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Regulation of Single Chain Urokinase Binding, Internalization, and Degradation by a Plasminogen Activator Inhibitor 1-Derived Peptide

1997; Elsevier BV; Volume: 272; Issue: 43 Linguagem: Inglês

10.1074/jbc.272.43.27053

ISSN

1083-351X

Autores

Litao Zhang, Dudley K. Strickland, Douglas B. Cines, Abd Al‐Roof Higazi,

Tópico(s)

Cell Adhesion Molecules Research

Resumo

The internalization and degradation of cell-associated urokinase type plasminogen activator (uPA) through the α2-macroglobulin receptor/low density lipoprotein-related receptor (α2MR/LRP) represent important steps in the control of plasmin formation. Complexes between two chain urokinase (tcuPA) and plasminogen activator type 1 are degraded rapidly whereas single chain urokinase (scuPA) is not, suggesting that α2MR/LRP requires specific epitopes in the serpin for effective function. We report an alternative mechanism that may contribute to this process. The binding of scuPA to LM-TK− cells that lack the uPA receptor was stimulated by the hexapeptide EEIIMD, corresponding to amino acids 350–355 of plasminogen activator type 1, which contacts the sequence RHRGGS, corresponding to amino acids 179–184 in uPA. EEIIMD increased theB max of scuPA binding 4-fold with the half-maximal effect achieved at a peptide concentration of 50 ॖm. Stimulation was dependent on the charge on the COOH-terminal amino acid but not on the NH2 terminus of the peptide. EEIIMD also stimulated the internalization and degradation of scuPA. Both the binding and internalization of scuPA in the presence of EEIIMD were blocked by recombinant, 39-kDa α2MR/LRP-associated protein as well as by an anti-α2MR/LRP antibody. EEIIMD also stimulated the binding of scuPA to purified α2MR/LRP. EEIIMD had no effect on the binding of tcuPA or of complexes between scuPA and its receptor. These results suggest that EEIIMD regulates the binding of scuPA with α2MR/LRP. These findings also suggest a potential mechanism by which scuPA can be cleared which is independent of activation by plasmin or binding to uPA receptor. The internalization and degradation of cell-associated urokinase type plasminogen activator (uPA) through the α2-macroglobulin receptor/low density lipoprotein-related receptor (α2MR/LRP) represent important steps in the control of plasmin formation. Complexes between two chain urokinase (tcuPA) and plasminogen activator type 1 are degraded rapidly whereas single chain urokinase (scuPA) is not, suggesting that α2MR/LRP requires specific epitopes in the serpin for effective function. We report an alternative mechanism that may contribute to this process. The binding of scuPA to LM-TK− cells that lack the uPA receptor was stimulated by the hexapeptide EEIIMD, corresponding to amino acids 350–355 of plasminogen activator type 1, which contacts the sequence RHRGGS, corresponding to amino acids 179–184 in uPA. EEIIMD increased theB max of scuPA binding 4-fold with the half-maximal effect achieved at a peptide concentration of 50 ॖm. Stimulation was dependent on the charge on the COOH-terminal amino acid but not on the NH2 terminus of the peptide. EEIIMD also stimulated the internalization and degradation of scuPA. Both the binding and internalization of scuPA in the presence of EEIIMD were blocked by recombinant, 39-kDa α2MR/LRP-associated protein as well as by an anti-α2MR/LRP antibody. EEIIMD also stimulated the binding of scuPA to purified α2MR/LRP. EEIIMD had no effect on the binding of tcuPA or of complexes between scuPA and its receptor. These results suggest that EEIIMD regulates the binding of scuPA with α2MR/LRP. These findings also suggest a potential mechanism by which scuPA can be cleared which is independent of activation by plasmin or binding to uPA receptor. Urokinase type plasminogen activator (uPA) 1The abbreviations used are: uPA, urokinase type plasminogen activator; scuPA, single chain urokinase type plasminogen activator; tcuPA, two chain urokinase type plasminogen activator; uPAR, urokinase type plasminogen activator receptor; PAI-1, plasminogen activator inhibitor type 1; α2MR/LRP, α2-macroglobulin receptor/low density lipoprotein-related receptor; suPAR, recombinant soluble urokinase type plasminogen activator receptor; rRAP, recombinant soluble 39-kDa α2MR/LRP-associated protein; BSA, bovine serum albumin; TBS, Tris-buffered saline. 