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GPI-anchored uPAR requires Endo180 for rapid directional sensing during chemotaxis.

Sturge J, Wienke D, East L, Jones GE, Isacke CM - J. Cell Biol. (2003)

Bottom Line: Endo180 expression was demonstrated to enhance uPA-mediated filopodia production and promote rapid activation of Cdc42 and Rac.Expression of a noninternalizing Endo180 mutant revealed that promotion of random cell migration requires receptor endocytosis, whereas the chemotactic response to uPA does not.From these studies, we conclude that Endo180 is a crucial link between uPA-uPAR and setting of the internal cellular compass.

View Article: PubMed Central - PubMed

Affiliation: The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London SW3 6JB, UK.

ABSTRACT
Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in cell guidance and chemotaxis during normal and pathological events. uPAR is GPI-anchored and the mechanism by which it transmits intracellular polarity cues across the plasma membrane during directional sensing has not been elucidated. The constitutively recycling endocytic receptor Endo180 forms a trimolecular complex with uPAR in the presence of uPA, hence its alternate name uPAR-associated protein. Here, we demonstrate that Endo180 is a general promoter of random cell migration and has a more specific function in cell chemotaxis up a uPA gradient. Endo180 expression was demonstrated to enhance uPA-mediated filopodia production and promote rapid activation of Cdc42 and Rac. Expression of a noninternalizing Endo180 mutant revealed that promotion of random cell migration requires receptor endocytosis, whereas the chemotactic response to uPA does not. From these studies, we conclude that Endo180 is a crucial link between uPA-uPAR and setting of the internal cellular compass.

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Endo189 expression does not alter ERK1/2 and Akt phosphorylation or cell surface LRP. (A) ERK1/2 and Akt phosphorylation in MCF-7 cells transfected with vector alone, Endo180 or Endo180(Ala1468/Ala1469) and stimulated with uPA. Blots show phosphorylated ERK1/2 and Akt together with total ERK1/2 and Akt as loading controls. (B) FACS® analysis of LRP cell surface expression levels in MCF-7 cells transfected with vector alone (green), wild-type Endo180 (red) or Endo180(Ala1468/Ala1469) (blue). Profiles in black represent vector transfected cells incubated with secondary antibody alone.
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fig5: Endo189 expression does not alter ERK1/2 and Akt phosphorylation or cell surface LRP. (A) ERK1/2 and Akt phosphorylation in MCF-7 cells transfected with vector alone, Endo180 or Endo180(Ala1468/Ala1469) and stimulated with uPA. Blots show phosphorylated ERK1/2 and Akt together with total ERK1/2 and Akt as loading controls. (B) FACS® analysis of LRP cell surface expression levels in MCF-7 cells transfected with vector alone (green), wild-type Endo180 (red) or Endo180(Ala1468/Ala1469) (blue). Profiles in black represent vector transfected cells incubated with secondary antibody alone.

