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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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Endo180 is a general requirement for growth factor–stimulated cell motility but a specific promoter of directionality up a uPA gradient. MDA-MB-231 cells were incubated with control or Endo180 siRNA oligonucleotides or left untreated and analyzed after 48 h. (A) FACS® analysis of Endo180 and uPAR expression levels in control (green) and Endo180 (red) siRNA treated cells. Black lines show binding of second antibody alone to control siRNA treated cells. Endo180 expression in control siRNA treated cells was identical to that in untreated cells (not depicted). (B) Mean migratory speed of unstimulated, uPA- or EGF-stimulated cells, or cells cultured in growth factor cocktail (Opti-MEM medium). ct, control siRNA; E, Endo180 siRNA. Migratory speed values are given as the mean speed for all cells analyzed over the 5-h period ± SEM. (C) Chemotactic response of siRNA or mAb treated cells to a gradient of uPA or EGF. (B and C) Data shown includes analysis of >50 cells pooled from at least three separate experiments. (C) Cell directionality was determined using the Rayleigh test and a horizon distance, which included 50% of all cells assayed for each treatment group. Analysis of the same data using 85% of cells is included in Fig. S1. This highly stringent approach discounts any bias in the calculation of directionality that might be caused by alterations in cell speed. Mean direction of cell migration (red arrow)/95% confidence interval (green wedge). (D) Epitope mapping of anti-Endo180 mAbs. Endo180-Fc constructs were resolved by 10% nonreducing SDS-PAGE and subject to Western blotting. The schematic diagram of Endo180 indicates mAb E1/183 binding to the cysteine-rich domain (CR) and mAb A51/58 binding to CTLD2.
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fig2: Endo180 is a general requirement for growth factor–stimulated cell motility but a specific promoter of directionality up a uPA gradient. MDA-MB-231 cells were incubated with control or Endo180 siRNA oligonucleotides or left untreated and analyzed after 48 h. (A) FACS® analysis of Endo180 and uPAR expression levels in control (green) and Endo180 (red) siRNA treated cells. Black lines show binding of second antibody alone to control siRNA treated cells. Endo180 expression in control siRNA treated cells was identical to that in untreated cells (not depicted). (B) Mean migratory speed of unstimulated, uPA- or EGF-stimulated cells, or cells cultured in growth factor cocktail (Opti-MEM medium). ct, control siRNA; E, Endo180 siRNA. Migratory speed values are given as the mean speed for all cells analyzed over the 5-h period ± SEM. (C) Chemotactic response of siRNA or mAb treated cells to a gradient of uPA or EGF. (B and C) Data shown includes analysis of >50 cells pooled from at least three separate experiments. (C) Cell directionality was determined using the Rayleigh test and a horizon distance, which included 50% of all cells assayed for each treatment group. Analysis of the same data using 85% of cells is included in Fig. S1. This highly stringent approach discounts any bias in the calculation of directionality that might be caused by alterations in cell speed. Mean direction of cell migration (red arrow)/95% confidence interval (green wedge). (D) Epitope mapping of anti-Endo180 mAbs. Endo180-Fc constructs were resolved by 10% nonreducing SDS-PAGE and subject to Western blotting. The schematic diagram of Endo180 indicates mAb E1/183 binding to the cysteine-rich domain (CR) and mAb A51/58 binding to CTLD2.