1The abbreviations used are: uPA, urokinase type plasminogen activator; scuPA, single chain urokinase type plasminogen activator; tcuPA, two chain urokinase type plasminogen activator; uPAR, urokinase type plasminogen activator receptor; PAI-1, plasminogen activator inhibitor type 1; α2MR/LRP, α2-macroglobulin receptor/low density lipoprotein-related receptor; suPAR, recombinant soluble urokinase type plasminogen activator receptor; rRAP, recombinant soluble 39-kDa α2MR/LRP-associated protein; BSA, bovine serum albumin; TBS, Tris-buffered saline. has been implicated in several biological processes including inflammation (1Gyetko M.R. Todd III, R.F. Wilkinson C.C. Sitrin R.G. J. Clin. Invest. 1994; 93: 1380-1387Crossref PubMed Scopus (288) Google Scholar, 2Gyetko M. Chen G.-H. McDonald R.A. Goodman R. Huffnagle G.B. Wilkinson C.C. Fuller J.A. Toews G.B. J. Clin. Invest. 1996; 97: 1818-1826Crossref PubMed Scopus (166) Google Scholar, 3Shapiro R.L. Duquette J.G. Nunes I. Roses D.F. Harris M.N. Wilson E.L. Rifkin D. Am. J. Pathol. 1997; 150: 359-369PubMed Google Scholar) and the development of tumor metastases (4Dano K. Behrendt N. Brunner N. Ellis V. Ploug M. Pyke C. Fibrinolysis. 1994; 8: 189-203Crossref Scopus (292) Google Scholar). Urokinase is synthesized as a single chain molecule (scuPA) which expresses little enzymatic activity (5Ellis V. Scully M.F. Kakkar V.V. J. Biol. Chem. 1987; 262: 14998-15003Abstract Full Text PDF PubMed Google Scholar, 6Petersen L.C. Lund L.R. Nielsen L.S. Dano K. Skriver L. J. Biol. Chem. 1988; 263: 11189-11195Abstract Full Text PDF PubMed Google Scholar, 7Husain S.S. Biochemistry. 1991; 30: 5707-5805Crossref PubMed Scopus (32) Google Scholar, 8Collen D. Zamarron C. Lijnen H.R. Hoylaerts M. J. Biol. Chem. 1986; 261: 1259-1266Abstract Full Text PDF PubMed Google Scholar). scuPA is activated when it is enzymatically converted to a two chain molecule (tcuPA), primarily by plasmin (9Robbins K.C. Summaria L. Hsieh B. Shah R.J. J. Biol. Chem. 1967; 242: 2333-2342Abstract Full Text PDF PubMed Google Scholar), or when it binds to its receptor (uPAR), in which case it remains as a single chain molecule (10Higazi A.A.-R. Cohen R.L. Henkin J. Kniss D. Schwartz B.S. Cines D.B. J. Biol. Chem. 1995; 270: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The enzymatic activity of tcuPA is regulated in plasma primarily by plasminogen activator inhibitor type 1 (PAI-1) (11Kruithof E.K.O. Enzyme. 1988; 40: 113-121Crossref PubMed Scopus (237) Google Scholar, 12Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Abstract Full Text PDF PubMed Google Scholar, 13Lijnen H.R. De Cock F. Collen D. Eur. J. Biochem. 1994; 224: 567-574Crossref PubMed Scopus (27) Google Scholar). The contact region between the two molecules includes the sequence RHRGGS (amino acids 179–184) in uPA and the sequence EEIIMD (amino acids 350–355) in PAI-1 which has been designated as the 舠docking site舡 for uPA (14Madison E.L. Goldsmith E.J. Gething M.-J.H. Sambrook J.F. Gerard R.D. J. Biol. Chem. 1990; 265: 21423-21426Abstract Full Text PDF PubMed Google Scholar, 15Adams D.S. Griffin L.A. Nachajko W.R. Reddy V.B. Wei C.M. J. Biol. Chem. 1991; 266: 8476-8482Abstract Full Text PDF PubMed Google Scholar). The postulated electrostatic interactions between the two regions play a crucial role in forming a stable uPA·PAI-1 complex. However, the scuPA·uPAR complex is less susceptible to inhibition by PAI-1 than is tcuPA or receptor-bound tcuPA (16Higazi A.A.-R. Mazar A. Wang J. Reilly R. Henkin J. Kniss D. Cines D. Blood. 1996; 87: 3545-3549Crossref PubMed Google Scholar, 17Ellis V. Wun T.C. Behrendt B. Ronne E. Dano K. J. Biol. Chem. 1990; 265: 9904-9908Abstract Full Text PDF PubMed Google Scholar), although the mechanism of this resistance, including alterations in the interaction involving the docking site, has not been established. In addition to inhibiting urokinase activity, PAI-1 also promotes the internalization and degradation of uPA through the α2-macroglobulin receptor/low density lipoprotein-related receptor (α2MR/LRP) (18Nykjaer A. Kjoller L. Cohen R.L. Lawrence D.A. Garni-Wagner B.A. Todd III, R.F. van Zonnefeld A.