments of the individual domains of uPAR were swapped with the corresponding sequences in the single domain homologue CD59, Li et al. (12Li Y. Lawrence D.A. Zhang L. J. Biol. Chem. 2003; 278: 29925-29932Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) reported that two segments in uPAR domain II (Asn172–Lys175 and Glu183–Asn186) 3The numbering of amino acid residues in PAI-1, uPA, uPAR, and vitronectin refers to the cDNA-derived sequences omitting the signal sequences. Nomenclature for the secondary structure elements in uPAR follows the conventions established for snake venom α-neurotoxins and are more explicitly clarified for the modular domains in uPAR (17Llinas P. Le Du M.H. Gårdsvoll H. Danø K. Ploug M. Gilquin B. Stura E.A. Ménez A. EMBO J. 2005; 24: 1655-1663Crossref PubMed Scopus (203) Google Scholar). 3The numbering of amino acid residues in PAI-1, uPA, uPAR, and vitronectin refers to the cDNA-derived sequences omitting the signal sequences. Nomenclature for the secondary structure elements in uPAR follows the conventions established for snake venom α-neurotoxins and are more explicitly clarified for the modular domains in uPAR (17Llinas P. Le Du M.H. Gårdsvoll H. Danø K. Ploug M. Gilquin B. Stura E.A. Ménez A. EMBO J. 2005; 24: 1655-1663Crossref PubMed Scopus (203) Google Scholar). were important for vitronectin binding but not for uPA binding. To gain further insight into the molecular details of this interaction, we have therefore probed the interaction between purified pro-uPA·uPAR complexes and vitronectin by using an almost complete alanine-scanning library of single-site uPAR mutants. We find that the functional epitope for this interaction encompasses a small region located outside the central uPA-binding cavity and involves residues in two loops connecting the central four-stranded β-sheet in uPAR domain I as well as residues in the linker peptide connecting uPAR domains I and II. Intriguingly, this functional epitope on uPAR for vitronectin binding resembles the corresponding binding site on PAI-1 for the somatomedin B-like (SMB) domain of vitronectin as both interactions employ arginine as the critical hot spot residue. Chemicals and Reagents—All reagents for time-resolved fluorescence measurements (DELFIA®) as well as the stable Eu3+-chelate used for specific protein conjugation (i.e. N1-(p-isothiocyanatobenzyl)diethylenetriamine-N1,N2,N3,N3-tetraacetic acid) were purchased from PerkinElmer Life Sciences. Protein Preparations—Monomeric human vitronectin purified from plasma was obtained from Molecular Innovations (Southfield, MI). Recombinant active PAI-1 produced in Escherichia coli was a kind gift from Dr. M. D. Andersen (NOVO-Nordisk, Denmark). The monoclonal anti-uPAR antibody R2 (30Rønne E. Behrendt N. Ellis V. Ploug M. Danø K. Høyer-Hansen G. FEBS Lett. 1991; 288: 233-236Crossref PubMed Scopus (179) Google Scholar), which also recognizes uPAR-vitronectin complexes (29Høyer-Hansen G. Behrendt N. Ploug M. Danø K. Preissner K.T. FEBS Lett. 1997; 420: 79-85Crossref PubMed Scopus (131) Google Scholar), was produced in-house and was labeled with Eu3+ using the stable Eu3+-chelate of N1-(p-isothiocyanatobenzyl)diethylenetriamine-N1,N2,N3,N3-tetraacetic acid at pH 9.3 and a molar ratio of 20:1. This procedure yielded a labeling density of 2–4 molecules of Eu3+ per R2. The monoclonal anti-uPA clone 5 as well as a mixture of four monoclonal anti-PAI-1 antibodies (clones 2, 3, 5, and 7) also prepared in-house were labeled with Eu3+ following the same procedure. Expression and Purification of Soluble Recombinant Human uPAR—Soluble forms of human uPAR were expressed and secreted by Chinese hamster ovary cells (CHO) or Drosophila melanogaster Schneider 2 (S2) cells, which were stably transfected with either suPAR (residues 1–277) (33Rønne E. Behrendt N. Ploug M. Nielsen H.J. Wöllisch E. Weidle U. Danø K. Høyer-Hansen G. J. Immunol. Methods. 1994; 167: 91-101Crossref PubMed Scopus (53) Google Scholar) or pMTC/uPAR (residues 1–283) (31Gårdsvoll H. Werner F. Søndergaard L. Danø K. Ploug M. Protein Expression Purif. 