Mentions: The intracellular mechanisms for generation of polarity and migration in eukaryotic cells are beginning to be elucidated. Phospholipid and small Rho GTPase signaling pathways converge to regulate recruitment of Arp2/3 complex by WASP/WAVE family scaffolding proteins, which rapidly rearrange the actin cytoskeleton to produce structures that change the polarity and orientation of a cell (Franz et al., 2002; Weiner, 2002). Much less is understood about how extracellular signals are linked to this intracellular machinery. To further elucidate the mechanism of Endo180-dependent chemotaxis, the effect of altered Endo180 expression on the temporal activation of the Rho-family GTPases Cdc42, Rac and RhoA was examined. These molecular switches have fundamental roles in cell migration and chemotaxis (Nobes and Hall, 1995; Allen et al., 1998; Jones, 2000) and their deregulation is strongly implicated in metastasis (Jaffe and Hall, 2002; Sahai and Marshall, 2002). More importantly, Rac and Cdc42 can regulate cell motility downstream of uPAR (Kjoller and Hall, 2001; Sturge et al., 2002). In Endo180, but not vector-alone, expressing MCF-7 cells uPA stimulation increased filopodia production (Fig. 4 A) and caused rapid and sustained activation of Cdc42 (Fig. 4 B). Consistent with these findings, uPA-stimulated activation of Cdc42 in MDA-MB-231 cells was blocked by Endo180 siRNA, but not control siRNA, treatment (Fig. 4 C), whereas EGF-stimulated Cdc42 activation was unaffected by Endo180 siRNA treatment (Fig. 4 C). Despite an impaired activation of Cdc42, vector alone expressing cells show a substantial activation of Rac in response to uPA, which together may account for their increased migration without directional sensing in a uPA gradient. Although these data also suggest that Rac activation is not dependent on Endo180, the rapidity of uPA-dependent Rac activation in Endo180 transfected cells points to a regulatory role for this receptor in stimulating Rac activity. Endo180(Ala1468/Ala1469) expression resulted in delayed Cdc42 activation, but the level of activation stimulated by uPA was similar to that seen in Endo180 expressing cells. In contrast, Rac activation was impaired in Endo180(Ala1468/Ala1469) expressing cells providing a molecular explanation for the ability of these cells to sense a uPA gradient without increasing their migratory speed. Based on these results, we propose that Endo180 plays an important regulatory role in coordinating the activation of the Cdc42 and Rac GTPases during uPA-induced chemotaxis. In agreement with a previous report (Jo et al., 2002), RhoA was not significantly activated by uPA in either the MCF-7 cell lines or MDA-MB-231 cells (unpublished data). uPA–uPAR signaling has been shown to activate the MAPK pathway (Nguyen et al., 1999) and Akt (protein kinase B; Sturge et al., 2002; Chandrasekar et al., 2003). However, wild-type Endo180 or Endo180(Ala1468/Ala1469) expression did not alter the time course of uPA-mediated extracellular signal–regulated kinase 1/2 (ERK1/2) phosphorylation in MCF-7 cells or enhance Akt phosphorylation (Fig. 5 A).


GPI-anchored uPAR requires Endo180 for rapid directional sensing during chemotaxis.

Sturge J, Wienke D, East L, Jones GE, Isacke CM - J. Cell Biol. (2003)

Endo189 expression does not alter ERK1/2 and Akt phosphorylation or cell surface LRP. (A) ERK1/2 and Akt phosphorylation in MCF-7 cells transfected with vector alone, Endo180 or Endo180(Ala1468/Ala1469) and stimulated with uPA. Blots show phosphorylated ERK1/2 and Akt together with total ERK1/2 and Akt as loading controls. (B) FACS® analysis of LRP cell surface expression levels in MCF-7 cells transfected with vector alone (green), wild-type Endo180 (red) or Endo180(Ala1468/Ala1469) (blue). Profiles in black represent vector transfected cells incubated with secondary antibody alone.
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Related In: Results  -  Collection

