Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The gene for CD59 [membrane inhibitor of reactive lysis (MIRL), protectin], a phosphatidylinositol-linked surface glycoprotein that regulates the formation of the polymeric C9 complex of complement and that is deficient on the abnormal hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria, consists of four exons spanning 20 kilobases. The untranslated first exon is preceded by a G+C-rich promoter region that lacks a consensus TATA or CAAT motif. The second exon encodes the hydrophobic leader sequence of the protein, and the third exon encodes the amino-terminal portion of the mature protein. The fourth exon encodes the remainder of the mature protein, including the hydrophobic sequence necessary for glycosyl-phosphatidylinositol anchor attachment. The structure of the CD59 gene is very similar to that encoding Ly-6, a murine glycoprotein with which CD59 has some structural similarity. The striking similarity in gene structure is further evidence that the two proteins belong to a superfamily of proteins that may also include the urokinase plasminogen-activator receptor and a squid glycoprotein of unknown function.
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PMID:Structure of the CD59-encoding gene: further evidence of a relationship to murine lymphocyte antigen Ly-6 protein. 138 3

The glycosylphosphatidylinositol (GPI)-anchored membrane protein urokinase plasminogen activator-receptor (uPA-R; CD87) is one of the key molecules involved in migration of leukocytes and tumor cells. uPA bound to uPA-R provides the cell proteolytic potential used for degradation of extracellular matrix. uPA-R is also involved in induction of cell adhesion and chemotaxis. Here, we provide a molecular explanation for these uPA-R-related cellular events. By size fractionation of monocyte lysate and affinity isolation on its natural ligand uPA, we demonstrate uPA-R as a component of a receptor complex of relatively large size. Reprecipitation and immunoblotting techniques allowed us to detect the protein tyrosine kinases (PTKs) p60fyn, p53/56lyn, p58/64hck, and p59fgr as components of this "uPA-R complex". Activation of monocytes even with enzymatically inactivated uPA resulted in induction of tyrosine phosphorylation, suggesting modulation of uPA-R-associated PTKs upon ligand binding. In spite of their presence in large complexes, we did not find the GPI-linked proteins CD14, CD58, and CD59 in the uPA-R complex, which indicates the presence of different receptor domains containing GPI-linked proteins in monocytes. However, we identified the leukocyte integrins LFA-1 and CR3 as components of the uPA-R complex as indicated by coisolation of these molecules, as well as by cocapping and comodulation of uPA-R and leukocyte integrins on the monocyte surface. The assemblage of uPA-R, PTKs and membrane spanning beta 2-integrins in one receptor complex indicates functional cooperation. In regard to the involvement of these molecules in pericellular proteolysis, signal transduction, as well as adhesion and chemotactic movement, we suggest uPA-R complex as a potential cellular device for cell migration.
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PMID:Urokinase plasminogen activator receptor, beta 2-integrins, and Src-kinases within a single receptor complex of human monocytes. 753 37

