Urokinase Regulates Vitronectin Binding by Controlling Urokinase Receptor Oligomerization
2002; Elsevier BV; Volume: 277; Issue: 31 Linguagem: Inglês
10.1074/jbc.m111736200
ISSN1083-351X
AutoresNicolai Sidénius, Annapaola Andolfo, Riccardo Fesce, Francesco Blasi,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoAdhesion of monocytes to the extracellular matrix is mediated by a direct high affinity interaction between cell-surface urokinase-type plasminogen activator (uPA) receptor (uPAR) and the extracellular matrix protein vitronectin. We demonstrate a tight connection between uPA-regulated uPAR oligomerization and high affinity binding to immobilized vitronectin. We find that binding of soluble uPAR (suPAR) to immobilized vitronectin is strictly ligand-dependent with a linear relationship between the observed binding and the concentration of ligand added. Nevertheless, a comparison of experimentally obtained binding curves to those generated using a simple equilibrium model suggests that the high affinity vitronectin-binding pro-uPA·suPAR complex contains two molecules of suPAR. In co-immunoprecipitation experiments, using different epitope-tagged suPAR molecules, suPAR/suPAR co-immunoprecipitation displayed a similar uPA dose dependence as that observed for vitronectin binding, demonstrating that the high affinity vitronectin-binding complex indeed contains oligomeric suPAR. Structurally, the kringle domain of uPA was found to be critical for the formation of the vitronectin-binding competent complex because the amino-terminal fragment, but not the growth factor-like domain, behaved as a full-length uPA. Our data represent the first demonstration of functional, ligand-induced uPAR oligomerization having extensive implications for glycosylphosphatidylinositol-anchored receptors in general, and for the biology of the uPA/uPAR system in particular. Adhesion of monocytes to the extracellular matrix is mediated by a direct high affinity interaction between cell-surface urokinase-type plasminogen activator (uPA) receptor (uPAR) and the extracellular matrix protein vitronectin. We demonstrate a tight connection between uPA-regulated uPAR oligomerization and high affinity binding to immobilized vitronectin. We find that binding of soluble uPAR (suPAR) to immobilized vitronectin is strictly ligand-dependent with a linear relationship between the observed binding and the concentration of ligand added. Nevertheless, a comparison of experimentally obtained binding curves to those generated using a simple equilibrium model suggests that the high affinity vitronectin-binding pro-uPA·suPAR complex contains two molecules of suPAR. In co-immunoprecipitation experiments, using different epitope-tagged suPAR molecules, suPAR/suPAR co-immunoprecipitation displayed a similar uPA dose dependence as that observed for vitronectin binding, demonstrating that the high affinity vitronectin-binding complex indeed contains oligomeric suPAR. Structurally, the kringle domain of uPA was found to be critical for the formation of the vitronectin-binding competent complex because the amino-terminal fragment, but not the growth factor-like domain, behaved as a full-length uPA. Our data represent the first demonstration of functional, ligand-induced uPAR oligomerization having extensive implications for glycosylphosphatidylinositol-anchored receptors in general, and for the biology of the uPA/uPAR system in particular. urokinase-type plasminogen activator uPA receptor soluble uPAR vitronectin bovine serum albumin phosphate-buffered saline growth factor-like domain amino-terminal fragment catalytic domain kringle domain Cell migration and invasion are important processes in many patho/physiological conditions such as tumor invasion, angiogenesis, and inflammation. Plasminogen activators, their inhibitors, and their cell-surface receptor(s) play central roles in these processes by regulating extracellular proteolysis, cell adhesion, and signal transduction. In tissues, extracellular proteolysis is controlled by the production of plasmin that is generated by plasminogen activators, mainly urokinase (uPA)1 (1Johnsen M. Lund L.R. Romer J. Almholt K. Dano K. Curr. Opin. Cell Biol. 1998; 10: 667-671Crossref PubMed Scopus (326) Google Scholar), which binds to