-J. Gliemann J. Andreasen P.A. J. Biol. Chem. 1994; 269: 25668-25676Abstract Full Text PDF PubMed Google Scholar). The complex formed between PAI-1 and tcuPA binds to α2MR/LRP with considerably higher affinity than does either component alone. The increased affinity of the complex results from an independent contribution of epitopes present in each ligand (18Nykjaer A. Kjoller L. Cohen R.L. Lawrence D.A. Garni-Wagner B.A. Todd III, R.F. van Zonnefeld A.-J. Gliemann J. Andreasen P.A. J. Biol. Chem. 1994; 269: 25668-25676Abstract Full Text PDF PubMed Google Scholar), but a possible effect of PAI-1 on the conformation of uPA itself has not been excluded (19Higazi A.A.-R. Upson R. Cohen R. McCrae K.R. Manuppello J. Bognacki J. Henkin J. Kounnas M. Strickland D. Preissner K.T. Lawler J. Cines D.B. Blood. 1996; 88: 542-551Crossref PubMed Google Scholar). In this context it is of interest that binding of scuPA to its receptor inhibits its binding to α2MR/LRP (18Nykjaer A. Kjoller L. Cohen R.L. Lawrence D.A. Garni-Wagner B.A. Todd III, R.F. van Zonnefeld A.-J. Gliemann J. Andreasen P.A. J. Biol. Chem. 1994; 269: 25668-25676Abstract Full Text PDF PubMed Google Scholar, 19Higazi A.A.-R. Upson R. Cohen R. McCrae K.R. Manuppello J. Bognacki J. Henkin J. Kounnas M. Strickland D. Preissner K.T. Lawler J. Cines D.B. Blood. 1996; 88: 542-551Crossref PubMed Google Scholar). Again, two mechanisms for this reduced affinity have been postulated. It has been proposed that the contact site between the A chain of scuPA and α2MR/LRP is shielded by uPAR (18Nykjaer A. Kjoller L. Cohen R.L. Lawrence D.A. Garni-Wagner B.A. Todd III, R.F. van Zonnefeld A.-J. Gliemann J. Andreasen P.A. J. Biol. Chem. 1994; 269: 25668-25676Abstract Full Text PDF PubMed Google Scholar). An alternative mechanism is that binding of scuPA to uPAR induces a conformational change that both promotes its binding to integrin ligands and leads to a loss of the epitope recognized by α2MR/LRP (19Higazi A.A.-R. Upson R. Cohen R. McCrae K.R. Manuppello J. Bognacki J. Henkin J. Kounnas M. Strickland D. Preissner K.T. Lawler J. Cines D.B. Blood. 1996; 88: 542-551Crossref PubMed Google Scholar). The latter possibility is consistent with the observation that soluble scuPA has a higher affinity for α2MR/LRP than does tcuPA (18Nykjaer A. Kjoller L. Cohen R.L. Lawrence D.A. Garni-Wagner B.A. Todd III, R.F. van Zonnefeld A.-J. Gliemann J. Andreasen P.A. J. Biol. Chem. 1994; 269: 25668-25676Abstract Full Text PDF PubMed Google Scholar) and with the loss of affinity for α2MR/LRP which occurs when the active site of tcuPA is occupied by diisopropyl fluorophosphate (18Nykjaer A. Kjoller L. Cohen R.L. Lawrence D.A. Garni-Wagner B.A. Todd III, R.F. van Zonnefeld A.-J. Gliemann J. Andreasen P.A. J. Biol. Chem. 1994; 269: 25668-25676Abstract Full Text PDF PubMed Google Scholar). To examine in greater detail the mechanism by which urokinase is recognized by α2MR/LRP we studied the effect of a hexapeptide derived from the docking site in PAI-1 on the cellular binding, internalization, and degradation of scuPA and tcuPA. The results of this investigation indicate that this peptide increases the recognition of scuPA, but not tcuPA, by α2MR/LRP. The data are compatible with the notion that scuPA is subject to 舠allosteric舡 regulation through an interaction with the docking site of PAI-1, as a result of which the molecule undergoes a conformational change that modulates its biologic activity. Recombinant scuPA and recombinant soluble urokinase receptor (suPAR) were purified as reported previously (16Higazi A.A.-R. Mazar A. Wang J. Reilly R. Henkin J. Kniss D. Cines D. Blood. 1996; 87: 3545-3549Crossref PubMed Google Scholar,20Higazi A.A.