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fig5: Endo189 expression does not alter ERK1/2 and Akt phosphorylation or cell surface LRP. (A) ERK1/2 and Akt phosphorylation in MCF-7 cells transfected with vector alone, Endo180 or Endo180(Ala1468/Ala1469) and stimulated with uPA. Blots show phosphorylated ERK1/2 and Akt together with total ERK1/2 and Akt as loading controls. (B) FACS® analysis of LRP cell surface expression levels in MCF-7 cells transfected with vector alone (green), wild-type Endo180 (red) or Endo180(Ala1468/Ala1469) (blue). Profiles in black represent vector transfected cells incubated with secondary antibody alone.
Mentions: The intracellular mechanisms for generation of polarity and migration in eukaryotic cells are beginning to be elucidated. Phospholipid and small Rho GTPase signaling pathways converge to regulate recruitment of Arp2/3 complex by WASP/WAVE family scaffolding proteins, which rapidly rearrange the actin cytoskeleton to produce structures that change the polarity and orientation of a cell (Franz et al., 2002; Weiner, 2002). Much less is understood about how extracellular signals are linked to this intracellular machinery. To further elucidate the mechanism of Endo180-dependent chemotaxis, the effect of altered Endo180 expression on the temporal activation of the Rho-family GTPases Cdc42, Rac and RhoA was examined. These molecular switches have fundamental roles in cell migration and chemotaxis (Nobes and Hall, 1995; Allen et al., 1998; Jones, 2000) and their deregulation is strongly implicated in metastasis (Jaffe and Hall, 2002; Sahai and Marshall, 2002). More importantly, Rac and Cdc42 can regulate cell motility downstream of uPAR (Kjoller and Hall, 2001; Sturge et al., 2002). In Endo180, but not vector-alone, expressing MCF-7 cells uPA stimulation increased filopodia production (Fig. 4 A) and caused rapid and sustained activation of Cdc42 (Fig. 4 B). Consistent with these findings, uPA-stimulated activation of Cdc42 in MDA-MB-231 cells was blocked by Endo180 siRNA, but not control siRNA, treatment (Fig. 4 C), whereas EGF-stimulated Cdc42 activation was unaffected by Endo180 siRNA treatment (Fig. 4 C). Despite an impaired activation of Cdc42, vector alone expressing cells show a substantial activation of Rac in response to uPA, which together may account for their increased migration without directional sensing in a uPA gradient. Although these data also suggest that Rac activation is not dependent on Endo180, the rapidity of uPA-dependent Rac activation in Endo180 transfected cells points to a regulatory role for this receptor in stimulating Rac activity. Endo180(Ala1468/Ala1469) expression resulted in delayed Cdc42 activation, but the level of activation stimulated by uPA was similar to that seen in Endo180 expressing cells. In contrast, Rac activation was impaired in Endo180(Ala1468/Ala1469) expressing cells providing a molecular explanation for the ability of these cells to sense a uPA gradient without increasing their migratory speed. Based on these results, we propose that Endo180 plays an important regulatory role in coordinating the activation of the Cdc42 and Rac GTPases during uPA-induced chemotaxis. In agreement with a previous report (Jo et al., 2002), RhoA was not significantly activated by uPA in either the MCF-7 cell lines or MDA-MB-231 cells (unpublished data). uPA–uPAR signaling has been shown to activate the MAPK pathway (Nguyen et al., 1999) and Akt (protein kinase B; Sturge et al., 2002; Chandrasekar et al., 2003). However, wild-type Endo180 or Endo180(Ala1468/Ala1469) expression did not alter the time course of uPA-mediated extracellular signal–regulated kinase 1/2 (ERK1/2) phosphorylation in MCF-7 cells or enhance Akt phosphorylation (Fig. 5 A).

Bottom Line: Endo180 expression was demonstrated to enhance uPA-mediated filopodia production and promote rapid activation of Cdc42 and Rac.Expression of a noninternalizing Endo180 mutant revealed that promotion of random cell migration requires receptor endocytosis, whereas the chemotactic response to uPA does not.From these studies, we conclude that Endo180 is a crucial link between uPA-uPAR and setting of the internal cellular compass.

View Article: PubMed Central - PubMed

Affiliation: The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London SW3 6JB, UK.

ABSTRACT
Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in cell guidance and chemotaxis during normal and pathological events. uPAR is GPI-anchored and the mechanism by which it transmits intracellular polarity cues across the plasma membrane during directional sensing has not been elucidated. The constitutively recycling endocytic receptor Endo180 forms a trimolecular complex with uPAR in the presence of uPA, hence its alternate name uPAR-associated protein. Here, we demonstrate that Endo180 is a general promoter of random cell migration and has a more specific function in cell chemotaxis up a uPA gradient. Endo180 expression was demonstrated to enhance uPA-mediated filopodia production and promote rapid activation of Cdc42 and Rac. Expression of a noninternalizing Endo180 mutant revealed that promotion of random cell migration requires receptor endocytosis, whereas the chemotactic response to uPA does not. From these studies, we conclude that Endo180 is a crucial link between uPA-uPAR and setting of the internal cellular compass.

Show MeSH
Related in: MedlinePlus