The Urokinase/PAI-2 Complex

The efficient inactivation of urokinase plasminogen activator (uPA) by plasminogen activator inhibitor type 2 (PAI-2) at the surface of carcinoma cells is followed by rapid endocytosis of the uPA-PAI-2 complex. We now show that one pathway of this receptor-mediated endocytosis is mediated via the low density lipoprotein receptor-related protein (LRP) in prostate cancer cells. Detailed biochemical analyses using ligand binding assays and surface plasmon resonance revealed a novel and distinct interaction mechanism between native, human LRP and uPA-PAI-2. As reported previously for PAI-1, inhibition of uPA by PAI-2 significantly increased the affinity of the complex for LRP (KD of 36 nm for uPA-PAI-2 versus 200 nm for uPA). This interaction was maintained in the presence of uPAR, confirming the validity of this interaction at the cell surface. However, unlike PAI-1, no interaction was observed between LRP and PAI-2 in either the stressed or the relaxed conformation. This suggests that the uPA-PAI-2-LRP interaction is mediated by site(s) within the uPA molecule alone. Thus, as inhibition of uPA by PAI-2 resulted in accelerated clearance of uPA from the cell surface possibly via its increased affinity for LRP, this represents a mechanism through which PAI-2 can clear proteolytic activity from the cell surface. Furthermore, lack of a direct interaction between PAI-2 and LRP implies that downstream signaling events initiated by PAI-1 may not be activated by PAI-2.

[1]  J. Whisstock,et al.  The Serpins Are an Expanding Superfamily of Structurally Similar but Functionally Diverse Proteins , 2001, The Journal of Biological Chemistry.

[2]  P. Andreasen,et al.  Very Low Density Lipoprotein Receptor Binds and Mediates Endocytosis of Urokinase-type Plasminogen Activator-Type-1 Plasminogen Activator Inhibitor Complex (*) , 1995, The Journal of Biological Chemistry.

[3]  J. Foekens,et al.  Plasminogen activator inhibitor-2: prognostic relevance in 1012 patients with primary breast cancer. , 1995, Cancer research.

[4]  R. Read,et al.  Structure of a serpin–protease complex shows inhibition by deformation , 2000, Nature.

[5]  P. Curmi,et al.  The crystal structure of plasminogen activator inhibitor 2 at 2.0 A resolution: implications for serpin function. , 1999, Structure.

[6]  D. Webb,et al.  Plasminogen Activator Inhibitor 1 Functions as a Urokinase Response Modifier at the Level of Cell Signaling and Thereby Promotes Mcf-7 Cell Growth , 2001, The Journal of cell biology.

[7]  P. Andreasen,et al.  Binding of urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein and very-low-density lipoprotein receptor involves basic residues in the inhibitor. , 1998, The Biochemical journal.

[8]  M. Ranson,et al.  Preclinical studies of targeted α therapy for breast cancer using 213Bi-labelled-plasminogen activator inhibitor type 2 , 2003, British Journal of Cancer.

[9]  S. Muhammad,et al.  Plasminogen Activator Inhibitor-1 Contains a Cryptic High Affinity Binding Site for the Low Density Lipoprotein Receptor-related Protein* , 1998, The Journal of Biological Chemistry.

[10]  F. Blasi,et al.  Recycling of the urokinase receptor upon internalization of the uPA:serpin complexes , 1997, The EMBO journal.

[11]  A. Salicioni,et al.  Low density lipoprotein receptor-related protein: regulation of the plasma membrane proteome , 2004, Thrombosis and Haemostasis.

[12]  R. Hammer,et al.  LDL receptor-related protein internalizes and degrades uPA-PAI-1 complexes and is essential for embryo implantation , 1992, Cell.

[13]  S. Pizzo,et al.  The role of Grp 78 in alpha 2-macroglobulin-induced signal transduction. Evidence from RNA interference that the low density lipoprotein receptor-related protein is associated with, but not necessary for, GRP 78-mediated signal transduction. , 2002, The Journal of biological chemistry.

[14]  M. Ranson,et al.  The topology of plasminogen binding and activation on the surface of human breast cancer cells , 2001, British Journal of Cancer.

