An alternatively spliced variant of mRNA for the human receptor for urokinase plasminogen activator (original) (raw)
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European Journal of Biochemistry, 1996
The amino-terminal fragment of human uPA (ATF; amino acids I-135), which contains the binding site for the uPA receptor (uPAR, CD87) was expressed in the yeast Saccharomyces cerevisiae. Recombinant yeast ATF, modified and extended by an amino-terminal in-frame insertion of a His, tract, was purified from total protein extracts by nickel chelate affinity chromatography and shown to be functionally active since it efficiently competes with uPA for binding to cell-surface-associated uPAR. The ATF expression plasmid served as a template for the construction of a series of site-directed mutants in order to define those amino acids that are important for binding to uPAR. All mutant ATF proteins but one (deletion of Ser26) were expressed in a stable form (about 20-30 ng/mg total protein) and the binding capacity of each mutant was tested by a uPA-ligand binding assay employing recombinant uPAR immobilized to a microtiter plate. Each of the 11 amino acids of loop B of the binding region of uPA (amino acids 20-30) were individually substituted with alanine. Lys23, Tyr24, Phe25, Ile28, and Trp30 were important determinants for uPAR binding. A systematic alanine scan was also performed with chemically synthesized linear peptides spanning amino acids 14-32 of ATE Comparable results to those with the yeast ATF mutants were obtained. In a different set of experiments, those amino acids of the uPARbinding region of uPA that are only conserved between man and baboon but not in other species were altered: whereas substitution of Thrl8 by alanine or Asn32 by serine had hardly any effect, replacement of Am22 by tyrosine and Trp30 by arginine (both positions are strictly conserved in other mammals) led to ATF variants incapable of interacting with human uPAR. Deletion of either Va120, Ser21, Lys23, His29 or Val20 plus Ser21, respectively, also generated non-reactive ATF mutants. Finally, Lys23 in ATF was substituted with certain amino acids: whereas the replacement of Lys23 by alanine, histidine or glutamine generated ATF variants with moderate uPAR-binding activity, the introduction of a negatively charged amino acid (exchange of Lys23 by glutamic acid) completely abolished uPAR-binding activity. The results presented for the ATF mutants and uPA-derived peptides may provide clues necessary to establish the nature of the physical interaction of uPA with its receptor and may help to develop uPA-derived peptide analogues as potential therapeutic agents to block tumor cell-associated UPNUPAR interaction.
European journal of biochemistry / FEBS, 1999
An 8.5-kb 5'-flanking region of the human urokinase-type plasminogen activator receptor (uPAR) gene was cloned and the detailed uPAR promoter region defined in an 188-bp fragment between bases -141 and +47 relative to the transcription-start site. 5'-Deletion to -100 and -60 in the region abolished its promoter activity, indicating that an 81-bp segment between -141 and -61, which contains a proximal AP-1 site at position -70, is required for uPAR promoter activity. Nuclear extracts from HCT116 cells contain proteins that specifically bind to the AP-1 site. Mutation of the AP-1 motif reduced uPAR promoter activity in comparison with the wild-types. Induction of uPAR expression by phorbol ester requires this AP-1 motif in colon cancer cells. Cotransfection with the c-jun and c-fos expression vectors stimulated the uPAR promoter activity four- to fivefold. These results demonstrate that the proximal AP-1 motif is responsible for approximately 50% of the basal expression of the...
The Journal of biological chemistry, 1990
The receptor for human urokinase-type plasminogen activator (u-PA) was purified from phorbol 12-myristate 13-acetate-stimulated U937 cells by temperature-induced phase separation of detergent extracts, followed by affinity chromatography with immobilized diisopropyl fluorophosphate-treated u-PA. The purified protein shows a single 55-60 kDa band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It is a heavily glycosylated protein, the deglycosylated polypeptide chain comprising only 35 kDa. The glycosylated protein contains N-acetyl-D-glucosamine and sialic acid, but no N-acetyl-D-galactosamine. Glycosylation is responsible for substantial heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines. The amino acid composition and the NH2-terminal amino acid sequence are reported. The protein has a high content of cysteine residues. The NH2-terminal sequence is not closely ...
Urokinase-type plasminogen activator up-regulates the expression of its cellular receptor
Febs Letters, 2000
The expression of the receptor for the urokinase-type plasminogen activator (uPAR) can be regulated by several hormones, cytokines, tumor promoters, etc. Recently, it has been reported that uPAR is capable of transducing signals, even though it is lacking a transmembrane domain and a cytoplasmatic tail. We now report that uPAR cell surface expression can be positively regulated by its ligand, uPA, in thyroid cells. The effect of uPA is independent of its proteolytic activity, since inactivated uPA or its aminoterminal fragment have the same effects of the active enzyme. The increase of uPAR on the cell surface correlates with an increase of specific uPAR mRNA. Finally, uPA up-regulates uPAR expression also in other cell lines of different type and origin, thus suggesting that the regulatory role of uPA on uPAR expression is not restricted to thyroid cells, but it occurs in different tissues, both normal and tumoral. ß
BMC Molecular Biology, 2009
Background Expression of the urokinase plasminogen activator receptor (UPAR) has been shown to have clinical relevance in various cancers. We have recently identified UPAR as an asthma susceptibility gene and there is evidence to suggest that uPAR may be upregulated in lung diseases such as COPD and asthma. uPAR is a key receptor involved in the formation of the serine protease plasmin by interacting with uPA and has been implicated in many physiological processes including proliferation and migration. The current aim was to determine key regulatory regions and splice variants of UPAR and quantify its expression in primary human tissues and cells (including lung, bronchial epithelium (HBEC), airway smooth muscle (HASM) and peripheral cells). Results Using Rapid Amplification of cDNA Ends (RACE) a conserved transcription start site (-42 to -77 relative to ATG) was identified and multiple transcription factor binding sites predicted. Seven major splice variants were identified (>5% total expression), including multiple exon deletions and an alternative exon 7b (encoding a truncated, soluble, 229aa protein). Variants were differentially expressed, with a high proportion of E7b usage in lung tissue and structural cells (55–87% of transcripts), whereas classical exon 7 (encoding the GPI-linked protein) was preferentially expressed in peripheral cells (~80% of transcripts), often with exon 6 or 5+6 deletions. Real-time PCR confirmed expression of uPAR mRNA in lung, as well as airway and peripheral cell types with ~50–100 fold greater expression in peripheral cells versus airway cells and confirmed RACE data. Protein analysis confirmed expression of multiple different forms of uPAR in the same cells as well as expression of soluble uPAR in cell supernatants. The pattern of expression did not directly reflect that seen at the mRNA level, indicating that post-translational mechanisms of regulation may also play an important role. Conclusion We have identified multiple uPAR isoforms in the lung and immune cells and shown that expression is cell specific. These data provide a novel mechanism for uPAR regulation, as different exon splicing may determine uPAR function e.g. alternative E7b results in a soluble isoform due to the loss of the GPI anchor and exon deletions may affect uPA (ligand) and/or integrin binding and therefore influence downstream pathways. Expression of different isoforms within the lung should be taken into consideration in studies of uPAR in respiratory disease.
