Báo cáo khoa học: Binding areas of urokinase-type plasminogen activator– plasminogen activator inhibitor-1 complex for endocytosis receptors of the low-density lipoprotein receptor family, determined by site-directed mutagenesis doc

Binding areas of urokinase-type plasminogen activator– plasminogen activator inhibitor-1 complex for endocytosis receptors of the low-density lipoprotein receptor family, determined by site-directed mutagenesis Sune Skeldal1, Jakob V Larsen1, Katrine E Pedersen1, Helle H Petersen1, Rikke Egelund1, Anni Christensen1, Jan K Jensen1,2, Jørgen Gliemann3 and Peter A Andreasen1,2 Department of Molecular Biology, University of Aarhus, Denmark Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Denmark Department of Medical Biochemistry, University of Aarhus, Denmark Keywords low-density lipoprotein receptor-related protein; plasminogen activator inhibitor 1; sorting protein-related receptor; urokinase plasminogen activator; very-low-density lipoprotein receptor Correspondence P A Andreasen, Department of Molecular Biology, University of Aarhus, Gustav Wied’s Vej 10C, 8000 Aarhus C, Denmark Fax: +45 86 12 31 78 Tel: +45 89 42 50 80 E-mail: pa@mb.au.dk (Received 17 July 2006, revised 20 September 2006, accepted 22 September 2006) doi:10.1111/j.1742-4658.2006.05511.x Some endocytosis receptors related to the low-density lipoprotein receptor, including low-density lipoprotein receptor-related protein-1A, very-lowdensity lipoprotein receptor, and sorting protein-related receptor, bind protease-inhibitor complexes, including urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and the uPA–PAI-1 complex The unique capacity of these receptors for high-affinity binding of many structurally unrelated ligands renders mapping of receptor-binding surfaces of serpin and serine protease ligands a special challenge We have mapped the receptor-binding area of the uPA–PAI-1 complex by site-directed mutagenesis Substitution of a cluster of basic residues near the 37-loop and 60-loop of uPA reduced the receptor-binding affinity of the uPA–PAI-1 complex approximately twofold Deletion of the N-terminal growth factor domain of uPA reduced the affinity 2–4-fold, depending on the receptor, and deletion of both the growth factor domain and the kringle reduced the affinity sevenfold The binding affinity of the uPA–PAI-1 complex to the receptors was greatly reduced by substitution of basic and hydrophobic residues in a-helix D and a-helix E of PAI-1 The localization of the implicated residues in the 3D structures of uPA and PAI-1 shows that they form a continuous receptor-binding area spanning the serpin as well as the A-chain and the serine protease domain of uPA Our results suggest that the 10–100-fold higher affinity of the uPA–PAI-1 complex compared with the free components depends on the bonus effect of bringing the binding areas on uPA and PAI-1 together on the same binding entity The low-density lipoprotein receptor (LDLR) family of endocytosis receptors has been implicated in binding and endocytosis of a large number of structurally unrelated proteins, including apolipoproteins, protease– inhibitor complexes, extracellular matrix proteins, and hormone carriers In mammals, this receptor family includes LDLR itself, low-density lipoprotein receptorrelated protein-1A (LRP-1A), LRP-1B, megalin or LRP-2, very-low-density lipoprotein receptor (VLDLR), and apolipoprotein E receptor-2 These Abbreviations a1-PI, a1-antiproteinase inhibitor; CTR, complement type repeat; HEK293T, human embryonic kidney cell line 293T; LDLR, low-density lipoprotein receptor; LRP, low-density lipoprotein receptor-related protein; PAI-1, plasminogen activator inhibitor 1; RAP, receptor-associated protein; RCL, reactive centre loop; sorLA, sorting protein-related receptor; SPD, serine protease domain; tPA, tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; VLDLR, very-low-density lipoprotein receptor FEBS Journal 273 (2006) 5143–5159 ª 2006 The Authors Journal compilation ª 2006 FEBS 5143 uPA–PAI-1 complex receptor binding S Skeldal et al receptors are constructed with the same types of domains, but with variable numbers of each type of domain The domains include complement-type repeats (CTRs), YWTD-repeat-containing b-propellers, and epidermal growth factor precursor domains The receptors also all have a transmembrane a-helix and a cytoplasmic C-terminal domain mediating endocytosis via clathrin-coated pits Generally, the CTRs are believed to mediate ligand binding The related receptor sorting protein-related receptor (sorLA), which in addition to other types of domains also contains CTRs, have a ligand repertoire overlapping that of the LDLR family One ligand common to all these receptors is the 40-kDa receptor-associated protein (RAP) [1] The crystal structures of the third domain of RAP in complex with a CTR pair from LDLR and of human rhinovirus serotype in complex with CTRs from VLDLR have recently been solved [2,3] Common to all of these structures is the fact that binding to the CTRs is mediated through basic and hydrophobic residues in the ligand Also, some inferences can be made from an X-ray structure analysis of LDLR crystals obtained at pH 5, in which the b-propeller bends back and makes contact with the CTR cluster in a way believed to mimic ligand binding [4] The fact that these receptors exhibit high-affinity binding of so many structurally unrelated ligands makes their ligand-binding potential a unique case of molecular recognition Particularly interesting ligands are the serine protease urokinase-type plasminogen activator (uPA), its primary serpin inhibitor, plasminogen activator inhibitor-1 (PAI-1), and the corresponding protease-serpin complex, which have been shown to bind to LRP-1A [5], megalin [6,7], VLDLR [8,9], LRP-1B [10], and sorLA [11] These receptors mediate endocytosis of uPA–PAI-1 complex accumulated on the cell surface by binding to the urokinase-type plasminogen activator receptor (uPAR), whereas endocytosis directly from the fluid phase is negligible [5] Although both free uPA and free PAI-1 exhibit a distinct affinity for these receptors, the uPA–PAI-1 complex binds to LRP-1A with an affinity much higher than that of the free components Thus, the Kd value for binding of the uPA–PAI-1 complex to these receptors is reported to be % nm [6,8,11,12], whereas that for binding of free PAI-1 is reported to be % 30 nm [11–13], and Kd values of 7–200 nm have been reported for binding of free u-PA [11,12,14] The molecular recognition between the receptors and the uPA–PAI-1 complex should therefore be seen in relation to the mechanism of protease–serpin complex formation X-ray crystal structure analyses have shown that serpins are globular proteins consisting of 5144 three b-sheets and nine a-helices [15] (Fig 