The endothelial cell (EC) urokinase receptor plays an important role in the localization and receptor-mediated activation of EC-bound plasminogen and hence surface-localized fibrinolysis. Thrombin induced a rapid (< 5 minute), time- (0 to 30 minutes) and dose- (0.1 to 8 U/mL) dependent decrease in the specific binding of 125I-labeled two-chain urokinase-type plasminogen activator (tcu-PA) or diisopropylfluoro-phosphate-tcu-PA to urokinase-type plasminogen activator receptor (u-PAR) in cultured ECs from various sources (range, 21% to 50%). The thrombin receptor activation peptide but not control peptide showed a similar but reduced decrease in the specific binding of 125I-labeled tcu-PA to u-PAR. Incubation of thrombin-treated cultures (10 to 12 hours) in complete medium restored 125I-labeled tcu-PA ligand binding to normal levels. u-PAR mRNA levels rapidly (1 hour) increased and peaked 10 to 12 hours after thrombin treatment as analyzed by reverse transcriptase-polymerase chain reaction. Decreased thrombin-induced 125I-labeled tcu-PA binding correlated with the time-dependent decrease in surface-localized plasmin generation, as measured by the direct activation of 125I-labeled Glu-plasminogen and quantification of the 20-kD light chains of 125I-labeled plasmin. After incubation with thrombin, plasmin generation was decreased 50% to 56% (125 to 152 fmol/3 to 3.5 x 10(4) cells). Isolation of metabolically labeled 35S-labeled u-PAR from the media of thrombin and phospholipase C-treated human aortic cultures yielded approximately 10- and approximately 12-fold more 55-kD M(r) and approximately 6-fold more 35-kD M(r) 35S-labeled u-PAR forms than control cultures, respectively. The u-PAR antigen forms (M(r), 54 kD) and the glycosyl-phosphatidylinositol-anchored protein CD59 (M(r), 20 kD) were also simultaneously identified by immunoprecipitation in the media of thrombin-treated cultures. This suggests that thrombin may release u-PAR and decrease u-PA ligand binding through a common pathway involving phospholipase C. These results establish a novel interrelation between thrombin and EC fibrinolysis and suggest that thrombin may also have an additional regulatory role in the net expression of surface-localized EC fibrinolytic activity.
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PMID:Thrombin decreases the urokinase receptor and surface-localized fibrinolysis in cultured endothelial cells. 774 51

The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that plays a central role in pericellular plasminogen activation. It contains 313 amino acid residues, including 28 cysteine residues in a pattern of three homologous repeats. The cysteine residue pattern suggests that uPAR belongs to a superfamily of proteins including CD59, murine Ly-6, and a variety of elapid snake venom toxins. A novel 1.7-kb uPAR cDNA was isolated that is missing exon 5 and that contains 380 bp not previously reported at the 5' end. This cDNA was used to probe a human genomic library from which three clones were isolated and analyzed. The uPAR gene consists of 7 exons spread over 23 kb of genomic DNA. Exons 2, 4, and 6 code for homologous domains within the mature protein, as do exons 3, 5, and 7; CD59-like homologous pairs are encoded by exons 2-3, 4-5, and 6-7, respectively. The structure of the gene for uPAR further confirms the relationship of this molecule to the superfamily containing CD59, Ly-6, and the elapid snake venom toxins.
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PMID:The structure of the urokinase-type plasminogen activator receptor gene. 804 31

CD59 (membrane inhibitor of reactive lysis, protectin) is a membrane protein whose functions include the inhibition of the insertion of the ninth component of complement into the target membrane. It belongs to a superfamily of proteins including Ly-6, elapid snake venom toxins, and urokinase receptor (UPAR); the members of the superfamily have a similar structure that includes four (in mammals five) disulfide bridges that maintain a three-dimensional conformation consisting of a central core, three finger-like "loops" extending from it and a small loop near the coboxyl end. We have used site directed mutagenesis to explore three aspects of the structure of CD59: 1) the role of the disulfide bridges in expression and function of the molecule; 2) the location of epitopes reacting with monoclonal antibodies to the molecule; and 3) the parts of the molecule that are critical to its function in inhibiting complement lysis. Mutant molecules in which the disulfides maintaining the finger-like loops (Cys3-Cys26, Cys19-Cys39, and Cys45-Cys63) were removed were not expressed on the cell surface. The mutation of the disulfide (Cys6-Cys13) resulted in no change in expression or function. The mutation of Cys64-Cys69 maintaining the small loop resulted in an expressed molecule with increased functional activity. The major epitope for 6 of 7 monoclonal antibodies was centered on Arg53 as the mutation 53Arg-->Ser resulted in a loss of interaction with these antibodies, as did the deletion of four nearby residues (Leu54-Asn57). The alteration 55Arg-->Ser resulted in loss of reactivity for some but not other antibodies. The reactivity with one monoclonal antibody, H19, was abrogated by the mutations 61Tyr-->Gly and 61Tyr-->Ala. Functional activity of the molecule was not adversely altered by mutations in the first and second loops; however, the 61Tyr-->Gly mutation was non-functional. The mutation of 61Tyr-->His diminished function but changes 61Tyr-->Ala and 61Tyr-->Phe had no effect on function. We conclude that the functional site of CD59 is located in this region of the molecule.
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PMID:Structure-function relationships of the complement regulatory protein, CD59. 907 80