a specific membrane receptor, uPAR. Fully processed human uPAR is a 45–55-kDa glycoprotein linked to the outer membrane leaflet by a glycosylphosphatidylinositol lipid anchor (2Ploug 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). The protein is composed of three homologous domains with a disulfide bonding pattern characteristic of the uPAR/Ly-6 superfamily (3Ploug M. Kjalke M. Rønne E. Weidle U. Høyer-Hansen G. Danø K. J. Biol. Chem. 1993; 268: 17539-17546Abstract Full Text PDF PubMed Google Scholar). Besides providing the cells with the means to perform directed extracellular matrix degradation, binding of uPA to uPAR has profound effects on cell adhesion, migration, and proliferation (4Ossowski L. Aguirre-Ghiso J.A. Curr. Opin. Cell Biol. 2000; 12: 613-620Crossref PubMed Scopus (355) Google Scholar, 5Chapman H.A. Curr. Opin. Cell Biol. 1997; 9: 714-724Crossref PubMed Scopus (421) Google Scholar, 6Blasi F. Immunol. Today. 1997; 18: 415-417Abstract Full Text PDF PubMed Scopus (240) Google Scholar). Although binding to uPAR is always required, these latter processes are often independent of the proteolytic activity of uPA, strongly suggesting that other protein interactions are involved. Indeed, several data indicate that a conformational change in uPAR is capable of profoundly modifying its biological properties. First, it has been shown that uPA binding to uPAR causes the appearance of novel binding sites for vitronectin (Vn) (7Waltz D.A. Chapman H.A. J. Biol. Chem. 1994; 269: 14746-14750Abstract Full Text PDF PubMed Google Scholar, 8Higazi A.A. Upson R.H. Cohen R.L. Manuppello J. Bognacki J. Henkin J. McCrae K.R. Kounnas M.Z. Strickland D.K. Preissner K.T. Lawler Cines D.B. Blood. 1996; 88: 542-551Crossref PubMed Google Scholar, 9Høyer-Hansen G. Behrendt N. Ploug M. Danø K. Preissner K.T. FEBS Lett. 1997; 420: 79-85Crossref PubMed Scopus (131) Google Scholar, 10Sidenius N. Blasi F. FEBS Lett. 2000; 470: 40-46Crossref PubMed Scopus (77) Google Scholar), thrombospondin (8Higazi A.A. Upson R.H. Cohen R.L. Manuppello J. Bognacki J. Henkin J. McCrae K.R. Kounnas M.Z. Strickland D.K. Preissner K.T. Lawler Cines D.B. Blood. 1996; 88: 542-551Crossref PubMed Google Scholar), uPAR-associated protein (11Behrendt N. Jensen O.N. Engelholm L.H. Mortz E. Mann M. Dano K. J. Biol. Chem. 2000; 275: 1993-2002Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), and the disappearance of binding sites for the α2-macroglobulin receptor (8Higazi A.A. Upson R.H. Cohen R.L. Manuppello J. Bognacki J. Henkin J. McCrae K.R. Kounnas M.Z. Strickland D.K. Preissner K.T. Lawler Cines D.B. Blood. 1996; 88: 542-551Crossref PubMed Google Scholar). Furthermore, uPA-induced chemotaxis (12Boyle M.D. Chiodo V.A. Lawman M.J. Gee A.P. Young M. J. Immunol. 1987; 139: 169-174PubMed Google Scholar, 13Gudewicz P.W. Gilboa N. Biochem. Biophys. Res. Commun. 1987; 147: 1176-1181Crossref PubMed Scopus (90) Google Scholar, 14Del Rosso M. Anichini E. Pedersen N. Blasi F. Fibbi G. Pucci M. Ruggiero M. Biochem. Biophys. Res. Commun. 1993; 190: 347-352Crossref PubMed Scopus (82) Google Scholar, 15Gyetko M.R. Todd R.F.R. Wilkinson C.C. Sitrin R.G. J. Clin. Invest. 1994; 93: 1380-1387Crossref PubMed Scopus (288) Google Scholar, 16Resnati M. Guttinger M. Valcamonica S. Sidenius N. Blasi F. Fazioli F. EMBO J. 1996; 15: 1572-1582Crossref PubMed Scopus (303) Google Scholar) can be mimicked by proteolytic cleavage of uPAR that generates uPAR fragments that act as potent inducers of chemotaxis in cells lacking endogenous uPAR (16Resnati M. Guttinger M. Valcamonica S. Sidenius N. Blasi F. Fazioli F. EMBO J. 1996; 15: 1572-1582Crossref PubMed Scopus (303) Google Scholar,17Fazioli F. Resnati M. Sidenius N. Higashimoto Y. Appella E. Blasi F. EMBO J. 1997; 16: 7279-7286Crossref PubMed Scopus (230) Google Scholar). However, although extensive evidence has been presented that uPAR entertains complex interactions with other proteins, very little is known about how these interactions are regulated at the molecular level. In this paper we have addressed the possibility that uPA binding influences uPAR oligomerization and that uPAR oligomerization is a major determinant for its interaction with other proteins. As a paradigm, we have employed the ability of uPAR to bind Vn, a function that has been shown previously to induce cell adhesion (7Waltz D.A. Chapman H.A. J. Biol. Chem. 1994; 269: 14746-14750Abstract Full Text PDF PubMed Google Scholar, 10Sidenius N. Blasi F. FEBS Lett. 2000; 470: 40-46Crossref PubMed Scopus (77) Google Scholar) and to change gene expression during the differentiation of human myeloid U937 cells (18Rao N.K. Shi G.P. Chapman H.A. J. Clin. Invest. 