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1.
Curr Drug Targets ; 12(12): 1711-28, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21707479

ABSTRACT

In the last two decades, the urokinase-type plasminogen activator receptor (uPAR) has been implicated in a number of human pathologies such as cancer, bacterial infections, and paroxysmal nocturnal hemoglobinuria. The primary function of this glycolipid-anchored receptor is to focalize uPA-mediated plasminogen activation at the cell surface, which is accomplished by its high-affinity interaction with the growth factor-like domain of uPA. Detailed insights into the molecular basis underlying the interactions between uPAR and its two bona fide ligands, uPA and vitronectin, have been obtained recently by X-ray crystallography and surface plasmon resonance studies. Importantly, these structural studies also define possible druggable target sites in uPAR for small molecules and provide guidelines for the development of reporter groups applicable for non-invasive molecular imaging of uPAR expression in vivo by positron emission tomography. In this review, we will discuss recent advances in our perception of the structure-function relationships of uPAR ligation and how these may assist translational research in preclinical intervention studies of uPAR function.


Subject(s)
Molecular Probes , Molecular Targeted Therapy , Receptors, Urokinase Plasminogen Activator/antagonists & inhibitors , Animals , Gene Silencing , Humans , Multimodal Imaging/methods , Positron-Emission Tomography/methods , Protein Conformation , Receptors, Urokinase Plasminogen Activator/chemistry , Receptors, Urokinase Plasminogen Activator/genetics , Receptors, Urokinase Plasminogen Activator/metabolism , Signal Transduction/drug effects , Species Specificity , Tomography, X-Ray Computed , Translational Research, Biomedical , Urokinase-Type Plasminogen Activator/antagonists & inhibitors , Urokinase-Type Plasminogen Activator/chemistry , Urokinase-Type Plasminogen Activator/metabolism , Vitronectin/chemistry , Vitronectin/metabolism
2.
Scand J Immunol ; 58(3): 298-305, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12950675

ABSTRACT

The urokinase receptor (uPAR) is a glycolipid-anchored cell surface glycoprotein that plays a central role in extracellular proteolysis during tissue remodeling processes including cancer invasion. Furthermore, uPAR is found on the surface of both dendritic cells (DCs) and T cells, and has been proposed to play a role in DC-induced T-cell activation and, therefore, in the induction of an immune response. In order to investigate the possibility of using DNA immunization for the generation of poly- and monoclonal antibodies to uPAR, we injected wild-type mice and mice deficient in uPAR (uPAR knockouts) intramuscularly with plasmid DNA encoding a carboxy-terminal truncated soluble form of the human uPAR. Multiple injections of 100 micro g of DNA resulted in a strong and specific antibody response in all mice irrespective of genotype. Antisera with a maximum titre of 32,000 were obtained, comparable with that obtained after immunization with recombinant uPAR. The subclass distribution of uPAR-specific antibodies in the sera demonstrated the induction of a mixed TH1/TH2 response, irrespective of the genotype of the mice. Our results demonstrate the possibility of generating high titre antibodies to uPAR by DNA immunization of wild-type as well as uPAR knockout mice, and that cell surface uPAR is not indispensable for the generation of a humoral immune response.


Subject(s)
Antibody Formation/immunology , Receptors, Cell Surface/immunology , Vaccines, DNA/immunology , Animals , Antibodies/blood , Antibody Formation/genetics , Blotting, Western , COS Cells , Chlorocebus aethiops , Enzyme-Linked Immunosorbent Assay , Female , Genetic Vectors/genetics , Humans , Immunization , Immunoglobulin G/biosynthesis , Immunoglobulin G/classification , Immunoglobulin G/immunology , Mice , Mice, Knockout , Plasmids/genetics , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/genetics , Receptors, Urokinase Plasminogen Activator , Transfection , Vaccines, DNA/genetics
3.
Biochem Soc Trans ; 30(2): 177-83, 2002 Apr.
Article in English | MEDLINE | ID: mdl-12023847

ABSTRACT

The ability to degrade the extracellular matrix by controlled proteolysis is an important property of malignant cancer cells, which enables them to invade the surrounding tissue and to gain access to the circulation by intravasation. One proteolytic system thought to be involved in these processes is urokinase-mediated plasminogen activation. Expression of a glycolipid-anchored receptor for urokinase-type plasminogen activator (uPA) targets this system to the cell surface. This receptor (uPAR) is composed of three homologous modules belonging to the Ly-6/uPAR/alpha-neurotoxin protein domain family. Integrity of the three-domain structure of uPAR is required for maintenance of its sub-nanomolar affinity for uPA, but the functional epitope for this interaction is primarily located in uPAR domain I. Using affinity maturation by combinatorial chemistry, we have recently identified a potent 9-mer peptide antagonist of the uPA-uPAR interaction having a high affinity for uPAR (K(d)< 1 nM). Photoaffinity labelling suggests that this peptide interacts with a composite binding site in uPAR involving both domains I and III. When tested in a chicken chorioallantoic membrane assay that was developed to quantify intravasation of human cells, this antagonist was able to reduce the intravasation of HEp-3 cancer cells by approx. 60%.