1) Threedimensional structures of a covalently coupled a1-antiproteinase inhibitor (a1-PI)–trypsin complex [16], a covalently coupled a1-PI–elastase complex [17], and several reversible complexes between serpins and proteases with the active-site Ser replaced by Ala [18–20] have been reported The structures support biochemical and biophysical evidence that complex formation is initiated by formation of a reversible docking complex, in which the P1–P1¢ bond in the surface-exposed reactive centre loop (RCL) interacts with the active site of the protease Next, the P1–P1¢ bond is cleaved, the P1 residue coupled to the active-site Ser of the protease by an ester bond, the N-terminal part of the RCL inserted as strand in b-sheet A, and the protease translocated to the opposite pole of the serpin [15] Serpins are thus attacked by the proteases as substrates, but the normal catalytic cycle stops at the acylenzyme intermediate stage From the available 3D structures of stable protease–serpin complexes [16,17], it was inferred that the catalytic mechanism is halted because of distortion of the active site of the protease The energy needed for the distortion stems from stabilization of the serpin in the ‘relaxed’ conformation by insertion of the RCL into b-sheet A, as opposed to the ‘stressed’, relatively unstable active conformation RCL insertion can also occur after abortive complex formation as the result of complete cleavage of the P1–P1¢ bond or by insertion of the uncleaved RCL in latent PAI-1 [15] Unfortunately, there is no X-ray crystal structure analysis of any receptor–protease–inhibitor complexes However, on the basis of the established inhibitory mechanism for serpins, there are several possible explanations of the increased receptor-binding affinity of the uPA–PAI-1 complex compared with the affinity of the individual components, including increased affinity associated with a conformational change in PAI-1, increased affinity associated with a conformational change in uPA, and ⁄ or an avidity effect of two or more binding sites being brought together on the same ligand We have now addressed this problem using site-directed mutagenesis, introducing mutations into both uPA and PAI-1, and studying binding to VLDLR, LRP-1A, and sorLA We initially chose basic residues for mutation on the basis of evidence for receptor binding involving interactions between basic residues in the ligands and acidic residues in the receptor [21] Binding of the uPA–PAI-1 complex to LRP1A and VLDLR was previously shown to require basic residues in a-helix D and a-helix E in the flexible joint-region of the inhibitor [13,22,23] We therefore mutated residues adjacent to this region in FEBS Journal 273 (2006) 5143–5159 ª 2006 The Authors Journal compilation ª 2006 FEBS S Skeldal et al uPA–PAI-1 complex receptor binding Fig The 3D structures of PAI-1 and uPA The figure shows ribbon diagrams of the structure of the active conformation of PAI-1 [53] (pdb file 1B3K) and the structure of the SPD of uPA [27] (pdb file 1LMW) The localization of several a-helices and b-strands are indicated The diagrams were constructed with the use of SWISSPDBVIEWER the uPA–PAI-1 complex, and we provide evidence that the complex has an extended receptor-binding area spanning uPA as well as PAI-1 Results Screening of the effects of Ala substitutions in PAI-1 on uPA–PAI-1 complex–receptor binding in microtiter wells For the initial screening, our strategy was to substitute basic and hydrophobic residues in PAI-1 with Ala, prepare complexes between the variants and 125I-uPA, measure the binding of 20 pm radioactive complexes to VLDLR, LRP-1A, or sorLA immobilized in microtiter wells, and express the binding of the Ala-substituted complexes relative to the binding of the wild-type complex This relatively simple strategy was based on several facilitating arrangements We used PAI-1 expressed in a human cell line, because of solubility problems with nonglycosylated, bacterially expressed PAI-1 The source of PAI-1 for the preparation of the complexes was not purified PAI-1, but serum-free conditioned medium from human embryonic kidney cell line 293T (HEK293T) transfected with the corresponding cDNA A well-established procedure [24] was used to re-activate the latent PAI-1 in the medium by the use of denaturation with SDS and refolding by removing the SDS by addition of an excess of BSA 125I-uPA–PAI-1 complexes were prepared by adding 125I-uPA to the medium and purifying the complexes by immuno-affinity chromatography [24] As a control for the integrity of the 125I-uPA–PAI-1 complexes during the binding assay, we measured the binding of all Ala-substituted complexes to parallel wells coated with 20 lgỈmL)1 monoclonal antibody to PAI-1 (mAb2) This antibody coat bound 64 ± 10% of all complexes, and the binding of variant complexes always varied less than 20% from the binding of the wild-type complex Binding of the uPA–PAI-1 complex to the receptors was assumed to follow the equation: FEBS Journal 273 (2006) 5143–5159 ª 2006 The Authors Journal compilation ª 2006 FEBS 5145 uPA–PAI-1 complex receptor binding S Skeldal et al ẵRL ẳ ẵRT ẵL=Kd ỵ ẵLị in which [RL] is receptor-bound ligand, [R]T is total receptor concentration, [L] is the concentration of free ligand, and Kd is the equilibrium dissociation constant With the 20 pm concentrations of 125I-labeled ligands used here and Kd values % nm (see above), [L] < Kd, and the above equation is reduced to: < ẵRL=ẵLị ẳ ẵRT =Kd Hence, the fractional uPA–PAI-1 complex–receptor binding is expected to be inversely proportional to the Kd value Control experiments (not shown) confirmed the linear relationship between [RL] and [L] We therefore used the amount of receptor-bound complex between 125I-labelled uPA and mutated PAI-1, as compared with the amount of receptor-bound complex between 125I-labeled uPA and wild-type PAI-1, as a measure of the effect of the substitution on the Kd value With the accuracy and background binding of this assay, we expected that binding of up to 10-fold less than the control value would be different from background For analyses of the binding of complexes of uPA with PAI-1 mutants, we first mutated groups of three residues to Ala If a triple mutation resulted in more than a twofold reduction in binding of the uPA–PAI-1 complex to the receptor compared with the wild-type complex, we investigated the effect of substituting each residue separately For single mutants, a receptor-binding reduction of more than 1.5-fold was considered to define the residues involved in receptor binding Residues for Ala substitutions were selected on the basis of their proximity to the previously implicated residues in a-helix D and a-helix E, namely Lys71, Arg78, Lys82, Lys90, Arg120 and Lys124 [13,22,23] With this strategy, out of