1995; 96: 465-474Crossref PubMed Scopus (65) Google Scholar). A soluble variant of uPAR (residues 1–277) was expressed and purified from culture supernatants of transfected CHO cells as described previously (16Resnati M. Guttinger M. Valcamonica S. Sidenius N. Blasi F. Fazioli F. EMBO J. 1996; 15: 1572-1582Crossref PubMed Scopus (303) Google Scholar). SuPAR/FLAG was purified from transiently transfected COS-7 cells as described previously (17Fazioli F. Resnati M. Sidenius N. Higashimoto Y. Appella E. Blasi F. EMBO J. 1997; 16: 7279-7286Crossref PubMed Scopus (230) Google Scholar). Pro-uPA, purified from eukaryotic cell culture supernatants, was a kind gift from Dr. Jack Henkin (Abbott). The amino-terminal fragments (ATF) of uPA and LMW-uPA were obtained from American Diagnostica. The growth factor-like domain of uPA (GFD, amino acids 1–48) was a kind gift from Dr. Steve Rosenberg. The exact molarity of suPAR and pro-uPA was established by amino acid analysis (Research Consortium Inc.) Urea-purified vitronectin was purchased from Promega. The monoclonal antibody R2 was kindly provided by Dr. Gunilla Høyer-Hansen (Finsen Laboratories, Copenhagen, Denmark). The monoclonal antibody M2 and thepNPP substrate were obtained from Sigma. Secondary antibodies were from Amersham Biosciences (peroxidase-conjugated) and Dako (alkaline phosphatase-conjugated). Binding assays were performed in 96-well plates (NUNC Maxisorb) coated with Vn (0.1 ml/well, 1 μg/ml in 0.05m phosphate buffer, pH 9.6) overnight at 4 °C and blocked with 0.15 ml of 2% BSA in PBS for 1 h. All subsequent incubations were performed with reagents diluted in dilution buffer (PBS containing 1% BSA) at room temperature on an orbital shaker. Initially, wells were incubated with suPAR and other reagents diluted to the indicated concentrations for 1 h at room temperature (Note: as described under "Results," the way that reagents are mixed is important in determining the level of Vn binding obtained. To minimize possible experimental variability caused by this fact, the reagents were always mixed from 2-fold concentrated stocks (when 2 reagents were to be tested together) or 3-fold concentrated stocks (when 3 reagents were to be tested together), etc. and allowed to sit for >30 min at room temperature before being transferred to the Vn-coated wells.) After extensive washing with PBS containing 0.1% Tween 20 (PBS-T), the wells were probed for bound suPAR or suPAR/FLAG (as indicated) using monoclonal antibodies (2 μg/ml) specific for the relevant molecule (R2 and M2, respectively) for 30 min at room temperature. After washing, the wells were probed for bound antibody by incubation for 30 min at room temperature with an alkaline phosphatase-conjugated rabbit anti-mouse antibody diluted 1:1000. After another round of washing, bound alkaline phosphatase activity was assayed using the chromogenic substrate p-nitrophenyl phosphate and quantified by measuring the absorbance of samples at 405 nm in an ELISA plate reader. The binding data from the experiments shown in Fig. 1 were fitted by minimizing square errors to a simple equilibrium model: {(i) L + R ↔ LR; (ii) RL + R ↔ LR2}, where L and R represent pro-uPA (ligand) and suPAR (receptor), respectively, and total suPAR binding to Vn is represented byB = a[LR2] +b[LR]. In order to fit the data it was necessary to allow for a ±25% error in the estimates of the relative concentrations of pro-uPA and suPAR, perhaps being due to loss of the reagent because of marginal unspecific binding to the substrate, or to other causes. For calculations of the predicted curves presented in Fig. 1, a concentration correction factor of 0.76 for pro-uPA was used. Computations were performed in the Mathlab software environment (Matworks, Natik, MA) on a personal computer. For immunoprecipitation analysis the indicated concentrations of suPAR, suPAR/FLAG, pro-uPA, ATF, or GFD were mixed in dilution buffer and incubated for 1 h at room temperature. SuPAR/FLAG was then immunoprecipitated by addition of 20 μl of M2-agarose beads (Sigma) and incubation under gentle agitation for 1 h. After extensive washing of the beads with PBS-T, bound proteins were eluted by boiling in non-reducing SDS-PAGE sample buffer, size-fractionated by 10% SDS-PAGE, and transferred to polyvinylidene difluoride membranes by semi-dry electroblotting (19Kyhse Andersen J. J. Biochem. Biophys. Methods. 