Subject(s)
Neoplasms/drug therapy , Neoplasms/metabolism , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Urokinase-Type Plasminogen Activator/chemistry , Urokinase-Type Plasminogen Activator/metabolism , Amino Acid Sequence , Animals , Humans , In Vitro Techniques , Models, Molecular , Molecular Sequence Data , Molecular Structure , Neoplasm Invasiveness , Oligopeptides/chemistry , Oligopeptides/pharmacology , Receptors, Cell Surface/drug effects , Receptors, Urokinase Plasminogen Activator , Urokinase-Type Plasminogen Activator/drug effects
4.
Biochemistry ; 40(40): 12157-68, 2001 Oct 09.
Article in English | MEDLINE | ID: mdl-11580291

ABSTRACT

The high-affinity interaction between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays an important role in pericellular plasminogen activation. Since proteolytic degradation of the extracellular matrix has an established role in tumor invasion and metastasis, the uPA-uPAR interaction represents a potential target for therapeutic intervention. By affinity maturation using combinatorial chemistry we have now developed and characterized a 9-mer, linear peptide antagonist of the uPA-uPAR interaction demonstrating specific, high-affinity binding to human uPAR (K(d) approximately 0.4 nM). Studies by surface plasmon resonance reveal that the off-rate for this receptor-peptide complex is comparable to that measured for the natural protein ligand, uPA. The functional epitope on human uPAR for this antagonist has been delineated by site-directed mutagenesis, and its assignment to loop 3 of uPAR domain III (Met(246), His(249), His(251), and Phe(256)) corroborates data previously obtained by photoaffinity labeling and provides a molecular explanation for the extreme selectivity observed for the antagonist toward human compared to mouse, monkey, and hamster uPAR. When human HEp-3 cancer cells were inoculated in the presence of this peptide antagonist, a specific inhibition of cancer cell intravasation was observed in a chicken chorioallantoic membrane assay. These data imply that design of small organic molecules mimicking the binding determinants of this 9-mer peptide antagonist may have a potential application in combination therapy for certain types of cancer.


Subject(s)
Combinatorial Chemistry Techniques , Epitopes/chemistry , Neoplasms/pathology , Receptors, Cell Surface/antagonists & inhibitors , Animals , Base Sequence , Cell Line , Cricetinae , DNA Primers , Humans , Mice , Oligopeptides/chemistry , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/immunology , Receptors, Urokinase Plasminogen Activator , Recombinant Proteins/chemistry , Recombinant Proteins/immunology
5.
J Immunol Methods ; 234(1-2): 107-16, 2000 Feb 03.
Article in English | MEDLINE | ID: mdl-10669775

ABSTRACT

The urokinase receptor (uPAR) is a glycolipid anchored cell surface glycoprotein that plays a central role in extracellular proteolysis during tissue remodeling processes and cancer invasion. By intramuscular (i.m.) injection of rabbits with plasmid DNA coding for a carboxy-terminally truncated secreted form of the murine uPAR (muPAR), specific anti-sera with a titer of 64,000, as measured by ELISA, have been obtained. Rabbits received a total of 10 monthly injections of 1 mg DNA in phosphate-buffered saline. The antibody titer peaked between the 5th and 7th injection and slowly declined after the 8th injection. After the final immunization the immune response persisted for at least 6 months without further injections. The antibodies generated by DNA immunization were useful for immunohistochemistry and immunoblotting, recognizing the antigen both in its native and in its reduced and alkylated form. Using the antibodies in immunoblotting muPAR was identified in lysates of peritoneal macrophages, spleen and lung tissue. Both the intact and cleaved form of muPAR were identified in lysates of a murine monocyte cell line P388D.1. No cross-reaction with human uPAR was observed. In immunohistochemical analysis of normal mouse lung tissue uPAR immunoreactivity was located in the alveoli and pulmonary vessels, whereas the bronchial epithelium was negative. These results demonstrate that DNA immunization of rabbits using i.m. injection is a very effective and easy method to raise polyclonal antibodies which can be used for characterization and localization of muPAR in mouse tissue.


