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Schwartz, Bradford S. Schwartz, Bradford S. Morrissey, James H. Ordal, George W. Fratti, Rutilio A. Nieves, Evelyn 2013-02-03T19:46:20Z 2013-02-03T19:46:20Z 2015-02-03T11:00:37Z 2012-12 2013-02-03T19:46:20Z 2012-12 The regulation of protease activity is essential for physiological events, such as angiogenesis, inflammation, wound healing, and tumor invasion. Urokinase plasminogen activator receptor (u-PAR) has been widely studied in both systemic and cellular processes. Systemic roles for u-PAR include angiogenesis, inflammation and cancer while cellular roles include cell proliferation, survival, adhesion, migration, and localizing activation of plasminogen (Pg) (Blasi, Behrendt et al. 1990; Ellis, Behrendt et al. 1991; Nguyen, Hussaini et al. 1998; Chapman, Wei et al. 1999). u-PAR exerts its effects through both proteolytic and non-proteolytic mechanisms that are inter-related. Both functions are directly affected by the activation of u-PAR through the binding of its cognate ligand, urokinase plasminogen activator (u-PA). u-PAR is a glycosidylphosphatidylinsotol (GPI)-anchored receptor that has the ability to activate Pg through localization of u-PA to the cell surface. In malignant cells, the increased glycosylation of u-PAR confers resistance to cleavage by two-chain urokinase plasminogen activator (tcu-PA) (Montuori, Rossi et al. 1999). Interestingly, the presence of highly glycosylated receptor prevents the tcu-PA from cleaving domain 1 (D1) by decreasing u-PA’s affinity for u-PAR. The central hypothesis of the work described here involves the sensitive balance that is created by the binding of u-PA to u-PAR for both the activation and functional regulation of these proteins. u-PAR has the ability to localize u-PA to the cell surface to initiate Pg activation. tcu-PA also has the ability to cleave D1 of u-PAR from the rest of the protein, which prevents localization of u-PA on the cell-surface. To explore and characterize this relationship, we engineered a mutant u-PAR that prevents cleavage of D1, and studied the effects on the activation of Pg, u-PAR dependent cellular migration and proliferation. Mutation of residues Arg 83 and Arg 89 in u-PAR leads to conformational changes that resemble the active conformation of u-PAR previously observed in crystal structures (Llinas, Le Du et al. 2005; Barinka, Parry et al. 2006; Huai, Mazar et al. 2006). Although our studies indicate that the initiation of the Pg activation cascade of our u-PAR mutants is essentially indistinguishable from wild type wt u-PAR, we also show an acceleration of the rate at which u-PA-PAI-1 complexes are cleared by 2-macroglobulin receptor/ low-density lipoprotein receptor-related protein (LRP) through u-PAR binding. In addition, we observe an increase in cell proliferation, cell migration and changes in cell morphology that correlates with an increase in ERK signaling. The u-PAR:LRP interaction we identified was remarkably novel, although one publication related u-PAR to LRP via binding the u-PA-PAI-1 complex, with u-PAR with minimal interaction via domain 3 of u-PAR (Czekay, Kuemmel et al. 2001). The u-PAR:LRP interaction we observed was supported by an increase in u-PAR detection when the mutant receptor was co-immunoprecipitated with an antibody against LRP. A similar increase was not observed with wt u-PAR. We also observed a direct impact on cell migration, adhesion and proliferation that are known to be u-PA dependent. The ability of u-PAR to aid in cellular motility in response to local environment is intriguing, in light of the fact that there is a correlation between plasma levels and the amount of intact su-PAR found in cancer patients (Bifulco, Longanesi-Cattani et al. 2011). The ability of highly glycosylated u-PAR molecules to increase proliferative events is also of interest. Over-glycosylation of u-PAR leads to resistance to cleavage by its cognate ligand, tcu-PA (Sier, Nicoletti et al. 2004). In addition, this effect is pronounced in highly metastatic anaplastic thyroid carcinoma (Montuori, Rossi et al. 1999). Studying the mechanisms used by u-PAR in vitro and in vivo provides great insight into which u-PAR activities are important in cancer. The expression of u-PAR in both neoplastic cells and tumor-associated cells from ovary, colon, lung, breast, endometrium, macrophages, endothelial cells, and fibroblasts indicates that u-PAR may be a useful therapeutic target since researchers have been able to correlate prognosis and u-PAR expression levels (Mazar 2001; Wang, Mao et al. 2001; Ge and Elghetany 2003; Sidenius and Blasi 2003; Mazzieri and Blasi 2005). Furthermore, u-PAR may be useful as a prognostic marker for cancer and to detect metastasis at an early stage. Generation of a non-cleavable u-PAR provides information regarding what relative roles are played by the intact and cleaved forms of the receptor. Item withdrawn by Mark Zulauf (zulauf@illinois.edu) on 2012-10-08T14:27:29Z Item was in collections: University of Illinois Theses & Dissertations (ID: 1) No. of bitstreams: 2 1_Nieves_Evelyn disseratation.docx: 11499338 bytes, checksum: 87a0082de358dbff933e4f3c300b6f2b (MD5) Nieves_Evelyn.pdf: 5773718 bytes, checksum: 9090325192a1534c0a0e5bcf493ffa5c (MD5) Made available in DSpace on 2013-02-03T19:46:20Z (GMT). No. of bitstreams: 3 Evelyn_Nieves.pdf: 5598230 bytes, checksum: c8016837e2ac0536c5b319e7df1c46f8 (MD5) 1_Nieves_Evelyn disseratation.docx: 11499338 bytes, checksum: 87a0082de358dbff933e4f3c300b6f2b (MD5) license.txt: 4060 bytes, checksum: 6efae6558a336b22b59ba0f4701c3240 (MD5) Restriction data tranferred 2014-07-01T11:12:06-05:00 Original Data Group with Access Administrator Release Date: 2015-02-03 13:47:48 UTC Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system Item marked as restricted to the 'Administrator' Group (id=1) by Seth Robbins (srobbins@illinois.edu) on 2013-02-03T19:47:56Z Item is restricted until 2015-02-03T19:47:48Z Limited Restriction Lifted for Item 42407 on 2015-02-03T11:00:37Z. http://hdl.handle.net/2142/42459 en This research was originally published in Journal Biological Chemistry. Evelyn C. Nieves and Naveen Manchanda. A cleavage-resistant urokinase plasminogen activator receptor exhibits dysregulated cell-surface clearance. J Biol Chem. 2010 April 23; 285(17): 12595–12603. © the American Society for Biochemistry and Molecular Biology. receptor internalization fibrinolysis plasminogen activation urokinase receptor Dissection of receptor functions through the generation of a tcu-PA cleavage resistant u-PAR: a u-PA independent active u-PAR text Biochemistry 1438 Biochemistry 0318 University of Illinois at Urbana-Champaign Dissertation Ph.D. PHD:Biochemistry -UIUC 10KS0318PHD