1984; 10: 203-209Crossref PubMed Scopus (2154) Google Scholar). After blocking with 5% non-fat dry milk in PBS-T, Western blots were developed by sequential incubations with biotinylated R2 and horseradish peroxidase-conjugated streptavidin, followed by chemiluminescent detection. suPAR, pro-uPA, and suPAR·pro-uPA complexes were analyzed by size-exclusion chromatography using a SuperdexTM 200 PC 3.2/30 column (Amersham Biosciences). A sample volume of 20 μl was loaded onto the column that was developed with PBS containing 0.5 m NaCl using a flow rate of 60 μl/min. The elution profile was recorded by measuring the absorbance of the eluted sample at 280 nm. The column was calibrated by measuring the retention times of several standard substances, calculating their corresponding K av values, and plotting theirK av values versus the logarithm of their molecular weights. The equation used to calculateK av was K av = (V e − V o )/(V t − V o ), where V e is the elution volume of the protein, V o the column void volume (= elution volume of blue dextran 2000), and V t the total bed volume (2.4 ml). The apparent molecular weights of pro-uPA, suPAR, and the 1:1 complex were determined by linear regression. The calibration molecules, their molecular weights, and their retention times (mean of two determinations ± S.D.) are as follows: thyroglobulin, 669 kDa, RT = 17.55 ± 0.08 min; ferritin, 440 kDa, 20.07 ± 0.02 min; catalase, 232 kDa, 22.41 ± 0.01 min; aldolase, 158 kDa, 22.83 ± 0.04 min; BSA, 67 kDa, 24.55 ± 0.02 min; ovalbumin, 43 kDa, 26.00 ± 0.04 min; chymotrypsinogen A, 25 kDa, 28.75 ± 0.01 min; ribonuclease A, 13.7 kDa, 28.75 ± 0.01 min; and blue dextran 2000, >2000 kDa, 14.79 ± 0.06 min. To study the mechanism of uPAR binding to Vn, we have exploited a simplein vitro binding assay in which Vn-coated plastic surfaces are incubated with suPAR in the presence or absence of the reagents to be tested. After removal of unbound reagents, bound suPAR was quantitated by sequential incubations with reagent-specific primary antibodies, secondary enzyme-conjugated antibodies, and finally a colorimetric substrate (see "Experimental Procedures"). In this assay, suPAR displays specific, high affinity, and ligand-dependent binding to Vn (Fig.1 A), indicating that the high affinity Vn-binding form of uPAR is a complex between uPAR and pro-uPA (7Waltz D.A. Chapman H.A. J. Biol. Chem. 1994; 269: 14746-14750Abstract Full Text PDF PubMed Google Scholar, 10Sidenius N. Blasi F. FEBS Lett. 2000; 470: 40-46Crossref PubMed Scopus (77) Google Scholar). However, although pro-uPA was clearly required for suPAR binding to Vn, it was also strongly inhibitory when present in excess of suPAR. This suggests that the mechanism of suPAR binding to immobilized Vn was more complex than a simple reaction in which one molecule of suPAR binds one molecule of pro-uPA forming a heterodimeric high affinity Vn-binding complex. To exclude the possibility that this unusual dose dependence of binding was an artifact caused by our detection system, we repeated the experiments using different antibodies to detect bound suPAR (a polyclonal rabbit antibody and two different mouse monoclonal antibodies), as well as using 125I-radiolabeled suPAR in which case no secondary reagent was required. However, independent of the method of detection, qualitatively identical binding curves were observed in all cases (results not shown), demonstrating that the behavior of the binding curves indeed reflects the ligand dependence of suPAR binding to immobilized Vn. To address the ligand dependence of suPAR binding to Vn, we further analyzed the extent of suPAR binding to immobilized Vn and its dependence on pro-uPA concentration (Fig. 1 B). No binding of suPAR was observed in the absence