-R. Mazar A. Wang J. Quan N. Griffin R. Reilly R. Henkin J. Cines D.B. J. Biol. Chem. 1997; 272: 5348-5353Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) and were the generous gifts of Dr. Jack Henkin (Abbott Laboratories; Abbott Park, IL). The peptides EEIIMD, REIIMD, and EEIIMR were synthesized by the Protein Chemistry Laboratory, Washington University, St. Louis. Glu-plasminogen, tcuPA, PAI-1, and the plasmin chromogenic substrate Spectrazyme PL were obtained from American Diagnostica Inc. (Greenwich, CT). Purified α2MR/LRP and recombinant soluble 39-kDa α2MR/LRP-associated protein (rRAP) were prepared as described previously (21Williams S.E. Aschcom J.D. Argraves W.S. Strickland D.K. J. Biol. Chem. 1992; 267: 9035-9040Abstract Full Text PDF PubMed Google Scholar). The LM-TK− cell line was obtained from the American Type Tissue Collection (Rockville, MD). scuPA, suPAR, and tcuPA were radiolabeled with 125I using IODO-BEADS (Pierce Chemical Co.) as described (22Barnathan E.S. Kuo A. Rosenfeld L. Kariko K. Leski M. Robbiati F. Nolli M.L. Henkin J. Cines D.B. J. Biol. Chem. 1990; 265: 2865-2872Abstract Full Text PDF PubMed Google Scholar). suPAR and scuPA were incubated with each other at a molar ratio of 1.25:1 for 1 h at 37 °C at 10 × the desired final concentration in ligand binding buffer (phosphate-buffered saline supplemented with 1.07 bovine serum albumin (BSA)). To form complexes with PAI-1, the inhibitor was added to scuPA, tcuPA, or complexes containing suPAR·scuPA and suPAR·tcuPA at a 1:1 molar ratio in binding buffer for 30 min at 37 °C. The complexes were diluted to the desired working concentration immediately before use. tcuPA (5 nm) was incubated in the absence or presence of EEIIMD or REIIMD (200 ॖm) for 30 min. The mixture was then added to a reaction mixture containing 25 nm PAI-1, 50 nmGlu-plasminogen, and 50 ॖm chromogenic substrate for the indicated times, and the O.D. at 405 nm was measured continuously (16Higazi A.A.-R. Mazar A. Wang J. Reilly R. Henkin J. Kniss D. Cines D. Blood. 1996; 87: 3545-3549Crossref PubMed Google Scholar). Binding of radiolabeled ligands to cells was measured as described (19Higazi A.A.-R. Upson R. Cohen R. McCrae K.R. Manuppello J. Bognacki J. Henkin J. Kounnas M. Strickland D. Preissner K.T. Lawler J. Cines D.B. Blood. 1996; 88: 542-551Crossref PubMed Google Scholar). Briefly, LM-TK−cells, resuspended in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 107 fetal calf serum, were plated in 96-well Falcon multiwell tissue culture dishes (Becton Dickinson) and grown to confluence at 37 °C overnight. The cells were prechilled for 1 h on ice and washed twice with prechilled binding buffer (phosphate- buffered saline and 1.07 BSA). 125I-Labeled ligands, with or without 100-fold molar excess unlabeled ligands, were added to the cells in the presence or absence of the aforementioned peptides for 2 h at 4 °C. The cells were washed four times with binding buffer, solubilized in 0.1 n NaOH, and the extract counted for radioactivity. In other experiments, binding of labeled ligands was performed in the absence or presence of 400 nmrRAP in Tris-buffered saline (TBS) containing 4 mmCa2+ or in the presence of affinity-purified IgG anti-α2MR/LRP antibody (100 ॖg/ml). To measure the binding of labeled ligands to α2MR/LRP, 96-well microtiter (Dynatech Immulon) was incubated with purified receptor or BSA (3 ॖg/ml) in TBS containing 4 mm Ca2+ overnight at 4 °C. The buffer was removed, and the unreactive sites were blocked with a solution containing TBS, 4 mm Ca2+, 0.057 Tween 20, and 37 BSA for 1 h and then incubated with the same buffer in the presence or absence of rRAP (200 nm) for another h. Binding of 125I-labeled ligands to immobilized α2MR/LRP was determined as described previously (19Higazi A.A.-R. Upson R. Cohen R. McCrae K.R. Manuppello J. Bognacki J. Henkin J. Kounnas M. Strickland D. Preissner K.T. Lawler J. Cines D.B. Blood. 1996; 88: 542-551Crossref PubMed Google Scholar,23Nykjaer A. Bengtsson-Olivecrona G. Lookene A. Moestrup S.K. Petersen C.M. Weber W. Beisigel U. Gliemann J. J. Biol. Chem. 1993; 268: 15048-15055Abstract Full Text PDF PubMed Google Scholar). LM-TK− cells were grown to confluence in 48-well Falcon multiwell tissue culture dishes (Becton Dickinson) overnight at 37 °C. The cells were prechilled on ice for 1 h, washed twice with binding buffer (TBS, 4 mmCa2+, 1.07 BSA), and incubated with the same buffer or with buffer supplemented with either 400 nm rRAP or 100 ॖg/ml IgG anti-α2MR/LRP for 1 h at room temperature. The binding buffer was removed. 