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GDNF promotes tubulogenesis of GFRalpha1-expressing MDCK cells by Src-mediated phosphorylation of Met receptor tyrosine kinase.

Popsueva A, Poteryaev D, Arighi E, Meng X, Angers-Loustau A, Kaplan D, Saarma M, Sariola H - J. Cell Biol. (2003)

Bottom Line: However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable.The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not.Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology, Institute of Biomedicine, University of Helsinki, FIN-00014 Helsinki, Finland.

ABSTRACT
Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are multifunctional signaling molecules in embryogenesis. HGF binds to and activates Met receptor tyrosine kinase. The signaling receptor complex for GDNF typically includes both GDNF family receptor alpha1 (GFRalpha1) and Ret receptor tyrosine kinase. GDNF can also signal independently of Ret via GFRalpha1, although the mechanism has remained unclear. We now show that GDNF partially restores ureteric branching morphogenesis in ret-deficient mice with severe renal hypodysplasia. The mechanism of Ret-independent effect of GDNF was therefore studied by the MDCK cell model. In MDCK cells expressing GFRalpha1 but no Ret, GDNF stimulates branching but not chemotactic migration, whereas both branching and chemotaxis are promoted by GDNF in the cells coexpressing Ret and GFRalpha1, mimicking HGF/Met responses in wild-type MDCK cells. Indeed, GDNF induces Met phosphorylation in several ret-deficient/GFRalpha1-positive and GFRalpha1/Ret-coexpressing cell lines. However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable. Met activation is mediated by Src family kinases. The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not. Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling.

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GFRα1-expressing, ret-deficient MDCK cells do not show a chemotactic response to GDNF. (A) In the Boyden chamber chemotaxis assay, the mock-transfected, GFRα1, and Ret/GFRα1 cells were exposed to GDNF (10 and 100 ng/ml), and wild-type MDCK were exposed to HGF (10 and 100 ng/ml). The number of cells was counted as described in Materials and methods. +/+, 100 ng/ml of GDNF or 50 ng/ml of HGF were added to both chambers to assay chemokinesis. The results represent the means ± SEM (n = 3). ***P < 0.001. (B) Chemoattraction assay on collagen matrix. Only Ret/GFRα1-expressing cells migrate toward GDNF-soaked beads. BSA-soaked agarose beads were used as negative control. Beads are marked by a white circle. Note that mock, GFRα1-expressing cells with GDNF-soaked beads and Ret/GFRα1-expressing cells with BSA-soaked bead form clusters of adherent cells (marked with arrowhead) after 3 d, whereas the Ret/GFRα1-expressing cells migrating toward the GDNF bead are scattered.
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fig3: GFRα1-expressing, ret-deficient MDCK cells do not show a chemotactic response to GDNF. (A) In the Boyden chamber chemotaxis assay, the mock-transfected, GFRα1, and Ret/GFRα1 cells were exposed to GDNF (10 and 100 ng/ml), and wild-type MDCK were exposed to HGF (10 and 100 ng/ml). The number of cells was counted as described in Materials and methods. +/+, 100 ng/ml of GDNF or 50 ng/ml of HGF were added to both chambers to assay chemokinesis. The results represent the means ± SEM (n = 3). ***P < 0.001. (B) Chemoattraction assay on collagen matrix. Only Ret/GFRα1-expressing cells migrate toward GDNF-soaked beads. BSA-soaked agarose beads were used as negative control. Beads are marked by a white circle. Note that mock, GFRα1-expressing cells with GDNF-soaked beads and Ret/GFRα1-expressing cells with BSA-soaked bead form clusters of adherent cells (marked with arrowhead) after 3 d, whereas the Ret/GFRα1-expressing cells migrating toward the GDNF bead are scattered.

Mentions: Similar to the HGF-induced chemotaxis of the wild-type MDCK cells (Stoker et al., 1987), the chemotactic migration of ret-transfected MDCK cells is stimulated by GDNF in the presence of soluble GFRα1 (Tang et al., 1998). Accordingly, GDNF was chemotactic to MDCK cells transfected with both ret and the GPI-anchored, membrane-bound form of gfrα1. In contrast, the GFRα1-expressing, ret-deficient MDCK cells did not show a chemotactic response to GDNF under the same conditions (Fig. 3 A).