of low concentrations of pro-uPA. With increasing pro-uPA, we found a linear increase in suPAR binding, the extent of which was dependent only on the pro-uPA concentration (in the initial tract the curves superimpose for all concentrations of suPAR). Binding reached a maximum at a pro-uPA concentration close to one-half of the suPAR concentration and then declined at higher pro-uPAs. This suggests the existence of an optimal pro-uPA:suPAR stoichiometry for Vn binding, possibly 1:2. At pro-uPA concentrations above those of suPAR, the curves settle to a plateau close to one-third of the maximum, suggesting the formation of complex with 1:1 stoichiometry, less efficient in binding Vn. Curves of suPAR binding to Vn for increasing concentrations of suPAR (Fig. 1 C) rise linearly at suPAR concentrations up to one-half pro-uPA concentration (and superimpose at all concentrations of pro-uPA). At higher suPAR concentrations the slopes increase (and more so for higher pro-uPA concentrations) until the curves rather abruptly plateau at suPAR concentrations about twice those of pro-uPA. This again points to an optimal 1:2 pro-uPA:suPAR stoichiometry for Vn binding. The region of linear increase in Vn binding (pro-uPA/suPAR >1/2) and the more than linear increase between stoichiometry 2:1 and 1:2 again point to lower binding efficiency for complexes with 1:1 stoichiometry. Qualitatively, the experimental data thus suggest that suPAR binding to Vn may be explained by the binding of two stoichiometrically different pro-uPA·suPAR complexes. A high affinity 1:2 complex forms when suPAR is in excess of pro-uPA, and a low affinity 1:1 complex preferentially forms when pro-uPA is in excess of suPAR. To address mathematically the validity of this mechanism, a simple equilibrium model was fit to the experimental data shown in Reactions 1 and 2, L+R↔KD(i)LRReaction 1 LR+R↔KD(ii)LR2Reaction 2 where L and R represent pro-uPA and suPAR, respectively, and total suPAR binding to Vn is represented by B =a[LR2] + b[LR]. The effectiveness ratio (b/a) represents the relative Vn binding activity of the LR and LR2 complexes. The curves predicted by this model were slightly sensitive to theK D values for Reactions 1 and 2, and best fits were obtained for low values of these parameters, K D (ii) <10−10 and K D (i) even lower (some 50-fold). This suggests that binding under the applied experimental conditions might constitute an almost irreversible reaction, and an equilibrium model might not be the best way to describe the system. However, most aspects of the experimental binding data represented in Fig. 1 were fully and quantitatively accounted for by this model. Experimental estimates are available in the literature for the affinity of the uPA/uPAR interaction (K D (i) ≈10−10m (20Ploug M. Ellis V. Danø K. Biochemistry. 1994; 33: 8991-8997Crossref PubMed Scopus (109) Google Scholar, 21Behrendt N. Rønne E. Danø K. J. Biol. Chem. 1996; 271: 22885-22894Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 22Ploug M. Rahbek Nielsen H. Nielsen P.F. Roepstorff P. Danø K. J. Biol. Chem. 1998; 273: 13933-13943Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 23Ploug M. Østergaard S. Hansen L.B. Holm A. Danø K. Biochemistry. 1998; 37: 3612-3622Crossref PubMed Scopus (80) Google Scholar, 24Bdeir 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)). Therefore, fits were recalculated by constraining K D (i) to this value (Fig. 1, B and C, dotted lines). The resulting estimate for K D (ii) was 0.77 nm, and an effectiveness ratio (b/a) of 0.061 was estimated for binding ofLR and LR 2 complexes to Vn. As can be seen in Fig. 1, crossing of the curves in B and the decrease to plateau in A are well predicted. Although the above binding model closely predicts the binding curves, it should be noted that it does not take into account the likely presence of different multimeric forms of Vn in the binding assay. In fact, it is possible that the high affinity pro-uPA·suPAR·Vn complex may have a 1:2:2 stoichiometry and the low affinity complex an 1:1:1 stoichiometry, etc. However, binding experiments using different preparations of Vn (native and denatured) resulted in identical binding curves (data not shown) suggesting that after adsorption to plastic Vn exposes the same