Mentions: For initial studies, the MDA-MB-231 breast cancer cells were used as they express Endo180, uPAR and EGF receptor, and respond chemotactically to uPA and EGF (Sturge et al., 2002). The staining of Endo180 in MDA-MB-231 cells (Fig. 1) is consistent with previous reports that 10–30% of this receptor is in clathrin-coated pits at the cell surface, whereas 70–90% is located in endosomal compartments (Isacke et al., 1990; Howard and Isacke, 2002). uPAR is also detected in a clustered distribution, although it has a limited overlap with Endo180. Upon uPA stimulation, which is known to promote uPA–uPAR-Endo180 complex formation (Behrendt et al., 2000), Endo180 and uPAR are translocated to the leading edge of polarized cells where they display a substantial degree of colocalization (Fig. 1). To directly investigate the role of Endo180 in these cells, a small interfering RNA (siRNA) approach was taken. Endo180 siRNA treatment resulted in a marked reduction in Endo180 expression (Fig. 2 A; see Fig. 4 C) but did not alter uPAR levels (Fig. 2 A), indicating that expression of these two receptors is not coordinately regulated at the cell surface. This down-regulation of Endo180 had no effect on the basal migratory speed of unstimulated cells but significantly (P < 0.001) reduced the motility of cells in response to uPA or EGF, or when cultured in the presence of a growth factor cocktail (Fig. 2 B). This suggests a general role for Endo180 in the motility of cells stimulated by pro-migratory factors. The most striking alteration in the behavior of Endo180 siRNA treated cells was in their differential ability to respond directionally in chemotactic gradients of uPA and EGF. As expected from previous studies (Sturge et al., 2002), untreated (not depicted) or control siRNA treated MDA-MB-231 cells migrated directionally up gradients of uPA (Fig. 2 C; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200302124/DC1) and EGF (Fig. 2 C). Endo180 siRNA treatment significantly inhibited the chemotactic response to uPA (Fig. 2 C; Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200302124/DC1) but had no impact on the chemotactic response to EGF (Fig. 2 C). To further investigate the structural interplay of Endo180 in the uPA–uPAR specific response, chemotaxis assays were performed in the presence of Endo180 mAbs E1/183 and A5/158. The four members of the mannose receptor family have a common structural organization of an NH2-terminal cysteine-rich domain, a fibronectin type II domain (FNII) and 8 or 10 C-type lectin-like domains (CTLDs), and single-particle electron microscopy has revealed that in Endo180 the NH2-terminal domains form a hairpin structure such that the cysteine-rich domain contacts CTLD2 (Rivera-Calzada et al., 2003). mAb A5/158 binds to CTLD2 and totally blocks directional migration up a uPA gradient, whereas mAb E1/183, which binds the cysteine-rich domain, partially blocks uPA-induced chemotaxis (Fig. 2 C; Fig. S1, see supplemental methods for a full definition of total and partial blockade, and available at http://www.jcb.org/cgi/content/full/jcb.200302124/DC1). The isotype matched mAb B3/25, which recognizes the endocytic transferrin receptor, had no effect on this response (Fig. 2 C). Together, these findings indicate that a specific biological function of Endo180 is to regulate uPA–uPAR mediated directionality and suggest that accessibility of CTLD2 is required for a full biological response.


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)

Endo180 is a general requirement for growth factor–stimulated cell motility but a specific promoter of directionality up a uPA gradient. MDA-MB-231 cells were incubated with control or Endo180 siRNA oligonucleotides or left untreated and analyzed after 48 h. (A) FACS® analysis of Endo180 and uPAR expression levels in control (green) and Endo180 (red) siRNA treated cells. Black lines show binding of second antibody alone to control siRNA treated cells. Endo180 expression in control siRNA treated cells was identical to that in untreated cells (not depicted). (B) Mean migratory speed of unstimulated, uPA- or EGF-stimulated cells, or cells cultured in growth factor cocktail (Opti-MEM medium). ct, control siRNA; E, Endo180 siRNA. Migratory speed values are given as the mean speed for all cells analyzed over the 5-h period ± SEM. (C) Chemotactic response of siRNA or mAb treated cells to a gradient of uPA or EGF. (B and C) Data shown includes analysis of >50 cells pooled from at least three separate experiments. (C) Cell directionality was determined using the Rayleigh test and a horizon distance, which included 50% of all cells assayed for each treatment group. Analysis of the same data using 85% of cells is included in Fig. S1. This highly stringent approach discounts any bias in the calculation of directionality that might be caused by alterations in cell speed. Mean direction of cell migration (red arrow)/95% confidence interval (green wedge). (D) Epitope mapping of anti-Endo180 mAbs. Endo180-Fc constructs were resolved by 10% nonreducing SDS-PAGE and subject to Western blotting. The schematic diagram of Endo180 indicates mAb E1/183 binding to the cysteine-rich domain (CR) and mAb A51/58 binding to CTLD2.
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Related In: Results  -  Collection