a total of 41 PAI-1 residues tested, we identified 11 of importance for binding of the uPA–PAI-1 complex to one, two, or all three receptors (Fig 2) However, the involvement of Lys124 in receptor binding could not be confirmed Screening of effects of Ala substitutions in uPA on uPA–PAI-1 complex–receptor binding in microtiter wells Besides the C-terminal, % 30-kDa serine protease domain (SPD), uPA contains an N-terminal, % 25kDa A-chain, consisting of a growth factor domain Fig Effect of Ala substitutions of PAI-1 residues on the binding of the uPA–PAI-1 complex to VLDLR, LRP-1A and sorLA in a solid-phase assay The binding of the variant complexes was expressed relative to the binding of wild-type complex in the same experiment Mean ± SD values are shown for at least three independent experiments As compared with the binding of wild-type complexes, the binding to the receptors of the complexes between uPA and the following PAI-1 mutants were reduced less than twofold when tested as triple mutants or less than 1.5-fold when tested individually, and these mutated residues were therefore considered to be unimportant in the binding: H4A, H5A; P6A; P7A; Y9A; Q58A; K67A; D69A; D70A; P75A; L77A; M85A; P87A; W88A; E92A; T96A; R103A; D104A; K106; L107A; Q109A; P113A; H114; F119A; S121A; K124A; Q125A; W141A; H145A; K178A °, Significantly different from binding observed with the corresponding wild-type (P < 0.01) 5146 FEBS Journal 273 (2006) 5143–5159 ª 2006 The Authors Journal compilation ª 2006 FEBS S Skeldal et al and a kringle domain [25] In agreement with previous reports on LRP-1A [12] and sorLA [11], we now demonstrate that a truncated version of uPA without the A-chain (LMW-uPA) has a sevenfold reduced affinity for VLDLR (Fig 3) Moreover, we demonstrated that a uPA variant with deletion of the growth factor domain (DGF-uPA) had 2–4-fold reduced binding to VLDLR, LRP-1A, and sorLA (see Fig 3) Suspecting the involvement of basic residues in the contact with the receptors, we measured the effect on receptor binding of substituting a cluster of three arginines (R108, R109, and R110) in the uPA kringle, but found no change in receptor binding after these mutations (Fig 3) At the moment, possible endocytosis receptorbinding residues in the kringle is not known We did not perform a mutational analysis of the growth factor domain, as this, under physiological conditions at the cell surface, is shielded from contact with the endocytosis receptors by binding to uPAR [5,12] We previously reported that a number of monoclonal antibodies against the SPD of uPA have epitopes localized in the 37-loop and 60-loop [26] (Fig 1) To preliminarily investigate whether a receptor-binding site exists in the SPD of uPA, we uPA–PAI-1 complex receptor binding studied the effect on uPA–PAI-1 complex–receptor binding of one such monoclonal antibody, mAb3689, with an epitope encompassing Arg179(36), His180(37), and Arg181(37a) in the 37-loop of uPA (amino-acid residues in the SPD of uPA will be referred to by a double numbering system, based on numbering from the N-terminus of the native protein with the chymotrypsin template numbering system in parentheses; residues in the N-terminal A-chain of uPA will be referred to by numbering from the N-terminus of the native protein [27]) The presence of the antibody reduced the binding significantly (Fig 3) We therefore studied the effect of Ala substitution of clusters of residues in the 37-loop and 60-loop on receptor binding and demonstrated that both sets of mutations reduced receptor binding 2–4fold (Fig 3) As a control, we found that mAb3689 did not reduce the binding of the variant with the mutations in its epitope in the 37-loop (Fig 3) Substitution of a number of other residues was without effect on receptor binding (Fig 3) We therefore concluded that two or more residues in the 37-loop and 60-loop of the SPD of uPA are part of the ligand– receptor interface Fig Effect of Ala substitutions of uPA residues on the binding of the uPA–PAI-1 complex to VLDLR, LRP-1A, and sorLA in a solid-phase assay The binding of the variant complexes was expressed relative to the binding of the wild-type complex in the same experiment Mean ± SD values are shown for at least three independent experiments Compared with binding of wild-type complexes, binding to the receptors of the complexes between PAI-1 and the following uPA mutants were reduced less than 1.5-fold, and the mutated residues were therefore considered unimportant for binding: K212(62)A; E213(62a)A; D194(63)A; I216(65)A; Y218(67)A; N227(76)A; Q229(78)A; E235(84)A; K264(110a)A; E265(110b)A; R267(110d)A; H402(241)A; K404(243)A °, Significantly different from binding observed with the corresponding wild-type complex (P < 0.01) ND, not determined FEBS Journal 273 (2006) 5143–5159 ª 2006 The Authors Journal compilation ª 2006 FEBS 5147 uPA–PAI-1 complex receptor binding S Skeldal et al Screening of effects of Ala substitutions in both uPA and PAI-1 on uPA–PAI-1 complex–receptor binding in microtiter wells To extend the results obtained by the microtiter wellbinding assays by Biacore binding analysis and assays of receptor-mediated endocytosis, we used a complex between a PAI-1 variant with low receptor affinity and a uPA variant with low receptor affinity We used a PAI-1 variant with a quadruple mutation in a-helix D, i.e K71A-R78A-Y81A-K82A This variant did not differ significantly from wild-type PAI-1 with respect to specific inhibitory activity (wild-type 69 ± 4% and mutant 59 ± 7% of the theoretical maximum), second-order rate constant for the reaction with uPA (wild-type 3.3 ± 0.7 · 106 m)1Ỉs)1 and mutant 3.7 ± 0.5 · 106 m)1Ỉs)1), and relative vitronectin binding (mutant 1.03 ± 0.11 times that of wild-type) We used the uPA variant with the triple mutation in the 37-loop, i.e R178(35)A-R179(36)A-R181(37a)A This variant did not differ significantly from wild-type uPA with respect to Km for hydrolysis of S-2444 (pyro-GluGly-Arg-p-nitroanilide), the Km values for wild-type and variant being 87 ± 11 lm and 85 ± 0.01 lm, respectively The resulting complex showed the expected more than 10-fold reduction in affinity for VLDLR and LRP-1A in microtiter well-binding assays (Fig 4) Surface plasmon resonance analysis of receptor binding of uPA–PAI-1 complexes We analysed receptor binding of the complexes between the quadruple a-helix D PAI-1 mutant (K71A-R78A-Y81A-K82A PAI-1) and the 37-loop uPA mutant [R178(35)A-R179(36)A-R181(37a)A uPA] by surface plasmon resonance In this case, nonradioactive wild-type and mutant uPA–PAI-1 complexes were prepared from PAI-1 purified by immuno-affinity chromatography and re-activated by denaturation with guanidinium chloride and refolding by dialysis, and uPA purified by immuno-affinity chromatography The complexes were purified