GDNF promotes tubulogenesis of GFRalpha1-expressing MDCK cells by Src-mediated phosphorylation of Met receptor tyrosine kinase.

Popsueva A, Poteryaev D, Arighi E, Meng X, Angers-Loustau A, Kaplan D, Saarma M, Sariola H - J. Cell Biol. (2003)

GFRα1-expressing, ret-deficient MDCK cells do not show a chemotactic response to GDNF. (A) In the Boyden chamber chemotaxis assay, the mock-transfected, GFRα1, and Ret/GFRα1 cells were exposed to GDNF (10 and 100 ng/ml), and wild-type MDCK were exposed to HGF (10 and 100 ng/ml). The number of cells was counted as described in Materials and methods. +/+, 100 ng/ml of GDNF or 50 ng/ml of HGF were added to both chambers to assay chemokinesis. The results represent the means ± SEM (n = 3). ***P < 0.001. (B) Chemoattraction assay on collagen matrix. Only Ret/GFRα1-expressing cells migrate toward GDNF-soaked beads. BSA-soaked agarose beads were used as negative control. Beads are marked by a white circle. Note that mock, GFRα1-expressing cells with GDNF-soaked beads and Ret/GFRα1-expressing cells with BSA-soaked bead form clusters of adherent cells (marked with arrowhead) after 3 d, whereas the Ret/GFRα1-expressing cells migrating toward the GDNF bead are scattered.
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Related In: Results  -  Collection

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fig3: GFRα1-expressing, ret-deficient MDCK cells do not show a chemotactic response to GDNF. (A) In the Boyden chamber chemotaxis assay, the mock-transfected, GFRα1, and Ret/GFRα1 cells were exposed to GDNF (10 and 100 ng/ml), and wild-type MDCK were exposed to HGF (10 and 100 ng/ml). The number of cells was counted as described in Materials and methods. +/+, 100 ng/ml of GDNF or 50 ng/ml of HGF were added to both chambers to assay chemokinesis. The results represent the means ± SEM (n = 3). ***P < 0.001. (B) Chemoattraction assay on collagen matrix. Only Ret/GFRα1-expressing cells migrate toward GDNF-soaked beads. BSA-soaked agarose beads were used as negative control. Beads are marked by a white circle. Note that mock, GFRα1-expressing cells with GDNF-soaked beads and Ret/GFRα1-expressing cells with BSA-soaked bead form clusters of adherent cells (marked with arrowhead) after 3 d, whereas the Ret/GFRα1-expressing cells migrating toward the GDNF bead are scattered.
Mentions: Similar to the HGF-induced chemotaxis of the wild-type MDCK cells (Stoker et al., 1987), the chemotactic migration of ret-transfected MDCK cells is stimulated by GDNF in the presence of soluble GFRα1 (Tang et al., 1998). Accordingly, GDNF was chemotactic to MDCK cells transfected with both ret and the GPI-anchored, membrane-bound form of gfrα1. In contrast, the GFRα1-expressing, ret-deficient MDCK cells did not show a chemotactic response to GDNF under the same conditions (Fig. 3 A).

Bottom Line: However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable.The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not.Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology, Institute of Biomedicine, University of Helsinki, FIN-00014 Helsinki, Finland.

ABSTRACT
Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are multifunctional signaling molecules in embryogenesis. HGF binds to and activates Met receptor tyrosine kinase. The signaling receptor complex for GDNF typically includes both GDNF family receptor alpha1 (GFRalpha1) and Ret receptor tyrosine kinase. GDNF can also signal independently of Ret via GFRalpha1, although the mechanism has remained unclear. We now show that GDNF partially restores ureteric branching morphogenesis in ret-deficient mice with severe renal hypodysplasia. The mechanism of Ret-independent effect of GDNF was therefore studied by the MDCK cell model. In MDCK cells expressing GFRalpha1 but no Ret, GDNF stimulates branching but not chemotactic migration, whereas both branching and chemotaxis are promoted by GDNF in the cells coexpressing Ret and GFRalpha1, mimicking HGF/Met responses in wild-type MDCK cells. Indeed, GDNF induces Met phosphorylation in several ret-deficient/GFRalpha1-positive and GFRalpha1/Ret-coexpressing cell lines. However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable. Met activation is mediated by Src family kinases. The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not. Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling.

Show MeSH
Related in: MedlinePlus