2004; 34: 284-295Crossref PubMed Scopus (54) Google Scholar). These proteins are secreted to the conditioned medium because of a deletion of the carboxyl-terminal signal sequence that is required for glycolipid anchoring (1Ploug M. Rønne E. Behrendt N. Jensen A.L. Blasi F. Danø K. J. Biol. Chem. 1991; 266: 1926-1933Abstract Full Text PDF PubMed Google Scholar, 2Ploug M. Curr. Pharm. Des. 2003; 9: 1499-1528Crossref PubMed Scopus (150) Google Scholar). Single-site alanine replacements were introduced into pMTC/uPAR by site-directed mutagenesis using a previously designed three-gene cassette approach (31Gårdsvoll H. Werner F. Søndergaard L. Danø K. Ploug M. Protein Expression Purif. 2004; 34: 284-295Crossref PubMed Scopus (54) Google Scholar), and the corresponding soluble uPAR mutants (244 in all) were expressed by S2 cells and immunoaffinity-purified as described (8Gårdsvoll H. Gilquin B. Le Du M.H. Ménez A. Jørgensen T.J. Ploug M. J. Biol. Chem. 2006; 281: 19260-19272Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Two additional uPAR variants with multiple mutations were generated in which the sequences 172NTTK175 and 183ELEN186 were swapped with the corresponding sequences found in the glycolipid-anchored, single domain homologue CD59, i.e. 65KKDL68 and 79GTSL82. In addition, selected positions were mutated to residues other than alanine. The following mutants were produced: R30E, R30D, R30K, W32F, W32Y, E37K, R58K, R58E, R58D, I63L, I63V, R91D, R91E, R91K, Y92F, and Y92W. All constructs were verified by DNA sequencing using an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA), and the identities of the individually purified uPAR mutants were also verified at the protein level by peptide-mass mapping using in-gel digestion of the reduced and alkylated protein followed by mass assignment and MS/MS-sequencing using an Autoflex™ TOF-TOF II (8Gårdsvoll H. Gilquin B. Le Du M.H. Ménez A. Jørgensen T.J. Ploug M. J. Biol. Chem. 2006; 281: 19260-19272Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). As judged from SDS-PAGE of ∼5 μg of reduced and alkylated sample, the purity of these uPAR mutant preparations was >95%. Concentrations of purified uPARWT were accurately quantified by amino acid composition analyses (32Barkholt V. Jensen A.L. Anal. Biochem. 1989; 177: 318-322Crossref PubMed Scopus (291) Google Scholar). Protein concentrations of the purified uPAR mutants were determined using an extinction coefficient E280nm1% of 9.2 (33Rønne E. Behrendt N. Ploug M. Nielsen H.J. Wöllisch E. Weidle U. Danø K. Høyer-Hansen G. J. Immunol. Methods. 1994; 167: 91-101Crossref PubMed Scopus (53) Google Scholar) except for those mutants involving Trp32 or Trp129 for which an estimated E280nm1% of 6.8 was used. Expression and Purification of Recombinant Human Pro-uPAS356A and Human Vitronectin Fragments—Human pro-uPA was produced by Drosophila S2 cells stably transfected with a pMTB/uPA (residues 1–411) vector. Expression of pro-uPAWT or the active site-mutated pro-uPAS356A was induced by 0.5 mm Cu2SO4 as described previously (31Gårdsvoll H. Werner F. Søndergaard L. Danø K. Ploug M. Protein Expression Purif. 2004; 34: 284-295Crossref PubMed Scopus (54) Google Scholar), but in this particular case the S2 media also contained 10 μg/ml aprotinin to prevent activation of pro-uPA. The pro-uPA was isolated by immunoaffinity chromatography using an immobilized anti-uPA monoclonal antibody (clone 6). The purity of this pro-uPA was >95%, and the preparation showed no evidence of conversion as judged by SDS-PAGE. The identity of the purified pro-uPA was verified by MS as described (8Gårdsvoll H. Gilquin B. Le Du M.H. Ménez A. Jørgensen T.J. Ploug M. J. Biol. Chem. 2006; 281: 19260-19272Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Protein concentration of a stock solution of pro-uPAS356A was accurately determined by amino acid composition analysis (32Barkholt V. Jensen A.L. Anal. Biochem. 1989; 177: 318-322Crossref PubMed Scopus (291) Google Scholar). Vitronectin fragment 1–121 was expressed in S2 cells using the expression vector pMTC/Vn-(1–121)/uPAR-DIII (34Gårdsvoll H. Hansen L.V. Jørgensen T.J. Ploug M. Protein Expression Purif. 