from unreacted uPA and PAI-1 by immuno-affinity chromatography VLDLR, LRP-1A, or sorLA was immobilized on Biacore chips, and wild-type and mutant uPA–PAI-1 complexes injected on to the chips at concentrations of 1.5–100 nm The time course of the binding obtained with VLDLR is shown in Fig Similar results were found with the two other receptors It is evident from the figure that the binding was reduced with the mutant uPA–wildtype PAI-1 complex, the wild-type uPA–mutant PAI-1 complex, and, in particular, with the mutant 5148 Fig Effect of Ala substitutions of both uPA and PAI-1 residues on binding of the uPA–PAI-1 complex to VLDLR, LRP-1A, and sorLA in a solid-phase assay Binding of wild-type and variant complexes to the receptors was estimated Binding of the variant complexes is expressed relative to that of the wild-type complex in the same experiment Mean ± SD values are shown for at least three independent experiments °, Significantly different from binding observed with the corresponding wild-type (P < 0.01) ND, not determined uPA–mutant PAI-1 complex Fitting the binding data to a Langmuir : binding model by the use of the biaevaluation 3.0 software (global fitting) resulted in a Kd value for binding of the wild-type uPA–wild-type PAI-1 complex to either of the three receptors of % nm, while Kd for the mutant uPA–mutant PAI-1 complex was increased more than 10-fold However, the fit to the simple : binding model was poor As appears from Fig 5, the rapid first phase of the association was followed by a second, slower phase, and a corresponding rapid dissociation phase, amounting to 20% of total binding, particularly with relatively high ligand concentrations We therefore carried out a more FEBS Journal 273 (2006) 5143–5159 ª 2006 The Authors Journal compilation ª 2006 FEBS S Skeldal et al uPA–PAI-1 complex receptor binding reliable estimation of the Kd values by determining the association rate constants from the initial rate of association and the dissociation rate constant from the slow phase of dissociation (see Experimental procedures) and calculated the Kd values as the ratio between the association and dissociation rate constants (Table 1) The Kd values obtained for the wild-type uPA–wild-type PAI-1 complex, % nm, were in good agreement with those reported previously [6,8,11,12] The Kd values of the complexes between mutant uPA and wild-type PAI-1 and wild-type uPA and mutant PAI-1 were increased 1.5–5-fold, mostly as the result of increased dissociation rate constants A particularly large change, corresponding to 10–30-fold increased Kd values, was observed for the complex with mutations in both uPA and PAI-1 This increase was the result of a decreased association rate constant and an increased dissociation rate constant The fold reduction in the Kd values determined by surface plasmon resonance did not differ significantly from those expected from the microtiter well-binding assays, although the average fold reductions of the Kd value were smaller in the Biacore experiments than expected from the microtiter well-binding assays Effects of Ala substitutions of uPA and PAI-1 on receptor-mediated endocytosis of the uPA–PAI-1 complex We measured the receptor-dependent degradation of the complexes between the quadruple a-helix D PAI-1 mutant (K71A-R78A-Y81A-K82A PAI-1) and the 37-loop uPA mutant [R178(35)A-R179(36)AR181(37a)A uPA] in cell lines expressing VLDLR and LRP-1A For VLDLR-mediated endocytosis, we used U937 cells These cells were previously shown to contain VLDLR-II mRNA, i.e a VLDLR variant without exon 16 encoding the O-linked sugar domain [28] Ligand blot analysis of membrane fragments from U937 cells revealed a RAP-binding membrane protein co-migrating with VLDLR-II (Fig 6) For LRP-1Amediated endocytosis, we used COS-1 cells, in which the only RAP-binding receptor detectable by RAP ligand blotting analysis is LRP-1A [29] In both cell lines, Fig Surface plasmon resonance analysis of binding of wild-type and variant uPA–PAI-1 complexes to VLDLR Binding was measured using chips with % 50 fmol ⁄ mm2 immobilized VLDLR The chips were superfused with the indicated complexes at concentrations of 12, 6, and 1.5 nM, followed by buffer alone at 480 s Mutant uPA ¼ R178(35)A-R179(36)A-R181(37a)A uPA Mutant PAI-1 ¼ K71A-R78A-Y81A-K82A PAI-1 FEBS Journal 273 (2006) 5143–5159 ª 2006 The Authors Journal compilation ª 2006 FEBS 5149 5150 ± 2.69 ± 0.26 ± 0.051 ± 5.4 ± 2.30 ± 0.74 ± 1.43 ± 2.54 ± 3.86 ± 18.9 ± 1.12 ± 0.60 ± 3.63 ± 0.368 ± 1.96 ± 0.56 ± 1.17 ± 0.83 Wild-type R178(35)A-R179 (36)A-R181(37a)A Wild-type R178(35)A-R179 (36)A-R181(37a)A K71A-R78AY81A-K82A K71A-R78AY81A-K82A Wild-type Wild-type a Significantly different from binding observed with wild-type uPA–wild-type PAI-1 complex (P < 0.01) b Values for the complex with mutations in both uPA and PAI-1, which are significantly different from the wild-type uPA–K71A-R78A-Y81A-K82A PAI-1 complex (P < 0.01) c Significantly different from the wild-type uPA–wild-type PAI-1 complex (P < 0.025) ± 3.72 ± 1.00 ± 3.24 ± 0.53 ± 3.31 ± 3.14 ± 0.99 0.480 (10) 0.704 (10)c 1.02 (9)a 5.66 (9)b 3.01 (10) 4.00 (10) 5.96 (9)a 8.43 (9)b 0.226 (4) 0.367 (3)c 1.02 (4)a 8.79 (4)b ± 1.15 4.59 (4) 5.27 (3) 10.3 (4)a 27.3 (4)b ± 6.0 11.5 (10) 9.30 (10) 7.51 (10) 1.80 (9)b 1.60 (8) 2.35 (9)a 4.07 (8)a 27.2 (6)b 4.35 (9) 5.90 (9)a 7.97 (9)a 9.76 (6) PAI-1 variant uPA variant 2.73 (10) 2.62 (10) 2.03 (9) 0.494 (9)b ± 1.22 ± 1.22 Kd (nM) k1 · 10)5 (M)1Ỉs)1) VLDLR binding k)1 · 104 (s)1) ± 0.47 k1 · 10)5 (M)1Ỉs)1) LRP-1 A binding k)1 · 104 (s)1) Kd (nM) ± 0.054 7.05 (10) 6.53 (10) 6.73 (10) 3.12 (9) ± 4.20 ± 1.37 Kd (nM) k1 · 10)5 (M)1Ỉs)1) SorLA binding k)1 · 104 (s)1) ± 0.39 ± 0.153 Table Effect of substitutions of PAI-1 residues on the accumulation of uPA–PAI-1 on U937 cell surface U937 cells were allowed to bind the respective uPA–PAI-1 mutant complexes for h on ice and subsequently washed before incubation for at either or 37 °C Cell surface-associated complex was then released with a low-pH buffer Cell surface-associated and internalized complexes are expressed as percentage of total amount of cell-associated complex Mean ± SD values are given for experiments performed in triplicate ± 0.241 S Skeldal et al Table Effect of Ala substitutions of uPA and PAI-1 residues on the binding of uPA–PAI-1 complex to VLDLR, LRP-1 A, and sorLA, as estimated by surface plasmon resonance The binding of wild-type and variant complexes to the receptors was estimated by Biacore Binding analyses were performed at several uPA–PAI-1 concentrations between 1.5 and 100 nM with the same receptor chips at two different days The k1, k)1, and Kd values were estimated for each separate binding curve from the initial rate of association and the rate of dissociation (as described in detail in Results and Experimental