Subject(s)
Antibody Formation , Receptors, Cell Surface/genetics , Vaccines, DNA/immunology , Animals , Antibodies/immunology , Antibody Affinity , Cell Line , Gene Expression , Genetic Vectors , Humans , Immunoblotting , Immunohistochemistry/methods , Mice , Rabbits , Receptors, Cell Surface/immunology , Receptors, Urokinase Plasminogen Activator , Staining and Labeling/methods , Time Factors
6.
J Biol Chem ; 274(53): 37995-8003, 1999 Dec 31.
Article in English | MEDLINE | ID: mdl-10608868

ABSTRACT

The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid anchored multidomain member of the Ly-6/uPAR protein domain superfamily. Studies by site-directed photoaffinity labeling, chemical cross-linking, and ligand-induced protection against chemical modification have highlighted the possible involvement of uPAR domain I and particularly loop 3 thereof in ligand binding (Ploug, M. (1998) Biochemistry 37, 16494-16505). Guided by these results we have now performed an alanine scanning analysis of this region in uPAR by site-directed mutagenesis and subsequently measured the effects thereof on the kinetics of uPA binding in real-time by surface plasmon resonance. Only four positions in loop 3 of uPAR domain I exhibited significant changes in the contribution to the free energy of uPA binding (DeltaDeltaG >/= 1.3 kcal mol(-1)) upon single-site substitutions to alanine (i.e. Arg(53), Leu(55), Tyr(57), and Leu(66)). The energetic impact of these four alanine substitutions was not caused by gross structural perturbations, since all monoclonal antibodies tested having conformation-dependent epitopes on this domain exhibited unaltered binding kinetics. These sites together with a three-dimensional structure for uPAR may provide an appropriate target for rational drug design aimed at developing new receptor binding antagonists with potential application in cancer therapy.


Subject(s)
Epitope Mapping , Receptors, Cell Surface/metabolism , Amino Acid Sequence , Base Sequence , DNA Primers , Humans , Kinetics , Ligands , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/genetics , Receptors, Urokinase Plasminogen Activator , Thermodynamics , Urokinase-Type Plasminogen Activator/metabolism
7.
FEBS Lett ; 431(2): 170-4, 1998 Jul 17.
Article in English | MEDLINE | ID: mdl-9708896

ABSTRACT

Large random hexa- and decapenta-peptide libraries were constructed and displayed on the surface of the filamentous phagemid pComb8. Panning of the hexa-peptide library on immobilized plasminogen activator inhibitor 1 (PAI-1) specifically selected a minor fraction of concatemers, indicating that binding to PAI-1 requires an extended amino acid sequence. Accordingly, the decapenta-peptide library exclusively yielded PAI-1 binding peptides of 15 amino acid residues. None of these phage-bound peptides prevented the interaction between PAI-1 and its target serine protease urokinase (u-PA). To isolate peptides that block the interaction between PAI-1 and u-PA, phages bound to immobilized PAI-1 were eluted by incubation with u-PA. Remarkably, this procedure resulted in elution of a unique phage type that harbors a concatemer of decapentamers, consisting of 49 amino acid residues with no obvious similarity to the primary sequence of PAI-1 or u-PA.


Subject(s)
Peptide Library , Peptides/metabolism , Plasminogen Activator Inhibitor 1/metabolism , Amino Acid Sequence , Bacteriophages , Binding Sites , Humans , Molecular Sequence Data , Sequence Homology, Amino Acid , Urokinase-Type Plasminogen Activator/metabolism
8.
Biol Chem Hoppe Seyler ; 376(5): 259-67, 1995 May.
Article in English | MEDLINE | ID: mdl-7662168

ABSTRACT

Degradation of the extracellular matrix plays a crucial role in cancer invasion. This degradation is accomplished by the concerted action of several enzyme systems, including generation of the serine protease plasmin by the urokinase pathway of plasminogen activation, different types of collagenases and other metalloproteinases, and other extracellular enzymes. The degradative enzymes are involved also in tissue remodelling under non-malignant conditions, and the main difference appears to be that mechanisms which regulates these processes under normal conditions are defective in cancer. Specific inhibitors have been identified for most of the proteolytic enzymes, e.g. plasminogen activator inhibitors (PAI's) and tissue inhibitors of metalloproteinases (TIMP's). It has been contemplated that these inhibitors counteracted the proteolytic activity of the enzymes, thereby inhibiting extracellular tissue degradation which in turn should prevent tumor cell invasion. This review focuses on plasminogen inhibitor type 1 (PAI-1). It is described that PAI-1 is not produced by the epithelial cancer cell but by the stromal cells in the tumors, suggesting a concerted action between stroma and tumor cells in the processes controlling proteolysis in cancer. The specific localization of PAI-1 to the tumor stroma and in many cases to areas surrounding the tumor vessels has lead us to suggest that PAI-1 serves to protect the tumor stroma from the ongoing uPA-mediated proteolysis. This hypothesis is supported by recent clinical data showing increased levels of PAI-1 in metastases as compared to the primary tumor as well as data demonstrating that high levels of PAI-1 in tumor extracts from breast, lung, gastric and ovarian cancer is associated with a shorter overall survival.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Neoplasms/physiopathology , Plasminogen Activator Inhibitor 1/physiology , Animals , Humans , Neoplasms/metabolism , Neoplasms/therapy , Plasminogen Activator Inhibitor 1/metabolism , Prognosis
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