[15]  A. Krüger,et al.  The urokinase plasminogen activator system as a novel target for tumour therapy , 2000 .

[16]  M. Brown,et al.  Low density lipoprotein receptor-related protein and gp330 bind similar ligands, including plasminogen activator-inhibitor complexes and lactoferrin, an inhibitor of chylomicron remnant clearance. , 1992, The Journal of biological chemistry.

[17]  D. Strickland,et al.  The Very Low Density Lipoprotein Receptor Mediates the Cellular Catabolism of Lipoprotein Lipase and Urokinase-Plasminogen Activator Inhibitor Type I Complexes (*) , 1995, The Journal of Biological Chemistry.

[18]  Richard G. W. Anderson,et al.  Platelet-derived Growth Factor Mediates Tyrosine Phosphorylation of the Cytoplasmic Domain of the Low Density Lipoprotein Receptor-related Protein in Caveolae* , 2002, The Journal of Biological Chemistry.

[19]  S. Moestrup,et al.  The human alpha 2-macroglobulin receptor contains high affinity calcium binding sites important for receptor conformation and ligand recognition. , 1990, Journal of Biological Chemistry.

[20]  S. L. Gonias,et al.  The low‐density lipoprotein receptor‐related protein‐1 associates transiently with lipid rafts , 2005, Journal of cellular biochemistry.

[21]  M. Baker,et al.  Increased plasminogen binding is associated with metastatic breast cancer cells: differential expression of plasminogen binding proteins. , 1998, British Journal of Cancer.

[22]  M. Duffy,et al.  The urokinase‐type plasminogen activator system in cancer metastasis: A review , 1997, International journal of cancer.

[23]  K. Danø,et al.  Inhibition of receptor-bound urokinase by plasminogen-activator inhibitors. , 1990, The Journal of biological chemistry.

[24]  P. Rettenberger,et al.  Ligand Binding Properties of the Very Low Density Lipoprotein Receptor , 1999, The Journal of Biological Chemistry.

[25]  R. Czekay,et al.  The Low Density Lipoprotein Receptor-related Protein Is a Motogenic Receptor for Plasminogen Activator Inhibitor-1* , 2004, Journal of Biological Chemistry.

[26]  M. Duffy,et al.  The urokinase plasminogen activator system: role in malignancy. , 2004, Current pharmaceutical design.

[27]  D. Strickland,et al.  Diverse roles for the LDL receptor family , 2002, Trends in Endocrinology & Metabolism.

[28]  J. Lawler,et al.  Internalization but not binding of thrombospondin‐1 to low density lipoprotein receptor‐related protein‐1 requires heparan sulfate proteoglycans , 2004, Journal of cellular biochemistry.

[29]  P. Andreasen,et al.  Lysosomal degradation of receptor-bound urokinase-type plasminogen activator is enhanced by its inhibitors in human trophoblastic choriocarcinoma cells. , 1990, Cell regulation.

[30]  J. Henkin,et al.  gp330 on type II pneumocytes mediates endocytosis leading to degradation of pro-urokinase, plasminogen activator inhibitor-1 and urokinase-plasminogen activator inhibitor-1 complex. , 1995, Journal of cell science.

[31]  M. Cubellis,et al.  Accessibility of receptor-bound urokinase to type-1 plasminogen activator inhibitor. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[32]  B. M. Mueller,et al.  The urokinase-type plasminogen activator receptor, a GPI-linked protein, is localized in caveolae , 1995, The Journal of cell biology.

[33]  D. Lawrence,et al.  Regions involved in binding of urokinase-type-1 inhibitor complex and pro-urokinase to the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Evidence that the urokinase receptor protects pro-urokinase against binding to the endocytic receptor. , 1994, The Journal of biological chemistry.

[34]  L. Orci,et al.  The receptor for urokinase type plasminogen activator polarizes expression of the protease to the leading edge of migrating monocytes and promotes degradation of enzyme inhibitor complexes , 1990, The Journal of cell biology.