The proteins that bind phospholipase A2 (PLA2) isozymes of Trimeresurus flavoviridis (habu snake, crotalinae) venom were fractionated from sera on four columns, each conjugated with one of four PLA2 isozymes. Five proteins, termed PLA2 inhibitors (PLI) I-V, were obtained as the binding components. The combinations of the binding components differed depending on the PLA2 isozymes. PLI-IV and PLI-V correspond to PLI-A and PLI-B, respectively, which were known to bind to a major [Asp49]PLA2, PLA2, and contained a segment similar to the carbohydrate-recognition domain of C-type lectins. PLI-I, which is a major component of inhibitory proteins against three basic PLA2 isozymes, PLA-B (a basic [Asp49]PLA2) and basic proteins I and II (both [Lys49]PLA2s), has been isolated, and its partial amino acid sequence has been determined. A cDNA encoding PLI-I was isolated from a T. flavoviridis liver cDNA library and sequenced. PLI-I cDNA encoded 200 amino acid residues, including a signal peptide of 19 amino acid residues. One sugar chain was predicted to occur at position 157. A gene coding for PLI-I was isolated. It is 9.6-kb long and consists of five exons and four introns. Comparison of the exon-intron structure of the PLI-I gene with those of genes encoding urokinase-type-plasminogen-activator receptor (uPAR), Ly-6, CD59 and neurotoxins showed that they have characteristic unit encoding approximately 90 amino acid residues, which is divided over two exons. This strongly suggests that the PLI-I gene belongs to the uPAR, Ly-6, CD59 and neurotoxin gene family. There are two types of structurally different inhibitors against PLA2 isozymes in T. flavoviridis serum with different evolutionary origins.
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PMID:Characterization and evolution of a gene encoding a Trimeresurus flavoviridis serum protein that inhibits basic phospholipase A2 isozymes in the snake's venom. 939 34

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem cell disorder characterized by the deficiency of all proteins anchored to the membrane by the glycosyl-phosphatidylinositol (GPI) anchor. The receptor for urokinase-type plasminogen activator (uPAR) also is attached to the cell membrane by a GPI anchor, and that soluble uPAR (suPAR) is present in plasma. In the present study, we measured uPAR, CD55, and CD59 on granulocytes by means of flow cytometry and suPAR in plasma by means of immunoradiometric assay. The subjects were 20 patients with PNH, 59 other patients with anemia, and 21 healthy individuals. In patients with PNH, both the mean fluorescence intensity and the positive percentage of fluorescence-activated granulocytes of uPAR, CD55, and CD59 were remarkably decreased, whereas in patients with other forms of anemia, except 2 patients with aplastic anemia, the results were not altered in comparison with those for the healthy individuals. The level of uPAR was reduced to the same extent as were those of CD55 and CD59 on the PNH-affected granulocytes. Some peak shape abnormalities (double peaks, peak tailing, or both) in the histogram of fluorescence intensity were also found in patients with PNH. The suPAR concentration of PNH plasma was 4.04+/-2.47 ng/mL, which was higher than that of the healthy individuals, 1.73+/-0.96 ng/mL (P < .01). The positive percentage of fluorescence-activated granulocytes was inversely associated with the plasma suPAR level in patients with PNH (r = -0.79, P < .01). Our data suggest that measurement of uPAR on granulocytes by means of flow cytometry and of suPAR in plasma by means of immunoradiometric assay are specific techniques for the diagnosis of PNH.
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PMID:Diagnostic significance of measurement of the receptor for urokinase-type plasminogen activator on granulocytes and in plasma from patients with paroxysmal nocturnal hemoglobinuria. 1204 78