procedures) In some individual experiments, the association or dissociation time courses could not be analysed, and hence the Kd values could not be calculated, because of irregular curve shapes caused by air bubbles, etc Means, standard deviations, and numbers of determinations are indicated, the latter in parentheses uPA–PAI-1 complex receptor binding °C, PAI-1 variant Inside 37 °C, Cell surface associated Inside Cell surface associated Wild-type 5.7 ± 0.1 94.3 ± 0.1 39.2 ± 1.1 60.8 ± 1.1 H5A-P6A-Q109A 5.0 ± 0.2 95.0 ± 0.2 35.8 ± 1.8 64.2 ± 1.8 H79A-F116A-R117A 4.5 ± 0.2a 95.5 ± 0.2a 20.6 ± 0.2a 79.4 ± 0.2a a Significantly different from the corresponding number for wildtype PAI-1 (P < 0.01) the receptor-mediated degradation of wild-type and variant complexes was reduced largely in parallel with receptor binding (Fig 6) Using another PAI-1 mutant, i.e a triple mutant containing three different substitutions each resulting in reduced receptor binding, we also showed that reduced complex–receptor binding was associated with increased accumulation of the complexes on the cell surface, compared with the wild-type complex Another triple mutant without reduced receptor binding did not result in accumulation on the cell surface (Table 2) It is not possible to study the endocytosis of variant complexes with truncations of the A-chain of uPA These variants not bind to uPAR and their endocytosis is therefore negligible Discussion In this work, we used site-directed mutagenesis to map the VLDLR, LRP-1A, and sorLA binding surfaces of the uPA–PAI-1 complex The mapped interaction surface spans both PAI-1 and uPA In PAI-1, residues His79, Tyr81, Met112, Phe116, Arg117, and Arg270 are implicated in the interaction surface Also, the previously reported reduced binding of the double mutants K82A-R120A and R78A-K124A [22] could be tracked back to reveal the importance of Arg120, as well as Lys82 and Arg78, in receptor binding, and confirm the previous reports of the importance of Lys71, Lys82, and Lys90 [13] and Arg78 [23] in a-helix D Of the 41 PAI-1 residues tested because of their proximity to a-helix D, 12 were Arg or Lys Of the 11 residues implicated in binding, seven were Arg or Lys Thus, basic residues constituted a much higher percentage of FEBS Journal 273 (2006) 5143–5159 ª 2006 The Authors Journal compilation ª 2006 FEBS S Skeldal et al uPA–PAI-1 complex receptor binding Fig Receptor-mediated degradation of uPA–PAI-1 wild-type and variant complexes in U937 and COS-1 cells U937 cells were incubated with 10 pM 125I-uPA–PAI-1 complex for 45 at 37 °C, by which time the amount of degraded complex was determined as the fraction of complex soluble in 7% trichloroacetic acid The degradation of wild-type 125I-uPA–PAI-1 complex was set equal to 1, and the degradation of mutant complexes expressed relative to that The figure shows mean ± SD for triple determinations in a typical experiment out of a total of three with U937 cells and two with COS-1 cells Insert, 125I-RAP ligand blotting analysis of a membrane preparation from U937 Lane 1, purified VLDLR type II [44]; lane 2, membranes from U937 cells The migration of molecular mass markers is indicated on the right the PAI-1 residues implicated in binding than of all the residues tested This finding agrees well with the hypothesis that ligand recognition by this receptor class relies on electrostatic interactions between basic residues in the ligands and acidic residues in the receptors [21] However, X-ray crystal structure analysis of LDLR at pH [4] and receptor–ligand complexes [2,3], site-directed mutagenesis of CTRs [30] (see below) and our results presented here (Fig 2) suggest that hydrophobic interactions may also be important Almost all the residues implicated in binding were localized in and around a-helix D and a-helix E, but, interestingly, substitution of Arg270, localized in b-strand 2C, % 2.7 nm from a-helix D, also affected binding, suggesting an even more extended binding surface (Fig 7) In uPA, we have shown that substitutions in and near the 37-loop and 60-loop caused a substantial reduction in binding Thus, our findings for the first time implicate the SPD of uPA in the binding of the complex to the receptors Obviously, we cannot exclude the possibility that additional residues in the SPD contribute to the binding In addition, we could confirm the previously implicated importance of the A-chain of uPA in the binding of the uPA–PAI-1 complex to the receptors Moreover, we demonstrated that deletion of the uPA growth factor domain resulted in reduced binding As the reduction was smaller than that caused by deletion of the entire A-chain, it seems likely that both the growth factor domain and the kringle contribute to the binding, but putative endocytosis receptor-binding residues in the kringle remain unknown The involvement of the growth factor domain readily explains the previously reported reduction of the affinity of the uPA–serpin complexes for LRP-1A and SorLA in the presence of the cellular receptor for uPA, uPAR [11,12,14], as uPAR binds to the growth factor domain of uPA [31] Binding to uPAR would thus shield endocytosis receptor-binding residues In the model of the complex between PAI-1 and the uPA SPD, constructed from the 3D structure of the a1-PI–trypsin complex, as determined by X-ray crystal structure analysis [16], the residues of the 37-loop and 60-loop of uPA studied here are relatively close to FEBS Journal 273 (2006) 5143–5159 ª 2006 The Authors Journal compilation ª 2006 FEBS 5151 uPA–PAI-1 complex receptor binding S Skeldal et al Fig The receptor binding surface in the uPA–PAI-1 complex The uPA–PAI-1 complex shown is a SWISSPDBVIEWER surface display of a model constructed from the structure of the a1-PI–trypsin complex [16] (pdb file 1EZX), by overlayering the a1-PI part of that structure with the structure of cleaved PAI-1 [54] (pdb file 9PAI) and the trypsin part of that structure with the structure of the SPD of uPA [27] (pdb file 1LMW) Although the relative orientation of uPA and PAI-1 in the model is realistic, it does not allow any predictions of exact distances between amino-acid residues The effects of Ala substitutions on the binding of 20 pM 125I-uPA–PAI-1 complex to VLDLR, LRP-1A, and sorLA individually are depicted in the model Ala substitution of residues colored red resulted in a more than fivefold reduction in binding Ala substitution of residues colored pink resulted in a 1.5–5-fold reduction in binding PAI-1 residue Tyr81 and uPA residue Arg178(35), which would have been colored pink, are not visible with the orientation of the structure used Ala substitution of residues colored blue resulted in a less than 1.5-fold reduction in binding when tested individually or less than twofold reduction when tested as part of triple