125I-scuPA was added alone or in the presence of each of the three peptides and in the presence or absence 400 nm rRAP or 100 ॖg/ml anti-α2MR/LRP for 2 h at 4 °C. Unbound ligands were removed, and cells were washed five times with binding buffer. Dulbecco's modified Eagle's medium containing 4 mmCa2+ in the presence and absence of 400 nm rRAP or 100 ॖg/ml anti-α2MR/LRP was added for 18 h at 37 °C. The internalization and degradation of scuPA were measured as described previously (24Kounnas M.Z. Henkin J. Argraves W.S. Strickland D.K. J. Biol. Chem. 1993; 268: 21862-21867Abstract Full Text PDF PubMed Google Scholar, 25Li H. Kuo A. Kochan J. Strickland D. Kariko K. Barnathan E.S. Cines D.B. J. Biol. Chem. 1994; 269: 8153-8158Abstract Full Text PDF PubMed Google Scholar). Briefly, to measure internalization, the cells were washed twice with the binding buffer and 50 mmglycine, 150 mm NaCl, pH 3.0, was added for 15 min at 4 °C to dissociate cell surface-bound ligands. The cells were dissolved by adding 0.1 n NaOH for 10 min, and the extract was counted. To measure degradation, the media were removed after an 18-h incubation with radiolabeled ligands at 37 °C, trichloroacetic acid was added to a final concentration of 107, the precipitated protein was separated by centrifugation, and the radioactivity in the supernatant was measured. The purpose of this study was to determine whether a small peptide, EEIIMD (amino acids 350–355), derived from the putative docking site of PAI-1 (14Madison E.L. Goldsmith E.J. Gething M.-J.H. Sambrook J.F. Gerard R.D. J. Biol. Chem. 1990; 265: 21423-21426Abstract Full Text PDF PubMed Google Scholar), would alter the cellular binding and clearance of uPA mediated by α2MR/LRP. To address these questions, we studied the effect of this peptide on the binding, internalization, and degradation of scuPA, scuPA·suPAR complex, and tcuPA by LM-TK− cells that express α2MR/LRP but lack endogenous uPAR (19Higazi A.A.-R. Upson R. Cohen R. McCrae K.R. Manuppello J. Bognacki J. Henkin J. Kounnas M. Strickland D. Preissner K.T. Lawler J. Cines D.B. Blood. 1996; 88: 542-551Crossref PubMed Google Scholar). The data in Fig.1 A indicate that minimal amounts of scuPA bind to LM-TK− cells, consistent with previous results (19Higazi A.A.-R. Upson R. Cohen R. McCrae K.R. Manuppello J. Bognacki J. Henkin J. Kounnas M. Strickland D. Preissner K.T. Lawler J. Cines D.B. Blood. 1996; 88: 542-551Crossref PubMed Google Scholar). However, binding of scuPA to these cells was stimulated by the peptide EEIIMD in a dose-dependent and saturable manner both with respect to scuPA concentration (Fig.1 A) as well as peptide concentration (Fig. 1 B). Stimulation was evident at all concentrations of scuPA tested, and the increase in scuPA binding in the presence of EEIIMD occurred in part as a result of a 4-fold increase in the B max (Fig.1 A). By Scatchard analysis, little specific binding of scuPA to LM-TK− cells was detected in the absence of EEIIMD (K d = >1 ॖm), whereas scuPA bound with a K d of approximately 35 nm in the presence of the peptide (not shown). Half-maximal stimulation was achieved at a peptide concentration ≈ 50 ॖm, with the near maximal effect being observed at ≈ 300 ॖmpeptide (Fig. 1 B). Studies were then performed to identify the requirements for EEIIMD to stimulate the binding of scuPA. It has been reported that substitution of glutamic acid by arginine at position 350 does not affect the capacity of PAI-1 to inhibit tcuPA, whereas substitution of aspartic acid for arginine at position 355 renders PAI-1 totally inactive (14Madison E.L. Goldsmith E.J. Gething M.-J.H. Sambrook J.F. Gerard R.D. J. Biol. Chem. 1990; 265: 21423-21426Abstract Full Text PDF PubMed Google Scholar). Therefore, we next examined how modification of these two amino acids individually would alter the stimulatory effect of the hexapeptide on the binding of scuPA to LM-TK− cells. The results shown in Fig. 1 A indicate that REIIMD was almost as potent as the parent sequence EEIIMD in this regard. In contrast, EEIIMR, with aspartic acid substituted for arginine at position 6, lost approximately 807 of the stimulatory capacity of the native peptide (Fig. 1). Experiments were then performed to examine the effect of EEIIMD on the binding of scuPA to LM-TK− cells in the presence of the parent PAI-1 molecule. Preincubation with PAI-1 increased the binding of 125I-scuPA minimally (Fig.2), consistent with the low affinity of the interaction between the two ligands (11Kruithof E.K.O. Enzyme. 1988; 40: 113-121Crossref PubMed Scopus (237) Google Scholar, 13Lijnen H.R. De Cock F. Collen D. Eur. J. Biochem. 