2012
The urokinase receptor (uPAR) is a modular receptor containing three LU domains. Results: Ligand-free uPAR is inherently flexible with a detached N-terminal domain (DI). Conclusion: Allosteric regulation of uPAR is driven by uPA-induced compaction of the intact receptor and a concomitant stabilization of DI. Significance: This flexibility and ligand-induced allostery are expected to impact future studies on uPAR function and targeted intervention.
Functional Analysis of the Cellular Receptor for Urokinase in Plasminogen Activation
Journal of Biological Chemistry
1 The abbreviations used are: DFP, diisopropyl fluorophosphate; uPA, urokinase-type plasminogen activator, both the activated two-chain protease and as a generic term; pro-uPA, single-chain form of uPA; uPAR, uPA receptor; s-uPAR, soluble recombinant uPAR residues 1-277; PAI-1, plasminogen activator inhibitor type-1; 6-AHA, 6-aminohexanoic acid; pNA, para-nitroaniline; AMC, 7-amido-4-methylcoumarin.
2000
The receptor for the serine protease urokinase-type plasminogen activator, uPAR (CD 87), plays an important role in tumor cell invasion and metastasis of solid malignant tumors. uPAR is a highly glycosylated, glycan lipid-anchored membrane protein, consisting of three homologous domains. Each individual domain is encoded by two exons: DI by exons 2+3, DII by exons 4+5, and DIII by exons 6+7. Beside the wild-type (wt) uPAR mRNA, two splice variants either lacking exon 5 (uPAR-del5) or both exons 4 and 5 (uPAR-del4/5) have been described. Previously, we studied expression of the mRNA variant uPAR-del4/5 and uPAR mRNA encompassing exons 2, 3, and 4 (i.e. uPAR-wt plus uPAR-del5) applying real-time RT-PCR assays for quantification of the mRNA concentration. In the present paper, we established two additional specific, robust and highly sensitive RT-PCR assays, based on the LightCycler technology, to specifically quantify either uPAR-wt or its splice variant, uPAR-del5. Expression of uPAR-wt and uPAR-del5 was analyzed in different human malignant cell lines (ovarian cancer cell lines OVMZ-6 and OVMZ-10; breast cancer cell lines MDA-MB 231, MDA-MB 231 BAG, MDA-MB 435, and aMCF-7; brain tumor cell line LN 18) as well as in a set of 174 breast cancer tissue samples. uPAR-del5 mRNA was found to be expressed very frequently at a rather low level (typically less than 1% of uPAR-wt mRNA). In tumor tissue from breast cancer patients, a statistically significant correlation between uPAR-del5 and uPAR-wt mRNA (r = 0.779; P < 0.001) was observed. There was no association between the expression level of either mRNA and clinical parameters such as nodal status, tumor size and grade. In estrogen receptor negative tumors, a significantly higher uPAR-del5 expression was found (P = 0.023). The two developed quantitative RT-PCR assays described here may aid further analysis of the function and clinical relevance of uPAR-wt and one of its splice variants, uPAR-del5, in malignant tumors. Key words: neoplasms; urinary plasminogen activator; RNA, messenger; reverse transcriptase polymerase chain reaction; RNA splicing Farthmann J et al. / Quantitative RT-PCR assays for uPAR-wt
Stem Cells, 1997
Several important functions have been assigned to the receptor for urokinase-type plasminogen activator, uPAR. As implied by the name, uPAR was first identified as a high affinity cellular receptor for urokinase plasminogen activator (uPA). It mediates the binding of the zymogen, pro-uPA, to the plasma membrane where trace amounts of plasmin will initiate a series of events referred to as "reciprocal zymogen activation" where plasmin converts pro-uPA to the active enzyme, uPA, which in turn converts plasma membrane-associated plasminogen to plasmin. This is an efficient machinery to generate broad-spectrum proteolytic activity which is spatially restricted to the plasma membrane, since plasmin that diffuses away from the plasma membrane is rapidly inactivated by circulating inhibitors (i.e., α 2 -antiplasmin). The system is controlled by a series of plasminogen activator inhibitors (PAIs), most importantly PAI-1 and PAI-2, providing means of temporally restricting the process of plasminogen activation.