mutants One structure is shown for binding to each receptor In addition, the complex model is also shown as a ribbon diagram in which b-strand 2C of PAI-1 is colored yellow and a-helix D and E of PAI-1 are colored green The 37-loop and 60-loop of uPA are shown in red and pink, respectively Also indicated is a ribbon diagram of the pH structure of the CTRs from LDLR [4] (pdb file N7D) 5152 FEBS Journal 273 (2006) 5143–5159 ª 2006 The Authors Journal compilation ª 2006 FEBS S Skeldal et al PAI-1, and it may be argued that the changed endocytosis receptor binding following substitution of these residues is due to a change in the relative orientations of uPA and PAI-1 However, in the two available crystal structure complexes [16,17], the corresponding residues remain fully surface-exposed Secondly, the substitution of these residues did not lead to changes in the stability of the complex, as evaluated by antibody-binding assays performed in parallel with microtiter well receptor-binding assays (data not shown) Thirdly, monoclonal antibodies with epitopes in the 37-loop and 60-loop remain accessible in the complex [26] Fourthly, in contrast with monoclonal antibodies binding near a-helix F of PAI-1, a monoclonal antibody to PAI-1 with an epitope near the expected interaction phase with uPA did not induce lability of the complex [32] Fifthly, the absence of any large structural effects of the triple 37-loop uPA mutation was established by demonstrating unchanged enzyme activity The most obvious interpretation of the observed effects of the substitutions of the residues in the 37 and 60 loops is that these residues are directly situated at the surface of the interaction between the uPA– PAI-1 complex and the endocytosis receptors In the model of the complex between PAI-1 and the uPA SPD, constructed from the 3D structure of the a1-PI–trypsin complex, as determined by X-ray crystal structure analysis [16], the residues involved in binding form a continuous, extended interaction surface spanning both PAI-1 and uPA (Fig 7) Together with the involvement of the uPA A-chain, these findings argue for the possibility that the high affinity of the complex, compared with that of the individual components, is due to an extended interaction surface assembled by complex formation Our results not exclude the possibility that conformational changes or exposure of ‘cryptic’ sites on complex formation [13,23] contribute to the increased affinity, but are more readily explained by an avidity effect caused by two or more binding sites being brought together on the same ligand Thus, it should be noted that the 37-loop of uPA does not seem to be in the part of uPA that changes conformation on complex formation [33] Another argument against the ‘cryptic’ site theory is that the 3D structures of the regions of cleaved a1-PI and trypsin-complexed a1-PI corresponding to the regions of PAI-1 implicated in endocytosis receptor binding are superimposable [16,34] Assuming that cleaved and complexed PAI-1 are as similar as cleaved and complexed a1-PI, the observation of a Kd for uPA–PAI-1 complex–LRP-1A binding of 0.4 nm, a Kd for active PAI-1–LRP-1A binding of 55 nm, and a Kd for cleaved PAI-1–LRP-1A binding of 41 nm [12] must uPA–PAI-1 complex receptor binding lead to the conclusion that conformational changes in PAI-1 are unlikely to be the only cause of the increased endocytosis receptor affinity upon complex formation Ligand binding by receptors of the LDLR family is critically dependent on the CTR clusters VLDLR and sorLA have only one such cluster, with eight or 11 CTRs, respectively LRP-1A has four clusters, of two, eight, 10, and 11 CTRs In general, high affinity and high specificity in ligand recognition by receptors of the LDLR receptor family is believed to rely on several individual CTRs making contact with several receptor recognition patches on the same ligand Each of the 217 known human CTRs comprises % 40 amino acids A conserved scaffold motif of only 12 amino acids is required for this common structure, with 28 nonconserved positions Receptors from the LDLR family alone accounts for 132 different CTRs Crystallographic and NMR studies of individual repeats have revealed that the sequence variability in short loop regions of each repeat results in a unique contour surface and charge density for each repeat [35] Several observations suggest that the binding sites for plasminogen activator– PAI-1 complexes are also assembled from combinations of CTRs Thus, although the interpretation of our present results with LRP-1A is hampered by the fact that both its CTR cluster II and IV bind to the uPA–PAI-1 complex [36–39], the results demonstrate that the uPA–PAI-1 complex has slightly different interaction surfaces with each of the three receptors tested (Fig 7) This observation is in agreement with conclusions from previous studies that VLDLR and LRP-1A have overlapping but not identical binding specificities in a series of serine protease–serpin complexes [40] By expression of VLDLR variants lacking specific complement-type repeats, it was found that the second CTR in the cluster of eight in this receptor was required for maximal affinity for the uPA–PAI-1 complex [41] The tPA–PAI-1 complex and PAI-1 were reported to bind to a fragment encompassing CTR3-CTR4-CTR5-CTR6-CTR7 of CTR cluster II of LRP-1A (the CTRs of the most N-terminal CTR cluster being numbered and 2) [38,42,43] The minimal functional unit capable of binding the uPA–PAI-1 complex to LRP-1A CTR cluster II was reported to be a two-CTR fragment, and the CTR5– CTR6 pair to be the pair mainly responsible for binding the uPA–PAI-1 complex [30] From the dimensions of the CTRs emerging from the X-ray crystal structure analysis of LDLR [4], the uPA SPD and the PAI-1 region around a-helix D and a-helix E would each make contact with a CTR To also accommodate the A-chain of uPA and the more distantly localized R270 of PAI-1, two additional CTRs would be needed Thus, whereas FEBS Journal 273 (2006) 5143–5159 ª 2006 The Authors Journal compilation ª 2006 FEBS 5153 uPA–PAI-1 complex receptor binding S Skeldal et al the individual components uPA and PAI-1 would each only be able to make contact with one or two CTRs, we propose that the high affinity of the complex is related to contacts with three or four CTRs In conclusion, we here add several new details to the map of the endocytosis receptor-binding surface of serine protease–serpin complexes and for the first time implicate the SPD of uPA in the binding The residues that mediate the binding affinity of the A-chain of uPA remain to be established In the long term, exact mapping of the interaction surface will require X-ray structure analysis of cocrystals between the uPA–PAI-1 complex and one of the receptors Experimental procedures