[35]  D. Knauer,et al.  Identification of a Binding Site in Protease Nexin I (PN1) Required for the Receptor Mediated Internalization of PN1-Thrombin Complexes* , 1997, The Journal of Biological Chemistry.

[36]  S. Moestrup,et al.  Purified alpha 2-macroglobulin receptor/LDL receptor-related protein binds urokinase.plasminogen activator inhibitor type-1 complex. Evidence that the alpha 2-macroglobulin receptor mediates cellular degradation of urokinase receptor-bound complexes. , 1992, The Journal of biological chemistry.

[37]  David R. Croucher,et al.  Kinetic analysis of plasminogen activator inhibitor type-2: urokinase complex formation and subsequent internalisation by carcinoma cell lines. , 2004, Experimental cell research.

[38]  M. Ranson,et al.  In Vitro Cytotoxicity of Bismuth-213 (213bi)-Labeled-Plasminogen Activator Inhibitor Type 2 (Alpha-PAI-2) on Human Breast Cancer Cells , 2004, Breast Cancer Research and Treatment.

[39]  J. Henkin,et al.  Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor mediates cellular uptake of pro-urokinase. , 1993, The Journal of biological chemistry.

[40]  L. Norkin,et al.  Bound simian virus 40 translocates to caveolin-enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae. , 1996, Molecular biology of the cell.

[41]  M. Baker,et al.  Immunological Detection of Conformational Neoepitopes Associated with the Serpin Activity of Plasminogen Activator Inhibitor Type-2* , 1998, The Journal of Biological Chemistry.

[42]  A. Vaheri,et al.  Prourokinase activation on the surface of human rhabdomyosarcoma cells: localization and inactivation of newly formed urokinase-type plasminogen activator by recombinant class 2 plasminogen activator inhibitor. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[43]  M. Cubellis,et al.  Receptor‐mediated internalization and degradation of urokinase is caused by its specific inhibitor PAI‐1. , 1990, The EMBO journal.

[44]  R. Sakthivel,et al.  The Low Density Lipoprotein Receptor-related Protein/α2-Macroglobulin Receptor Regulates Cell Surface Plasminogen Activator Activity on Human Trophoblast Cells* , 1998, The Journal of Biological Chemistry.

[45]  S. Moestrup,et al.  Receptor‐mediated endocytosis of plasminogen activators and activator/inhibitor complexes , 1994, FEBS letters.

[46]  M. Baker,et al.  Biological and clinical aspects of plasminogen activator inhibitor type 2. , 1995, Blood.

[47]  C. Glabe,et al.  Cell surface APP751 forms complexes with protease nexin 2 ligands and is internalized via the low density lipoprotein receptor-related protein (LRP) , 1996, Brain Research.

[48]  W. Stoorvogel,et al.  Endocytosed Transferrin Receptors Recycle via Distinct Dynamin and Phosphatidylinositol 3-Kinase-dependent Pathways* , 2002, The Journal of Biological Chemistry.

[49]  P. Curmi,et al.  Interaction between the P14 Residue and Strand 2 of β-Sheet B Is Critical for Reactive Center Loop Insertion in Plasminogen Activator Inhibitor-2* , 2001, The Journal of Biological Chemistry.

[50]  M. Ranson,et al.  213Bi-PAI2 conjugate selectively induces apoptosis in PC3 metastatic prostate cancer cell line and shows anti-cancer activity in a xenograft animal model , 2002, British Journal of Cancer.

[51]  S. Moestrup,et al.  Epithelial glycoprotein-330 mediates endocytosis of plasminogen activator-plasminogen activator inhibitor type-1 complexes. , 1993, The Journal of biological chemistry.

[52]  R. Czekay,et al.  Direct binding of occupied urokinase receptor (uPAR) to LDL receptor-related protein is required for endocytosis of uPAR and regulation of cell surface urokinase activity. , 2001, Molecular biology of the cell.

[53]  S. L. Gonias,et al.  The Very Low Density Lipoprotein Receptor Regulates Urokinase Receptor Catabolism and Breast Cancer Cell Motility in Vitro * , 1999, The Journal of Biological Chemistry.