suPAR binding epitopes independently of its original state of oligomerization. Pro-uPA has also been reported previously (25Moser T.L. Enghild J.J. Pizzo S.V. Stack M.S. Biochem. J. 1995; 307: 867-873Crossref PubMed Scopus (46) Google Scholar) to interact directly with Vn, and we therefore addressed the alternative possibility that pro-uPA in some way competes for an overlapping binding site on Vn. However, for several reasons this could not explain the inhibitory effect. First, the inhibitory effect of pro-uPA (Fig. 1) occurs at concentrations well below its apparent K m value for Vn (97 nm (25Moser T.L. Enghild J.J. Pizzo S.V. Stack M.S. Biochem. J. 1995; 307: 867-873Crossref PubMed Scopus (46) Google Scholar)). Second, the minimal concentration of pro-uPA required to observe inhibition was not fixed but correlated with the concentration of suPAR (Fig. 1). Third, preincubation of immobilized Vn with pro-uPA failed to prevent the concomitant binding of pro-uPA·suPAR complexes (Fig.2 A,columns F–I). Another possibility is that pro-uPA causes the release of pro-uPA·suPAR complexes from Vn. This might, for example, occur by proteolytic cleavage of suPAR by trace amounts of active two-chain uPA present in the pro-uPA preparation. However, even this did not explain the inhibitory effect as pro-uPA·suPAR complexes bound to Vn were resistant to release by an excess of free pro-uPA (Fig. 2 A, columns J–M). Taken together the data suggest that suPAR binding to Vn is inhibited by elevated pro-uPA due to a shift from binding-competent to less effective pro-uPA·suPAR complexes for binding to Vn. To test this possibility we performed binding experiments using pre-formed pro-uPA·suPAR complexes containing optimal amounts of pro-uPA and suPAR (Fig. 2 B). After a 1-h incubation, these pre-formed complexes were supplemented with increasing concentrations of pro-uPA, incubated for another hour, and then assayed for Vn binding activity (Fig. 2 B, columns F–H). For comparison, we analyzed the reaction having the same final concentrations of both components, but prepared without the two-step addition of pro-uPA (Fig.2 B, columns B–E). Indeed, the capacity of excess pro-uPA to inhibit suPAR binding to Vn was strongly reduced when the pro-uPA·suPAR complexes were allowed to form before the addition of the excess pro-uPA. These data demonstrate that what determines the level of suPAR binding to Vn is the ratio between suPAR and pro-uPA and not the absolute concentration of the two proteins. Consequently, the pro-uPA·suPAR complexes that bind with high affinity to Vn cannot be simple 1:1 complexes. A fundamental prediction of the model used to explain the binding data is the existence of complexes between pro-uPA and two (or more) molecules of suPAR. To address this possibility directly, we performed co-immunoprecipitation experiments using two different epitope-tagged suPAR preparations (Fig. 3). We had previously constructed, expressed, and purified a suPAR variant in which a short peptide epitope had been engineered onto Pro-274 of uPAR generating a recognition epitope for the monoclonal anti-FLAG antibody M2 (10Sidenius N. Blasi F. FEBS Lett. 2000; 470: 40-46Crossref PubMed Scopus (77) Google Scholar, 17Fazioli F. Resnati M. Sidenius N. Higashimoto Y. Appella E. Blasi F. EMBO J. 1997; 16: 7279-7286Crossref PubMed Scopus (230) Google Scholar). The addition of this epitope involved the removal of the three carboxyl-terminal amino acids (Asp-Leu-Asp, amino acids 275–277) present on "wild-type" suPAR (amino acids 1–277), and resulted in the complete destruction of the recognition epitope for the monoclonal anti-uPAR antibody R2. In contrast to the differential recognition by monoclonal antibodies, these two suPAR variants (from here on termed suPAR and suPAR/FLAG) display indistinguishable pro-uPA and Vn binding (Fig. 3 and results not shown). We mixed the two forms of suPAR and immunoprecipitated the mixture with the M2 anti-FLAG antibody. When the immunoprecipitate was blotted with R2 antibody, R2 did not recognize any protein in the M2 precipitate (Fig. 3 A, lane 7). However, in the presence of pro-uPA, suPAR was readily identified in the M2 immunoprecipitate (Fig. 3 A, lane 8). The specificity of the co-immunoprecipitation procedure was verified