1994; 224: 567-574Crossref PubMed Scopus (27) Google Scholar, 26Kruithof E.K.O. Tran-Thang C. Ransijn A. Bachmann F. Blood. 1984; 64: 907-913Crossref PubMed Google Scholar, 27Andreasen P.A. Nielsen L.S. Kristensen P. Grondahl-Hansen J. Skriver L. Dano K. J. Biol. Chem. 1986; 261: 7644-7651Abstract Full Text PDF PubMed Google Scholar, 28Manchanda N. Schwartz B.S. J. Biol. Chem. 1995; 270: 20032-20035Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The stimulatory effect of EEIIMD on the binding of scuPA was identical in the presence or absence of PAI-1, suggesting that the peptide was better able to gain access to the corresponding site in scuPA than was the larger PAI-1 molecule and that PAI-1 did not displace the peptide from scuPA. The complex between scuPA and its receptor suPAR (scuPA·suPAR) as well as tcuPA are both active enzymes (10Higazi A.A.-R. Cohen R.L. Henkin J. Kniss D. Schwartz B.S. Cines D.B. J. Biol. Chem. 1995; 270: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) but differ in their susceptibility to PAI-1 (16Higazi A.A.-R. Mazar A. Wang J. Reilly R. Henkin J. Kniss D. Cines D. Blood. 1996; 87: 3545-3549Crossref PubMed Google Scholar). This led us to question whether the docking site in the scuPA·suPAR complex is available to PAI-1 as has been reported for tcuPA. To address this question, we compared the effect of PAI-1 and EEIIMD on the binding of scuPA·suPAR and tcuPA to LM-TK− cells. PAI-1 and EEIIMD each caused a minimal increase in the binding of scuPA·suPAR to LM-TK− cells (Fig. 3). On the other hand, PAI-1 clearly stimulated the binding of tcuPA·suPAR as reported by others (18Nykjaer A. Kjoller L. Cohen R.L. Lawrence D.A. Garni-Wagner B.A. Todd III, R.F. van Zonnefeld A.-J. Gliemann J. Andreasen P.A. J. Biol. Chem. 1994; 269: 25668-25676Abstract Full Text PDF PubMed Google Scholar, 29Nykjaer A. Petersen C.M. Moller B. Jensen P.H. Moestrup S.K. Holtet T.L. Etzerodt M. Thogersen H.C. Munch M. Andreasen P.A. Gliemann J. J. Biol. Chem. 1992; 267: 14543-14546Abstract Full Text PDF PubMed Google Scholar), whereas EEIIMD had no effect (Fig. 3). EEIIMD also failed to stimulate the binding of tcuPA to LM-TK− cells (not shown). The failure of EEIIMD to stimulate the binding of tcuPA, tcuPA·suPAR, or scuPA·suPAR complexes to the LM-TK−cells could occur as a result of the inability of the peptide to bind to the docking site, or the effect of EEIIMD may be highly dependent on the conformational state of uPA. To address the question of whether EEIIMD can bind to the docking site, we measured the capacity of the peptide to compete with the plasminogen activator inhibitor activity of PAI-1 using tcuPA. The data shown in Fig.4 indicate that EEIIMD inhibits the activity of PAI-1, whereas the control peptide EEIIMR did not (not shown).Figure 4Effect of EEIIMD on the plasminogen inhibitor activity of PAI-1. tcuPA (5 nm) was incubated in the absence or presence of EEIIMD or REIIMD (200 ॖm) for 30 min. The mixture was then added to a reaction mixture containing 25 nm PAI-1, 50 nm Glu-plasminogen, and 50 ॖm chromogenic substrate for the indicated times, and the O.D. at 405 nm was measured continuously. The results are representative of three experiments so performed.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It is well described that complexes between urokinase and PAI-1 are internalized and degraded by binding to α2MR/LRP (25Li H. Kuo A. Kochan J. Strickland D. Kariko K. Barnathan E.S. Cines D.B. J. Biol. Chem. 1994; 269: 8153-8158Abstract Full Text PDF PubMed Google Scholar, 29Nykjaer A. Petersen C.M. Moller B. Jensen P.H. Moestrup S.K. Holtet T.L. Etzerodt M. Thogersen H.C. Munch M. Andreasen P.A. Gliemann J. J. Biol. Chem. 1992; 267: 14543-14546Abstract Full Text PDF PubMed Google Scholar). Therefore, we asked whether EEIIMD increased the binding, internalization, and degradation of scuPA through this pathway. In support of this hypothesis, EEIIMD stimulated the binding of scuPA to purified α2MR/LRP and binding, to the purified protein was inhibited by rRAP (Fig.5 A). In addition, the binding of scuPA to LM-TK− cells in the presence of EEIIMD was inhibited by 400 nm rRAP and by affinity-purified anti-α2MR/LRP IgG by approximately 70 and 857, respectively (Fig. 5 B). Furthermore, EEIIMD stimulated the internalization