2007; 52: 384-394Crossref PubMed Scopus (36) Google Scholar). This construction covers vitronectin residues 1–121 followed by an enterokinase cleavage sequence (DDDDK) and finally a carboxyl-terminal tag comprising uPAR domain III (uPAR-DIII residues 182–283) to assist subsequent immunoaffinity purification (34Gårdsvoll H. Hansen L.V. Jørgensen T.J. Ploug M. Protein Expression Purif. 2007; 52: 384-394Crossref PubMed Scopus (36) Google Scholar). The following single-site alanine replacements (F13A, V15A, D22A, E23A, L24A, S26A, Y27A, Y28A, and Q29A) as well as the double substitutions (T50A/T57A and T50E/T57E) were introduced into pMTC/Vn-(1–121)/uPAR-DIII as described previously (35Picard V. Ersdal-Badju E. Lu A. Bock S.C. Nucleic Acids Res. 1994; 22: 2587-2591Crossref PubMed Scopus (216) Google Scholar). All constructs were verified by DNA sequencing. These fusion proteins were purified by immunoaffinity chromatography using an immobilized anti-uPAR antibody (R2), as outlined previously for the purification of uPAR, and used as uncleaved fusion proteins. Human GFD-(1–48) and SMB-(1–47) domains fused to a carboxyl-terminal His6 tag were expressed by stably transfected Pichia pastoris strain X-33 using the yeast expression vector pPICZα. The secreted domains were purified by Ni2+-nitrilotriacetic acid affinity chromatography followed by reversed-phase chromatography as described previously (8Gårdsvoll H. Gilquin B. Le Du M.H. Ménez A. Jørgensen T.J. Ploug M. J. Biol. Chem. 2006; 281: 19260-19272Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Assessing uPAR Binding to Immobilized Vitronectin by Dissociation-enhanced Time-resolved Fluorescence—White Maxisorb fluoroplates (Nunc, Roskilde, Denmark) were coated overnight at 4 °C with 1 μg/μl human vitronectin in 0.5 m carbonate buffer, pH 9.6. Excess binding sites were blocked by a brief incubation with SuperBlock™ (Pierce) diluted to 50% (v/v) in 0.04 m NaH2PO4 and 0.3 m NaCl, pH 7.4. Complexes between 10 nm pro-uPAS356A and 100 nm uPAR were preformed by incubation for 30 min at room temperature, and the interaction with immobilized vitronectin was subsequently allowed to proceed for 60 min at room temperature on an orbital shaker. All incubations and dilutions were performed in DELFIA® assay buffer (50 mm Tris-buffered saline, pH 7.8, containing 0.5% (w/v) BSA, bovine immunoglobulin, 0.04% (v/v) Tween 40, and 20 μm diethylenetriaminepentaacetic acid). After washing the fluoroplates six times, they were incubated for 60 min with 0.6 μg/ml of the Eu3+-labeled R2 monoclonal anti-uPAR antibody in DELFIA® assay buffer followed by six additional washes. Finally, the Eu3+ attached to the receptor-bound R2 was dissociated from its chelator by incubation in DELFIA® enhancement solution for 5 min. The fluorescence of the free Eu3+ was measured by time-resolved fluorescence using a Fluostar Galaxy fluorometer (PerkinElmer Life Sciences) with excitation set at 340 nm and reading emission at 615 nm with a 400-μs delay and a 400-μs acquisition window. In a modified version of this binding assay, fusion proteins containing derivatives of vitronectin linked to uPAR domain III (Vn-(1–121)/uPAR-DIII) were immobilized on white Maxisorb fluoroplates using a 2-fold dilution series in coating buffer covering 0.12 to 1 μg/ml. After blocking excess binding sites as described above, the coated wells were either incubated with preformed pro-uPA·uPAR complexes (100 nm uPAR preincubated with 10 nm pro-uPAS356A as described above), 10 nm active PAI-1, or with buffer alone. In these experiments the DELFIA® assay buffer included an additional 0.1% (v/v) Tween 20 and 0.25 m NaCl. The level of immobilized fusion protein was assessed by adding 0.6 μg/ml of the Eu3+-labeled monoclonal anti-uPAR antibody R2 to wells incubated with buffer only. Quantification of the pro-uPA-uPAR interaction with the immobilized vitronectin fusion proteins was accomplished by incubating the relevant wells with the Eu3+-labeled monoclonal anti-uPA antibody clone 5 and measuring the released Eu3+ as described above. PAI-1 binding was measured by a similar approach after incubation with a Eu3+-labeled mixture of four monoclonal anti-PAI-1 antibodies. Surface Plasmon Resonance Studies—All real time interaction studies were carried out on a Biacore 3000™ instrument (Biacore, Uppsala, Sweden) using 