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fig2: Endo180 is a general requirement for growth factor–stimulated cell motility but a specific promoter of directionality up a uPA gradient. MDA-MB-231 cells were incubated with control or Endo180 siRNA oligonucleotides or left untreated and analyzed after 48 h. (A) FACS® analysis of Endo180 and uPAR expression levels in control (green) and Endo180 (red) siRNA treated cells. Black lines show binding of second antibody alone to control siRNA treated cells. Endo180 expression in control siRNA treated cells was identical to that in untreated cells (not depicted). (B) Mean migratory speed of unstimulated, uPA- or EGF-stimulated cells, or cells cultured in growth factor cocktail (Opti-MEM medium). ct, control siRNA; E, Endo180 siRNA. Migratory speed values are given as the mean speed for all cells analyzed over the 5-h period ± SEM. (C) Chemotactic response of siRNA or mAb treated cells to a gradient of uPA or EGF. (B and C) Data shown includes analysis of >50 cells pooled from at least three separate experiments. (C) Cell directionality was determined using the Rayleigh test and a horizon distance, which included 50% of all cells assayed for each treatment group. Analysis of the same data using 85% of cells is included in Fig. S1. This highly stringent approach discounts any bias in the calculation of directionality that might be caused by alterations in cell speed. Mean direction of cell migration (red arrow)/95% confidence interval (green wedge). (D) Epitope mapping of anti-Endo180 mAbs. Endo180-Fc constructs were resolved by 10% nonreducing SDS-PAGE and subject to Western blotting. The schematic diagram of Endo180 indicates mAb E1/183 binding to the cysteine-rich domain (CR) and mAb A51/58 binding to CTLD2.
Mentions: For initial studies, the MDA-MB-231 breast cancer cells were used as they express Endo180, uPAR and EGF receptor, and respond chemotactically to uPA and EGF (Sturge et al., 2002). The staining of Endo180 in MDA-MB-231 cells (Fig. 1) is consistent with previous reports that 10–30% of this receptor is in clathrin-coated pits at the cell surface, whereas 70–90% is located in endosomal compartments (Isacke et al., 1990; Howard and Isacke, 2002). uPAR is also detected in a clustered distribution, although it has a limited overlap with Endo180. Upon uPA stimulation, which is known to promote uPA–uPAR-Endo180 complex formation (Behrendt et al., 2000), Endo180 and uPAR are translocated to the leading edge of polarized cells where they display a substantial degree of colocalization (Fig. 1). To directly investigate the role of Endo180 in these cells, a small interfering RNA (siRNA) approach was taken. Endo180 siRNA treatment resulted in a marked reduction in Endo180 expression (Fig. 2 A; see Fig. 4 C) but did not alter uPAR levels (Fig. 2 A), indicating that expression of these two receptors is not coordinately regulated at the cell surface. This down-regulation of Endo180 had no effect on the basal migratory speed of unstimulated cells but significantly (P < 0.001) reduced the motility of cells in response to uPA or EGF, or when cultured in the presence of a growth factor cocktail (Fig. 2 B). This suggests a general role for Endo180 in the motility of cells stimulated by pro-migratory factors. The most striking alteration in the behavior of Endo180 siRNA treated cells was in their differential ability to respond directionally in chemotactic gradients of uPA and EGF. As expected from previous studies (Sturge et al., 2002), untreated (not depicted) or control siRNA treated MDA-MB-231 cells migrated directionally up gradients of uPA (Fig. 2 C; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200302124/DC1) and EGF (Fig. 2 C). Endo180 siRNA treatment significantly inhibited the chemotactic response to uPA (Fig. 2 C; Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200302124/DC1) but had no impact on the chemotactic response to EGF (Fig. 2 C). To further investigate the structural interplay of Endo180 in the uPA–uPAR specific response, chemotaxis assays were performed in the presence of Endo180 mAbs E1/183 and A5/158. The four members of the mannose receptor family have a common structural organization of an NH2-terminal cysteine-rich domain, a fibronectin type II domain (FNII) and 8 or 10 C-type lectin-like domains (CTLDs), and single-particle electron microscopy has revealed that in Endo180 the NH2-terminal domains form a hairpin structure such that the cysteine-rich domain contacts CTLD2 (Rivera-Calzada et al., 2003). mAb A5/158 binds to CTLD2 and totally blocks directional migration up a uPA gradient, whereas mAb E1/183, which binds the cysteine-rich domain, partially blocks uPA-induced chemotaxis (Fig. 2 C; Fig. S1, see supplemental methods for a full definition of total and partial blockade, and available at http://www.jcb.org/cgi/content/full/jcb.200302124/DC1). The isotype matched mAb B3/25, which recognizes the endocytic transferrin receptor, had no effect on this response (Fig. 2 C). Together, these findings indicate that a specific biological function of Endo180 is to regulate uPA–uPAR mediated directionality and suggest that accessibility of CTLD2 is required for a full biological response.

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