and degradation of scuPA by LM-TK− cells (Fig. 6), and the EEIIMD-induced internalization/degradation were inhibited 70 and 807, respectively, by 400 nm rRAP and anti-α2MR/LRP IgG (Fig.6).Figure 6Effect of EEIIMD on the internalization and degradation of scuPA. Prechilled LM-TK− cells were incubated with BSA (10 ॖg/ml) or BSA supplemented with 400 nm rRAP or 100 ॖg/ml IgG anti-α2MR/LRP for 1 h at RT. 125I-scuPA was added in the absence or presence of each peptide and in the absence or presence of 100-foldm excess unlabeled scuPA for 2 h at 4 °C. Unbound ligands were removed, and the cells were washed five times with the binding buffer. Dulbecco's modified Eagle's medium (200 ॖl) or Dulbecco's modified Eagle's medium supplemented with 400 nm rRAP or 100 ॖg/ml IgG anti-α2MR/LRP was added for 18 h at 37 °C. The cells were washed and incubated with 50 mm, 150 mm NaCl, pH 3.0, to dissociate surface-bound ligands. The cells were then solubilized in 0.1n NaOH, and the radioactivity in the cell extract was counted. The medium was removed from replicate cells, and trichloroacetic acid was added (final concentration 107). After centrifugation, the radioactivity in the acid-soluble extract was counted. Control refers to BSA alone; Ab refers to anti-α2MR/LRP. □, no peptide addition; ▨, +EEIIMD. The mean ± S.D. of three or four such experiments are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Binding of scuPA to its receptor promotes its catalytic activity (10Higazi A.A.-R. Cohen R.L. Henkin J. Kniss D. Schwartz B.S. Cines D.B. J. Biol. Chem. 1995; 270: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), dampens the inhibitory capacity of PAI-1 compared with tcuPA (16Higazi A.A.-R. Mazar A. Wang J. Reilly R. Henkin J. Kniss D. Cines D. Blood. 1996; 87: 3545-3549Crossref PubMed Google Scholar), alters its regulation by peptide substrates of plasmin (30Higazi A.A.-R. Cines D.B. Thromb. Res. 1996; 84: 243-252Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar), and promotes its binding to vitronectin (19Higazi A.A.-R. Upson R. Cohen R. McCrae K.R. Manuppello J. Bognacki J. Henkin J. Kounnas M. Strickland D. Preissner K.T. Lawler J. Cines D.B. Blood. 1996; 88: 542-551Crossref PubMed Google Scholar, 31Wei W. 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, 32Wei 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, 33Deng G. Curriden S.A. Wang S. Rosenberg S. Loskutoff D.J. J. Cell Biol. 1996; 134: 1563-1571Crossref PubMed Scopus (429) Google Scholar, 34Stefansson S. Lawrence D.A. Nature. 1996; 383: 441-443Crossref PubMed Scopus (604) Google Scholar, 35O'Reilly M.S. Holmgren L. Chen C. Folkman J. Nature Med. 1996; 2: 689-692Crossref PubMed Scopus (1146) Google Scholar). The results of the current study identify another mechanism by which the function of scuPA can be altered. The binding of scuPA to LM-TK− cells is stimulated by the PAI-1-derived peptide EEIIMD. The observations that the increase in binding of scuPA is accompanied by its internalization and degradation and that these consequences are prevented by rRAP as well as by a specific anti-α2MR/LRP antibody are consistent with the involvement of α2MR/LRP in this process. This contention is supported further by the finding that EEIIMD increases the binding of scuPA to purified α2MR/LRP and that this augmentation is also inhibited by rRAP. Therefore, these results suggest a potential mechanism by which scuPA can be cleared which is independent of receptor binding or proteolytic activation (10Higazi A.A.-R. Cohen R.L. Henkin J. Kniss D. Schwartz B.S. Cines D.B. J. Biol. Chem. 1995; 270: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Although it has been postulated that binding of tcuPA·PAI-1 to α2MR/LRP is mediated by independent epitopes in each molecule (18Nykjaer A. Kjoller L. Cohen R.L. Lawrence D.A. Garni-Wagner B.A. Todd III, R.F. van Zonnefeld A.