Regulation of Urokinase Receptor Proteolytic Function by the Tetraspanin CD82

School of Biological Science, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2005; 280(15):14811-8. DOI: 10.1074/jbc.M414189200
Source: PubMed


The high affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored cellular receptor (uPAR) promotes plasminogen activation and the efficient generation of pericellular proteolytic activity. We demonstrate here that expression of the tetraspanin CD82/KAI1 (a tumor metastasis suppressor) leads to a profound effect on uPAR function. Pericellular plasminogen activation was reduced by approximately 50-fold in the presence of CD82, although levels of components of the plasminogen activation system were unchanged. uPAR was present on the cell surface and molecularly intact, but radioligand binding analysis with uPA and anti-uPAR antibodies revealed that it was in a previously undetected cryptic form unable to bind uPA. This was not due to direct interactions between uPAR and CD82, as they neither co-localized on the cell surface nor could be co-immunoprecipitated. However, expression of CD82 led to a redistribution of uPAR to focal adhesions, where it was shown by double immunofluorescence labeling to co-localize with the integrin alpha(5)beta(1), which was also redistributed in the presence of CD82. Co-immunoprecipitation experiments showed that, in the presence of CD82, uPAR preferentially formed stable associations with alpha(5)beta(1), but not with a variety of other integrins, including alpha(3)beta(1). These data suggest that CD82 inhibits the proteolytic function of uPAR indirectly, directing uPAR and alpha(5)beta(1) to focal adhesions and promoting their association with a resultant loss of uPA binding. This represents a novel mechanism whereby tetraspanins, integrins, and uPAR, systems involved in cell adhesion and migration, cooperate to regulate pericellular proteolytic activity and may suggest a mechanism for the tumor-suppressive effects of CD82/KAI1.

