An Uncleavable uPAR Mutant Allows Dissection of Signaling Pathways in uPA-dependent Cell Migration

Abstract

Urokinase-type plasminogen activator (uPA) binding to uPAR induces migration, adhesion, and proliferation through multiple interactions with G proteins-coupled receptor FPRL1, integrins, or the epidermal growth factor (EGF) receptor (EGFR). At least two forms of uPAR are present on the cell surface: full-length and cleaved uPAR, each specifically interacting with one or more transmembrane proteins. The connection between these interactions and the effects on the signaling pathways activation is not clear. We have exploited an uPAR mutant (hcr, human cleavage resistant) to dissect the pathways involved in uPA-induced cell migration. This mutant is not cleaved by proteases, is glycosylphosphatidylinositol anchored, and binds uPA with a normal K d . Both wild-type (wt) and hcr-uPAR are able to mediate uPA-induced migration, are constitutively associated with the EGFR, and associate with ␣3␤1 integrin upon uPA binding. However, they engage different pathways in response to uPA. wt-uPAR requires both integrins and FPRL1 to mediate uPA-induced migration, and association of wt-uPAR to ␣3␤1 results in uPAR cleavage and extracellular signal-regulated kinase (ERK) activation. On the contrary, hcr-uPAR does not activate ERK and does not engage FPRL1 or any other G protein-coupled receptor, but it activates an alternative pathway initiated by the formation of a triple complex (uPAR-␣3␤1-EGFR) and resulting in the autotyrosine phosphorylation of EGFR. INTRODUCTIONThe serine protease urokinase-type plasminogen activator (uPA) and its high-affinity cell surface receptor (uPAR) play an important role in a number of physiological as well as pathological extracellular degradation processes, where cell migration is required, such as inflammatory responses and tumor invasion (Blasi and Carmeliet, 2002). uPAR was first identified as a major player in the regulation of pericellular proteolysis by modulating and concentrating the uPA activity at the required sites of the cell surface . However, new evidence revealed that uPA binding to uPAR also induces proteolysis-dependent and -independent intracellular signaling affecting cell adhesion, migration, and proliferation in a variety of cells (Chapman, 1997;Ossowski and Aguirre-Ghiso, 2000;Preissner et al., 2000;Blasi and Carmeliet, 2002;Kjøller, 2002).uPAR is a heavily glycosylated glycosylphosphatidylinositol (GPI)-anchored protein formed by three cysteine-rich LY6-like extracellular domains (LU domains D1, D2, and D3) connected by short linker regions (Ploug and Ellis, 1994). The three consecutive three-finger domains of uPAR are organized in an almost circular manner and generate a deep internal cavity for the interaction with uPA. The receptor-binding domain of uPA is engaged in this central cavity, leaving the whole external surface available for other interactions (Llinas et al., 2005). Identified interactors include signaling molecules such as various integrins, the G protein-coupled receptor FPRL1, the epidermal growth factor (EGF) receptor (EGFR), the mannose-6-phosphate recep...

Mannose 6-Phosphate/Insulin-like Growth Factor 2 Receptor Limits Cell Invasion by Controlling αVβ3 Integrin Expression and Proteolytic Processing of Urokinase-type Plasminogen Activator Receptor

“…Resistance to uPAR cleavage in M6P/IGF2R knockdown cells might also have important implications for the nature of uPA signaling in these cells. Using a cleavageresistant mutant of uPAR Mazzieri et al (2006) showed that reduced cleavage of uPAR favored epidermal growth factor Figure 8. uPAR cleavage but not uPAR internalization and turnover is dependent on M6P/IGF2R expression.…”

“…The cleavage was almost completely blocked by the uPA inhibitor amiloride, a mixture of the serine protease inhibitors aprotinin and PMSF, and partially blocked by addition of PAI-1 (Supplemental Figure S4), indicating that the cleavage is mediated or initiated by active uPA. uPAR seems to be more cleaved when dimerized within lipid rafts (Cunningham et al, 2003) and complexed with integrins (Mazzieri et al, 2006). To what extend these observations explain the role of M6P/IGF2R in uPAR cleavage needs future investigations.…”

A direct link between expression of urokinase plasminogen activator receptor, growth rate and oncogenic transformation in mouse embryonic fibroblasts

“…Available data in cultured cancer cells, however, indicate that uPAR overexpression activates cell proliferation by interacting with integrins and with growth factor receptor tyrosine kinases like the EGF and the PDGF receptors (Liu et al, 2002;Kiyan et al, 2005). However, peptide a325 that dissociates uPAR-a3b1 and -a5b1 complexes (Wei et al, 2001;Mazzieri et al, 2006) had no effect on wt MEFs growth rate or ERK activation (data not shown). It is known that overexpression of uPAR in Hep3 cells, for example, modifies the response of the EGF-R to EGF, so that EGF-R was activated in the absence of EGF (Liu et al, 2002).…”

“…On the other hand, direct experimental demonstration of uPAR/EGF-R functional interaction in vivo is still outstanding. In cell culture, on the other hand, different uPAR conformations are able to differentiate between different interacting proteins, that is, EGF-R or integrins (Mazzieri et al, 2006). A detailed mutation study of uPAR will uPAR regulates cell growth in primary embryo fibroblasts R Mazzieri et al probably allow to describe accurately the molecular mechanism (and interactors) involved in its role in growth control.…”

Soluble Urokinase Receptor Is Released Selectively by Glioblastoma Cells That Express Epidermal Growth Factor Receptor Variant III and Promotes Tumor Cell Migration and Invasion*

“…A number of studies have demonstrated that suPAR binds directly to membrane proteins, such as FPRL1, and triggers cell signaling (6,(27)(28)(29). The nature of the signaling response may be determined by whether suPAR is cleaved between domain-1 and the domain-2-3 region, revealing the epitope, SRSRY, involved in FPRL1 binding (6,27,30). suPAR has been implicated in hematopoietic stem cell mobilization (31) and in the development of focal segmental glomerulosclerosis (32).…”