10 mm HEPES, 150 mm NaCl, 3 mm EDTA, and 0.005% (v/v) surfactant P-20 at pH 7.4 as running buffer. Human recombinant pro-uPAS356A (0.25 to 1 μg/ml) was immobilized covalently on a carboxymethylated dextran matrix (CM5 sensor chip) using N-hydroxysuccinimide/N-ethyl-N′-[3-(diethylamino)propyl]carbodiimide as described previously (11Gårdsvoll H. Danø K. Ploug M. J. Biol. Chem. 1999; 274: 37995-38003Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). To obtain comparative evaluations of the kinetics for the uPA-uPAR interaction, 2-fold serial dilutions of the various uPAR mutants (2–200 nm in running buffer) were analyzed in parallel for uPA binding at a flow rate of 50 μl/min at 20 °C. Compared with previous studies (8Gårdsvoll H. Gilquin B. Le Du M.H. Ménez A. Jørgensen T.J. Ploug M. J. Biol. Chem. 2006; 281: 19260-19272Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 11Gårdsvoll H. Danø K. Ploug M. J. Biol. Chem. 1999; 274: 37995-38003Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), in this study we have used a very high flow rate to minimize effects from mass transport limitations on the assessment of the kinetic rate constants. After each run the sensor chip was regenerated by two consecutive injections of 0.1 m acetic acid in 0.5 m NaCl. The kinetic rate constants, kon and koff, were derived from these real time interaction analyses by fitting the association and dissociation phases to a bimolecular interaction model using the BIAevaluation 4.1 software (Biacore, Uppsala, Sweden), as described in detail previously (8Gårdsvoll H. Gilquin B. Le Du M.H. Ménez A. Jørgensen T.J. Ploug M. J. Biol. Chem. 2006; 281: 19260-19272Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). For direct affinity measurements of the SMB-uPAR interaction, purified human uPARWT, uPARW32A, and uPARR91A (2.5 μg/ml) were immobilized by conventional amine chemistry in separate flow cells on a single CM5 sensor chip with coupling yields ranging from 1,500 to 2,000 RU. 2-Fold serial dilutions of either GFD-(1–48)-His6 (0.5–125 nm) or SMB-(1–47)-His6 (0.02–80 μm) were analyzed in parallel for uPAR binding at a flow rate of 50 μl/min at 4 °C. Kinetic rate constants for GFD binding were derived by fitting the data to a single binding site model using BIAevaluation 4.1 software (8Gårdsvoll H. Gilquin B. Le Du M.H. Ménez A. Jørgensen T.J. Ploug M. J. Biol. Chem. 2006; 281: 19260-19272Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). KD and Rmax values for the SMB-uPAR interaction were calculated from the equilibrium binding isotherm by nonlinear curve fitting assuming a model with saturation of a single binding site as shown in Equation 1, Req=Rmax[SMB]KD+[SMB]Eq.1 The equilibrium binding (Req) and binding capacity (Rmax) of the sensor chip are determined in resonance units (RU), but these can be transformed into binding densities using the approximation that 1000 RU equals 1 ng/mm2. For affinity measurements of the interaction between SMB and preformed pro-uPA·uPAR complexes, the immobilized uPAR mutants were initially saturated by 200-μl injections of 200 nm solutions of pro-uPAS356A, ATF-(1–143), or GFD-(1–48) before 67 μl of 2-fold serial dilution series of SMB were injected (up to 10 μm). Quantification of the Interaction between Immobilized Vitronectin and Pro-uPA·uPAR Complexes Using Time-resolved Fluorescence—Cellular attachment and adhesion studies to vitronectin are often conducted with immobilized protein in microtiter plates because the adsorbed vitronectin in such studies is considered a suitable in vitro surrogate for the natural, matrix-embedded protein. Several experiments employing either cell cultures (21Waltz D.A. Chapman H.A. J. Biol. Chem. 1994; 269: 14746-14750Abstract Full Text PDF PubMed Google Scholar, 27Sidenius N. Blasi F. FEBS Lett. 2000; 470: 40-46Crossref PubMed Scopus (77) Google Scholar) or purified components (25Sidenius N. Andolfo A. Fesce R. Blasi F. J. Biol. Chem. 2002; 277: 27982-27990Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 29Høyer-Hansen G. Behrendt N. Ploug M. Danø K. Preissner K.T. FEBS Lett. 1997; 420: 79-85Crossref PubMed Scopus (131
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