medium containing soluble VLDLR was poured on to an RAP-coupled Sepharose column, recirculated for 24 h, and washed The equilibration and washing buffer was Hepesbuffered saline (10 mm Hepes, pH 7.4, 0.14 m NaCl) with mm CaCl2 and mm MgCl2 Elution was carried out with 0.1 m acetic acid (pH 3.5) ⁄ 0.5 m NaCl ⁄ 10 mm EDTA Fractions were neutralized immediately with 0.1 vol m Tris (pH 9) ⁄ 25 mm CaCl2 Relevant fractions were selected on the basis of 125I-RAP-blots, and pooled The receptor preparations were concentrated with a Centriplus YM-10 spin column, dialysed into NaCl ⁄ Pi (10 mm sodium phosphate, pH 7.4, 0.14 m NaCl), and stored at )80 °C Full-length LRP-1A was purified from human placentas as described [45] Mature, truncated soluble human sorLA (residues 54–2107), lacking the N-terminal propeptide and the C-terminal cytoplasmic and transmembrane part, was produced in CHO cells and purified as described [46] Cell lines U937 cells were cultured in RPMI 1640 medium COS-1 and HEK293T cells were cultured in Dulbecco modified Eagle’s medium Both media were supplemented with 10% fetal bovine serum, 100 mL)1 penicillin and 100 lgỈmL)1 streptomycin The medium for HEK293T cells also contained 1% nonessential amino acids Cells were maintained in a humidified atmosphere with 5% CO2 at 37 °C CHO cells were cultured as described [44] Receptors A stop codon succeeding the codon encoding amino acid 772 was introduced into human VLDLR type-I cDNA, i.e cDNA containing all exons of the human VLDLR gene (kindly provided by A Soutar, University College London, UK) [44] The resulting cDNA, encoding soluble VLDLR-I without the cytoplasmic domain and transmembrane region (residues 1–772, sVLDLR), was cloned into the plasmid pcDNA3.1- and controlled by DNA sequencing Transient transfection by the calcium phosphate precipitation method and expression of soluble VLDLR were carried out by standard methods [26] Briefly, HEK293T cells were grown to full confluence and split : and allowed to grow for an additional 24 h Cells were then split : and seeded on gelatine-coated dishes After 24 h, chloroquine was added directly to the cultures to a final concentration of 25 lm The cultures were then incubated for h Then 30 lg DNA and water were mixed to a final volume of 1752 lL, and 248 lL m CaCl2 was added This mixture was added dropwise to mL 42 mm Hepes, pH 7.05, containing 274 mm NaCl, 10 mm KCl, 1.5 mm Na2HPO4, and 11 mm d-(+)-glucose This solution was then added dropwise to the conditioned medium of one 15-cm culture dish with HEK293T cells After 7–11 h of incubation, the medium was replaced with serum-free medium, which was harvested 48 h later This was repeated twice The conditioned 5154 Proteases, inhibitors, and their complexes Human wild-type uPA and uPA mutants were expressed recombinantly in and purified from conditioned media of HEK293T cells, as previously described [26] Recombinant uPA N-terminally truncated at Lys136 (LMW-uPA) was a gift from W Gunzler, Grunenthal Company, Aachen, Geră ă many uPA variants were characterized with respect to Km for hydrolysis of S-2444 (pyro-Glu-Gly-Arg-p-nitroanilide) as previously described [47] Human wild-type PAI-1 and PAI-1 mutants were expressed recombinantly in and purified from conditioned media of HEK293T cells, as previously described [24] Some PAI-1 variants were characterized with respect to specific inhibitory activity, second-order rate constant for the reaction with uPA, and relative vitronectin affinity, as described previously [26,48] The numbering of PAI-1 residues was as described previously [49] 125 I-labeling of uPA, LMW-uPA, and RAP was performed as described [12] Complexes between 125I-uPA and purified PAI-1 were prepared as described [40] In some cases, complexes between wild-type 125I-uPA and PAI-1 variants were prepared by the use of conditioned media from cells transfected with the corresponding PAI-1 cDNA In these cases, the conditioned media were incubated for h at room temperature with 0.1% SDS, followed by an eightfold dilution with ice-cold 0.114 m Tris ⁄ HCl, pH 7.4, containing 1.14% BSA, in order to re-activate the latent PAI-1 125I-uPA was then added in an amount corresponding to a 15-fold molar excess of PAI-1 over 125I-uPA In experiments performed to compare different PAI-1 variants, aliquots of one and the same preparation of 125I-uPA were added to separate portions of conditioned medium with the different PAI-1 variants The 125I-uPA–PAI-1 complexes were allowed to form during 30 of incubation at 37 °C The 125I-uPA–PAI-1 complexes were purified by pouring the uPA–PAI-1 mixture on to an anti-uPA FEBS Journal 273 (2006) 5143–5159 ª 2006 The Authors Journal compilation ª 2006 FEBS S Skeldal et al mAb6-coupled Sepharose column followed by an anti-PAI1 mAb2-coupled Sepharose column Both columns were equilibrated and washed in washing buffer (0.1 m Tris, m NaCl, 1% BSA) and eluted with 0.1 m acetic acid (pH 2.9) ⁄ m NaCl, and fractions immediately neutralized with 0.1 vol m Tris (pH 9) ⁄ m NaCl 125I-LMW-uPA– PAI-1 complex was produced and purified in the same way as uPA–PAI-1 except that an anti-uPA mAb2-coupled Sepharose column was used instead of an anti-uPA mAb6coupled Sepharose column To prepare nonradioactive wild-type and mutant uPA– PAI-1 complex, recombinant PAI-1 expressed in and purified from HEK293T cells in the latent form (see above) were denatured and refolded by the use of guanidinium chloride [24] The re-activated PAI-1 was mixed with purified uPA also expressed in and purified from HEK293T cells (see above) The complexes formed were purified by immuno-affinity chromatography on two successive columns coupled with monoclonal antibodies against PAI-1 and uPA, respectively [12] Antibodies and other proteins Anti-PAI-1 mAb2, anti-uPA mAb2, anti-uPA mAb6, and anti-uPA mAb3689 have previously been described [26,50] Recombinant RAP was a gift from M Etzerodt, Department of Molecular Biology, University of Aarhus [5] Solid-phase binding assays Receptors were coated on to microtiter wells overnight in 50 mm NaHCO3, pH 9.6, with a receptor density adjusted to obtain 5–10% binding of wild-type 125I-uPA–PAI-1 complex, corresponding to receptor concentrations in the coating buffer of % lgỈmL)1 The wells were blocked with 10% BSA in binding buffer (10 mm Hepes, pH 7.8, 140 mm NaCl, mm CaCl2, mm MgCl2) for h, washed, and incubated overnight at °C with 20 pm 125I-ligand Washing and incubation with ligands were performed in binding buffer with 1% BSA When the effect of monoclonal antibodies on binding was investigated, the antibodies were added at a concentration of 20 lgỈmL)1 After the incubation, the supernantants were removed for c-counting and the wells washed Bound ligand was released from the wells with 10% SDS and taken for c-counting Ligand binding was expressed