by immunoprecipitation of samples in which one, two, or all three reactants had been omitted (Fig. 3 A, lanes 1–6). In these samples no R2 reactive material was observed. We next compared the pro-uPA dose dependence of suPAR co-immunoprecipitation (Fig. 3 B) and Vn binding (Fig.3 C). In these experiments an exact correlation between suPAR oligomerization, as evidenced by co-immunoprecipitation, and Vn binding was observed, demonstrating that the generation of the high affinity Vn-binding complex involves pro-uPA-induced suPAR oligomerization. As the suPAR:suPAR co-immunoprecipitation experiments demonstrated the existence of higher order suPAR·pro-uPA complexes in the absence of Vn, we next sought to identify these complexes by size exclusion chromatography. To this end we subjected purified suPAR, pro-uPA, and mixtures of the two proteins to analytical gel filtration on a SuperdexTM 200 column (Fig.4 A). Analyzed individually, both suPAR (dotted curves) and pro-uPA (gray curves) filtered as single peaks with highly reproducible retention times (24.80 ± 0.07 (n = 3) and 27.67 ± 0.04 (n = 3) min, respectively). When mixtures of the suPAR and pro-uPA were analyzed (black curves), a single new peak (retention time, 22.91 ± 0.02 min (n = 5)), corresponding to the 1:1 pro-uPA·suPAR complex was observed. No other complexes could be identified. Furthermore, no apparent difference in the retention time of the pro-uPA·suPAR complex could be observed when the complex was formed in the presence of excess pro-uPA (22.91 ± 0.01 min (n = 2)) or in the presence of excess suPAR (22.92 ± 0.01 min (n = 2)). To exclude that the failure to detect ternary complexes by gel filtration was caused by a lack of resolution, the column performance was validated using mixtures of reference proteins (Fig. 4 B). In these experiments an excellent resolution was observed with a linear correlation (correlation coefficient 0.99) between the K avand the log-transformed molecular weights of the reference proteins (see "Experimental Procedures"). Based on the calibrations curve, the relative molecular masses of pro-uPA, suPAR, and the 1:1 complex were calculated to be 28.1 ± 0.6, 75.2 ± 1.1, and 144.2 ± 2.2 kDa, respectively. It thus appears that in contrast to pro-uPA, which behaves as a very compact molecule, both suPAR and the 1:1 complex may have an extended (non-spherical) shape in solution. Within the presented series of gel filtrations, the maximal difference in retention time between independent runs of the same protein (11 different proteins) was 0.13 min. A reduction in the retention time for the 1:1 pro-uPA·suPAR complex of twice this size (0.26 min) translated into a molecular mass increase of 13.4 kDa (9.2%). We therefore concluded that if the ternary complex had formed stoichiometrically, we would have observed it even if its molecular size was only marginally different from that of the 1:1 complex. The gel filtrations presented in Fig. 4 were all performed in the presence of 0.5 m NaCl to prevent interactions between the solid phase and pro-uPA which was otherwise observed at physiological salt concentrations (data not shown). This is unlikely to explain the failure to detect higher order complexes as suPAR binding to Vn was unaffected under these conditions (data not shown). Although the use of physiological salt concentrations did not allow a quantitative recovery of injected pro-uPA, both suPAR and pro-uPA·suPAR complexes filtered normally under these conditions (not shown). However, even under these conditions no material eluting prior to the 1:1 complex was ever observed (not shown). The gel filtration experiments thus indicated that either the higher order pro-uPA·suPAR complexes are only marginally larger that the 1:1 complex or not abundant enough for detection using this method. In any case the gel filtration experiments allowed us to compare the relative abundance of free pro-uPA, suPAR, and pro-uPA·suPAR complexes, with the Vn binding activity. To this end, aliquots of the samples applied to the gel filtration column were diluted and assayed for their Vn binding activity (Fig. 4 C). Samples containing excess suPAR (filtrations I and II) displayed approximately twice the Vn bindin
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