The receptor for urokinase-type plasminogen activator (uPAR) plays important roles in a number of physiological and pathological processes by virtue of its interactions with urokinase-type plasminogen activator (uPA), vitronectin (Vn), and several other proteins. The uPA binding site spans all three domains (D1 to D3) of uPAR. However, the nature of the Vn binding site within uPAR is still not clear. In this study, we conducted homolog-scanning mutagenesis on uPAR by switching 14 individual segments of 4-8 residues to their counterpart sequences of a uPAR homolog CD59. All 14 mutants were well expressed, reacted with a panel of monoclonal antibodies, and exhibited correct molecular weights. Of these 14 mutants, six mutants were defective in both uPA and Vn binding. Most importantly, we found two unique mutants uPAR(Asn172-Lys175) and uPAR(Glu183-Asn186) within the D2 domain, which displayed differential ligand binding activity: both had high affinity uPA binding, but completely lost Vn binding, indicating that these two sequences constitute a novel Vn binding site. Indeed, two peptides, P1 (153CPGSNGFHNNDTFHFLKC) and P2 (171CNTTKCNEGPILELENLPQ), derived from the sequences of the identified uPA and Vn binding pockets within D2, respectively, behaved like bona fide ligand binding sites: peptide P1 bound uPA but not Vn, whereas peptide P2 bound Vn and inhibited uPAR-mediated cell adhesion, but did not interact with uPA. Altogether, our data demonstrated that uPAR D2 contains two distinct ligand binding sites for uPA and Vn. Such information will help us better understand the complex roles of uPAR in cell adhesion, migration, and tumor metastasis.
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PMID:Sequences within domain II of the urokinase receptor critical for differential ligand recognition. 1276 Dec 27

The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor as a convenient C-terminal fusion partner for various recombinant extracellular human proteins used in basic cancer research. The stability of this cystein-rich domain, which structure adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for expression of recombinant proteins. Up to 20mg of intact fusion protein were expressed by stably transfected Drosophila S2 cells per liter of culture using this strategy. Purification of these secreted fusion proteins from the conditioned serum free medium of S2 cells was accompanied by an efficient one-step immunoaffinity chromatography procedure using the immobilized anti-uPAR monoclonal antibody R2. An optional enterokinase cleavage site is included between the various recombinant proteins and the linker region of the tag, which enables generation of highly pure preparations of tag-free recombinant proteins. Using this system we successfully produced soluble and intact recombinant forms of extracellular proteins such as CD59, C4.4A and vitronectin, as well as a number of truncated domain constructs of these proteins. In conclusion, the present tagging system offers a convenient general method for the robust expression and efficient purification of a variety of recombinant proteins.
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PMID:A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor. 1721 41

Extracellular domains of some cellular receptors expressed in the organisms at different levels of development belong to three-fingered protein (TFP) fold. The Homo sapiens genome encodes at least 45 genes containing from one to three TFP domains (TFPDs), namely diverse paralogues of the Ly6 gene, CD59 and the receptors of activins, bone morphogenetic proteins, Mullerian inhibiting substance and transforming growth factor-beta. C4.4a and urokinase/plasminogen activatory receptor contain two and three TFPD repeats, respectively. These diverse proteins have a low overall sequence similarity with each other and their hydrophobicity levels vary to a considerable degree. It is suggested that sequence differentiation within the TFPD led to distinct groups of proteins whose attributes were optimized to fit both the physicochemical properties specific to their functional microenvironment and selective targeting of their highly diversified extracellular cofactors.
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PMID:The three-fingered protein domain of the human genome. 1882 Oct 57


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