as bound divided by free ligand and corrected for nonspecific binding, i.e the radioactivity recovered from wells without receptor As a control for the integrity of the complexes, the binding of all Ala-substituted complexes to parallel wells coated with 20 lgỈmL)1 anti-PAI-1 mAb2 was shown not to differ more than 20% from that of the wild-type The binding of variant uPA–PAI-1 complexes to the receptors was corrected by the use of the small variations in antibody binding, assumed to be caused by a dissociation of small fractions of the complexes, by dividing the actually observed binding uPA–PAI-1 complex receptor binding by the factor by which antibody binding of each variant complex differed from that of the wild-type complex Ligand degradation and internalization experiments For ligand degradation experiments with U937 cells, the cells were washed three times with serum-free medium supplemented with 0.5% BSA and incubated for 45 at 37 °C in 0.5 mL of the same buffer with 10 pm 125I-labeled ligand and a cell density of 10 · 106 cellsỈmL)1 The cultures were then made 7% with respect to trichloroacetic acid and centrifuged Supernatants and pellets were c-counted separately For experiments with COS-1 cells, the cells were cultured in 24-well cell culture dishes to a confluence % 80%, washed three times with serum-free medium supplemented with 0.5% BSA, and incubated for h at 37 °C with 0.3 mL of the same buffer with 10 pm 125I-labeled ligand The medium was then removed, and the cells solubilized with 1% SDS BSA was added to the cell extracts to a final concentration of 1% Cell medium and cell extracts were then made 7% with respect to trichloroacetic acid and centrifuged Supernatant and pellets were c-counted separately Ligand degradation was defined as nontrichloroacetic acid-precipitable radioactivity versus total added radioactivity The degradation in incubations with 100 nm RAP was scored as nonspecific degradation, not mediated by RAP-binding receptors, and subtracted from all other data [51] Separate control experiments showed that there was linearity between time and amount of ligand degraded with the incubation times used here (not shown) To evaluate the distribution of 125I-uPA–PAI-1 complexes between the cell surface and intracellular vesicles, we washed U937 cells three times with serum-free medium containing 0.5% BSA and cell density adjusted to 10 · 106 cellsỈmL)1 in the same buffer Then 0.5 mL cell suspension was incubated with 10 pm 125I-uPA–PAI-1 for h on ice with gentle rocking and then washed three times in ice-cold washing buffer Cells where then incubated at 37 °C or kept on ice for and subsequently quickly placed on ice Cell surface-associated ligand was released by washing cells three times with an ice-cold buffer comprising 0.2 m acetic acid, pH 2.6, and 0.1 m NaCl The remaining radioactivity was defined as internalized ligand Internalized and cell surface-associated ligand were expressed as a percentage of the total radioactivity 125 I-RAP blotting analysis of membrane fragments from U937 cells Membrane fragments from U937 cells were purified as described previously [44] Ligand blotting analysis with 125 I-RAP was carried out as previously described [52], using VLDLR type-II as a molecular mass marker [44] FEBS Journal 273 (2006) 5143–5159 ª 2006 The Authors Journal compilation ª 2006 FEBS 5155 uPA–PAI-1 complex receptor binding S Skeldal et al Surface plasmon resonance analysis References Surface plasmon resonance measurements were performed on a Biacore 3000 instrument (Biacore, Uppsala, Sweden) equipped with CM5 sensor chips The receptors were immobilized to densities of % 50 fmolỈmm)2, and samples for binding (40 lL) were injected at lLỈmin)1 at 25 °C in 10 mm Hepes, pH 7.4, containing 150 mm NaCl, 1.5 mm CaCl2, mm EGTA, and 0.01% P20 Various concentrations of uPA–PAI-1 complex were injected for After discontinuation of the injection, dissociation of bound ligand was followed for another Binding was expressed in relative response units as the response obtained from the flow cell containing immobilized receptor minus the response obtained when using an activated but uncoupled chip The Kd values were calculated as the ratio between the dissocation rate constants (k)1 values) and the association rate constants (k1 values) The dissocation rate constants were estimated from semilogarithmic plots of the relative amount of uPA–PAI-1 complex bound to the chip versus time during the dissociation phase The data were fitted to biphasic dissociation kinetics, to distinguish between slowly dissociating, high-affinity binding (rate constant k)1), and low affinity, rapidly dissociating binding (rate constant k)2): Bu G & Schwartz AL (1998) RAP, a novel type of ER chaperone Trends Cell Biol 8, 272–276 Fisher C, Beglova N & Blacklow SC (2006) Structure of an LDLR-RAP complex reveals a general mode for ligand recognition by lipoprotein receptors Mol Cell 22, 277–283 Verdaguer N, Fita I, Reithmayer M, Moser R & Blaas D (2004) X-ray structure of a minor group human rhinovirus bound to a fragment of its cellular 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cell surface and inhibits cell migration J Biol Chem 277, 42366 42371 Relative binding ẳ A expk1 tị þ B expðÀkÀ2 tÞ ð1Þ To estimate the association rate constants (k1 values), we estimated the initial rates of association by linear regression analysis The initial rates were converted into k1 values by the following equation: Initial rate ¼ k1 ½RT Š½uPA À PAI-1 complexŠ ð2Þ Here, the [RT] values were calculated from the response units bound to the chips when exposed to 100 nm wild-type uPA– wild-type PAI-1 complex, after correction for the rapidly dissociating fraction of % 20% In all cases, the initial association rates showed the expected relationship with the uPA– PAI-1 complex concentration The Kd values were then calculated as k)1 ⁄ k1 Statistical analysis Results were analysed by Student’s t-test Acknowledgements We acknowledge the Danish Cancer Research Foundation, The Interdisciplinary Nanoscience Centre of Aarhus University (iNANO), the Danish Cancer Society, the Danish 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Journal compilation ª 2006 FEBS 5159 ... bound 64 ± 10% of all complexes, and the binding of variant complexes always varied less than 20% from the binding of the wild-type complex Binding of the uPA–PAI-1 complex to the receptors was... the uPA– PAI-1 complex and the endocytosis receptors In the model of the complex between PAI-1 and the uPA SPD, constructed from the 3D structure of the a1-PI–trypsin complex, as determined by. .. sorLA [11] These receptors mediate endocytosis of uPA–PAI-1 complex accumulated on the cell surface by binding to the urokinase-type plasminogen activator receptor (uPAR), whereas endocytosis