-J. Gliemann J. Andreasen P.A. J. Biol. Chem. 1994; 269: 25668-25676Abstract Full Text PDF PubMed Google Scholar), such a mechanism is unlikely to explain the stimulatory effect of EEIIMD on scuPA binding in that it is unlikely that the peptide is large enough to accommodate both proteins simultaneously. Further, EEIIMD bound to the docking site in tcuPA, as shown by inhibition of PAI-1 activity, but did not increase its binding to α2MR/LRP. Therefore, the observation that EEIIMD increased the B max for scuPA suggests that the peptide induces previously unrecognized cellular binding sites that are not available in the native molecule which are recognized by α2MR/LRP, i.e. that the docking sequence to which EEIIMD binds functions as an allosteric site. Consistent with this notion, whereas EEIIMD stimulated the binding of scuPA, the peptide had no effect on the binding of either the scuPA·suPAR complex or tcuPA. These results are consistent with the reported differences in the ability of scuPA, the scuPA·suPAR complex, and tcuPA to interact with α2MR/LRP (18Nykjaer A. Kjoller L. Cohen R.L. Lawrence D.A. Garni-Wagner B.A. Todd III, R.F. van Zonnefeld A.-J. Gliemann J. Andreasen P.A. J. Biol. Chem. 1994; 269: 25668-25676Abstract Full Text PDF PubMed Google Scholar). The observation that EEIIMD regulates the affinity with which scuPA binds to α2MR/LRP indicates that the docking site for PAI-1 is accessible to the peptide. This finding suggests that the resistance of scuPA to PAI-1 may occur because docking of the serpin does not facilitate its binding to the catalytic site as occurs in tcuPA (14Madison E.L. Goldsmith E.J. Gething M.-J.H. Sambrook J.F. Gerard R.D. J. Biol. Chem. 1990; 265: 21423-21426Abstract Full Text PDF PubMed Google Scholar) because of an inappropriate distance between the sites in scuPA compared with tcuPA, differences in the orientation of the docking and catalytic sites in the two molecules, or a failure of the serpin to interact with the catalytic site. It is likely that EEIIMD fails to stimulate the cellular binding of tcuPA, although it binds to the docking site, because the peptide is unable to induce the site recognized by α2MR/LRP. A similar mechanism may also account for the failure of EEIIMD to induce the binding of the other active form of uPA, scuPA·suPAR. Alternatively, the binding site for α2MR/LRP on scuPA may be induced and shielded by its receptor, consistent with the capacity of uPAR to block the interaction of tcuPA·PAI-1 and scuPA with α2MR/LRP (18Nykjaer A. Kjoller L. Cohen R.L. Lawrence D.A. Garni-Wagner B.A. Todd III, R.F. van Zonnefeld A.-J. Gliemann J. Andreasen P.A. J. Biol. Chem. 1994; 269: 25668-25676Abstract Full Text PDF PubMed Google Scholar, 19Higazi A.A.-R. Upson R. Cohen R. McCrae K.R. Manuppello J. Bognacki J. Henkin J. Kounnas M. Strickland D. Preissner K.T. Lawler J. Cines D.B. Blood. 1996; 88: 542-551Crossref PubMed Google Scholar). Thus, conversion of scuPA to tcuPA is associated with the loss of the ability of EEIIMD to induce the structure recognized by α2MR/LRP, presumably as a result of the loss of coordinate interaction between different portions of the molecule. Support for this interpretation comes from the observations that uPAR has little or no effect on the enzymatic activity of tcuPA (10Higazi A.A.-R. Cohen R.L. Henkin J. Kniss D. Schwartz B.S. Cines D.B. J. Biol. Chem. 1995; 270: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) or its susceptibility to inactivation by PAI-1 (10Higazi A.A.-R. Cohen R.L. Henkin J. Kniss D. Schwartz B.S. Cines D.B. J. Biol. Chem. 1995; 270: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 17Ellis V. Wun T.C. Behrendt B. Ronne E. Dano K. J. Biol. Chem. 1990; 265: 9904-9908Abstract Full Text PDF PubMed Google Scholar). These results provide additional support for the concept that the conversion of scuPA to tcuPA is the first step in its inactivation and degradation. Irrespective of the sequence of events, the capacity of EEIIMD to stimulate the binding of scuPA provides additional support for the notion that the biologic activity of scuPA can be regulated through its docking site, as a result of which the internalization and degradation of the protein are accelerated. Thus, it is not necessary that scuPA be converted to tcuPA for the protein to be recognized by α2MR/LRP (18Nykjaer A. Kjoller L. Cohen R.L. Lawrence D.A. Garni-Wagner B.A. Todd III, R.F. van Zonnefeld A.-J. Gliemann J. Andreasen P.A. J. Biol. Chem. 1994; 269: 25668-25676Abstract Full Text PDF PubMed Google Scholar). It remains to be established whether there exists a physiologic analog for the activity of EEIIMD described in this study.

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