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    • "Integrins, GPCRs and growth factor receptors are found to physically interact with uPAR and are assumed to serve as the co-receptors of uPAR. Among them, integrins are the most studied and are considered as the most significant co-receptors associated with uPAR signaling 85, 86. The interaction of uPAR with integrins was originally found in uPAR immunoprecipitates of human monocytes 87, and αMβ2 (MAC1) being the first reported uPAR-interacting integrin 88. uPAR-αMβ2 integrin interaction is capable of simultaneously increasing the binding of αMβ2 integrin to its ligand fibrinogen and to promote adhesion to vitronectin in an uPA-independent mechanism 89-91. "
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    ABSTRACT: Elevated level of urokinase receptor (uPAR) is detected in various aggressive cancer types and is closely associated with poor prognosis of cancers. Binding of uPA to uPAR triggers the conversion of plasminogen to plasmin and the subsequent activation of metalloproteinases. These events confer tumor cells with the capability to degrade the components of the surrounding extracellular matrix, thus contributing to tumor cell invasion and metastasis. uPA-uPAR interaction also elicits signals that stimulate cell proliferation/survival and the expression of tumor-promoting genes, thus assisting tumor development. In addition to its interaction with uPA, uPAR also interacts with vitronectin and this interaction promotes cancer metastasis by activating Rac and stimulating cell migration. Although underlying mechanisms are yet to be fully elucidated, uPAR has been shown to facilitate epithelial-mesenchymal transition (EMT) and induce cancer stem cell-like properties in breast cancer cells. The fact that uPAR lacks intracellular domain suggests that its signaling must be mediated through its co-receptors. Indeed, uPAR interacts with diverse transmembrane proteins including integrins, ENDO180, G protein-coupled receptors and growth factor receptors in cancer cells and these interactions are proven to be critical for the role of uPAR in tumorigenesis. Inhibitory peptide that prevents uPA-uPAR interaction has shown the promise to prolong patients' survival in the early stage of clinical trial. The importance of uPAR's co-receptor in uPAR's tumor-promoting effects implicate that anti-cancer therapeutic agents may also be developed by disrupting the interactions between uPAR and its functional partners.
    Theranostics 06/2013; 3(7):487-495. DOI:10.7150/thno.4218 · 8.02 Impact Factor
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    • "Reports suggest that uPAR interacts with integrins to confer specificity to the activated signaling pathway [28], [29]. In addition, uPAR forms a complex with ligands such as uPA and vitronectin that enhances the binding and assembly of various other ligands to integrins and subsequently activates downstream signaling [30]–[32]. However, considering the frequency of recurrence in patients who receive radiation therapy, we were primarily interested in determining the mechanism of radiation-induced cell adhesion and invasion in medulloblastoma. "
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    ABSTRACT: Despite effective radiotherapy for the initial stages of cancer, several studies have reported the recurrence of various cancers, including medulloblastoma. Here, we attempt to capitalize on the radiation-induced aggressive behavior of medulloblastoma cells by comparing the extracellular protease activity and the expression pattern of molecules, known to be involved in cell adhesion, migration and invasion, between non-irradiated and irradiated cells. We identified an increase in invasion and migration of irradiated compared to non-irradiated medulloblastoma cells. RT-PCR analysis confirmed increased expression of uPA, uPAR, focal adhesion kinase (FAK), N-Cadherin and integrin subunits (e.g., α3, α5 and β1) in irradiated cells. Furthermore, we noticed a ∼2-fold increase in tyrosine phosphorylation of FAK in irradiated cells. Immunoprecipitation studies confirmed increased interaction of integrin β1 and FAK in irradiated cells. In addition, our results show that overexpression of uPAR in cancer cells can mimic radiation-induced activation of FAK signaling. Moreover, by inhibiting FAK phosphorylation, we were able to reduce the radiation-induced invasiveness of the cancer cells. In this vein, we studied the effect of siRNA-mediated knockdown of uPAR on cell migration and adhesion in irradiated and non-irradiated medulloblastoma cells. Downregulation of uPAR reduced the radiation-induced adhesion, migration and invasion of the irradiated cells, primarily by inhibiting phosphorylation of FAK, Paxillin and Rac-1/Cdc42. As observed from the immunoprecipitation studies, uPAR knockdown reduced interaction among the focal adhesion molecules, such as FAK, Paxillin and p130Cas, which are known to play key roles in cancer metastasis. Pretreatment with uPAR shRNA expressing construct reduced uPAR and phospho FAK expression levels in pre-established medulloblastoma in nude mice. Taken together, our results show that radiation enhances uPAR-mediated FAK signaling and by targeting uPAR we can inhibit radiation-activated cell adhesion and migration both in vitro and in vivo.
    PLoS ONE 09/2010; 5(9):e13006. DOI:10.1371/journal.pone.0013006 · 3.23 Impact Factor
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    • "Despite the controversy surrounding whether uPAR and integrins interact directly, many studies show that uPAR signaling requires integrin co-receptors. Furthermore, some non-integrin co-receptors of uPAR cooperate with integrins in signaling or influence uPAR-integrin interactions [19], [20]. Additionally, uPA-uPAR binding results in the expression of cathepsin B [21], another important protease involved in ECM degradation, and significantly higher levels of cathepsin B has been found in high-grade glioblastomas [22], [23]. "
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    ABSTRACT: Involvement of MMP-9, uPAR and cathepsin B in adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well established. In the present study, MMP-9, uPAR and cathepsin B genes were downregulated in glioma xenograft cells using shRNA plasmid constructs and we evaluated the involvement of integrins and changes in their adhesion, migration and invasive potential. MMP-9, uPAR and cathepsin B single shRNA plasmid constructs were used to downregulate these molecules in xenograft cells. We also used MMP-9/uPAR and MMP-9/cathepsin B bicistronic constructs to evaluate the cumulative effects. MMP-9, uPAR and cathepsin B downregulation significantly inhibits xenograft cell adhesion to several extracellular matrix proteins. Treatment with MMP-9, uPAR and cathepsin B shRNA of xenografts led to the downregulation of several alpha and beta integrins. In all the assays, we noticed more prominent effects with the bicistronic plasmid constructs when compared to the single plasmid shRNA constructs. FACS analysis demonstrated the expression of alphaVbeta3, alpha6beta1 and alpha9beta1 integrins in xenograft cells. Treatment with bicistronic constructs reduced alphaVbeta3, alpha6beta1 and alpha9beta1 integrin expressions in xenograft injected nude mice. Migration and invasion were also inhibited by MMP-9, uPAR and cathepsin B shRNA treatments as assessed by spheroid migration, wound healing, and Matrigel invasion assays. As expected, bicistronic constructs further inhibited the adhesion, migration and invasive potential of the xenograft cells as compared to individual treatments. Downregulation of MMP-9, uPAR and cathespin B alone and in combination inhibits adhesion, migration and invasive potential of glioma xenografts by downregulating integrins and associated signaling molecules. Considering the existence of integrin inhibitor-resistant cancer cells, our study provides a novel and effective approach to inhibiting integrins by downregulating MMP-9, uPAR and cathepsin B in the treatment of glioma.
    PLoS ONE 07/2010; 5(7):e11583. DOI:10.1371/journal.pone.0011583 · 3.23 Impact Factor
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