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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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GDNF induces phosphorylation of Met. (A and B) Dose-dependent phosphorylation of Met by GDNF in GFRα1- and Ret/GFRα1-expressing MDCK cells. Met was activated in 15 min after GDNF application. The bottom panels show the reprobing of the same filter with anti-Met antibodies. The numbers below the lanes indicate the fold of induction of Met tyrosine kinase. (C) Phosphorylation of Met in mock-transfected MDCK cells. Concentrations of GDNF and HGF are given in ng/ml. 30 μg of total proteins were incubated with 10 μl of immobilized phosphotyrosine mAbs, and immunocomplexes were washed and analyzed as described in Materials and methods. (D) Dose-dependent activation of Ret by GDNF in Ret/GFRα1-expressing MDCK cells. The bottom panel shows the reprobing of the same filter with anti-Ret antibodies. The numbers below the lanes indicate the fold of induction of Ret tyrosine kinase. IP, immunoprecipitation; WB, Western blotting; P-tyr, phosphotyrosine. The results are representative of three independent experiments.
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fig4: GDNF induces phosphorylation of Met. (A and B) Dose-dependent phosphorylation of Met by GDNF in GFRα1- and Ret/GFRα1-expressing MDCK cells. Met was activated in 15 min after GDNF application. The bottom panels show the reprobing of the same filter with anti-Met antibodies. The numbers below the lanes indicate the fold of induction of Met tyrosine kinase. (C) Phosphorylation of Met in mock-transfected MDCK cells. Concentrations of GDNF and HGF are given in ng/ml. 30 μg of total proteins were incubated with 10 μl of immobilized phosphotyrosine mAbs, and immunocomplexes were washed and analyzed as described in Materials and methods. (D) Dose-dependent activation of Ret by GDNF in Ret/GFRα1-expressing MDCK cells. The bottom panel shows the reprobing of the same filter with anti-Ret antibodies. The numbers below the lanes indicate the fold of induction of Ret tyrosine kinase. IP, immunoprecipitation; WB, Western blotting; P-tyr, phosphotyrosine. The results are representative of three independent experiments.

Mentions: Since both GDNF and HGF promoted branching of MDCK cells and Met is the only receptor known to promote tubule formation in these cells (Santos et al., 1993), we suggested that GDNF may induce Met phosphorylation. In 15 min, GDNF indeed evoked Met phosphorylation in GFRα1- and Ret/GFRα1-expressing MDCK cells but not in wild-type MDCK cells. Saturation was reached at 0.1 pg/ml (Fig. 4, A and B) . The same concentration of GDNF also induced rapid Met phosphorylation in human neuroblastoma SHEP cells (unpublished data), which express GFRα1 but no Ret (Poteryaev et al., 1999). GDNF activated Met in GFRα1- and Ret/GFRα1-expressing MDCK cells already in 15 min (Fig. 4, A and B), and the activation lasted at least 2 h (unpublished data). In the mock-transfected MDCK cells, only HGF phosphorylated Met (Fig. 4 C). In Ret/GFRα1-expressing MDCK cells, Ret was phosphorylated already at 0.1 pg/ml of GDNF, and saturation was reached at 10 ng/ml (Fig. 4 D).


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)

GDNF induces phosphorylation of Met. (A and B) Dose-dependent phosphorylation of Met by GDNF in GFRα1- and Ret/GFRα1-expressing MDCK cells. Met was activated in 15 min after GDNF application. The bottom panels show the reprobing of the same filter with anti-Met antibodies. The numbers below the lanes indicate the fold of induction of Met tyrosine kinase. (C) Phosphorylation of Met in mock-transfected MDCK cells. Concentrations of GDNF and HGF are given in ng/ml. 30 μg of total proteins were incubated with 10 μl of immobilized phosphotyrosine mAbs, and immunocomplexes were washed and analyzed as described in Materials and methods. (D) Dose-dependent activation of Ret by GDNF in Ret/GFRα1-expressing MDCK cells. The bottom panel shows the reprobing of the same filter with anti-Ret antibodies. The numbers below the lanes indicate the fold of induction of Ret tyrosine kinase. IP, immunoprecipitation; WB, Western blotting; P-tyr, phosphotyrosine. The results are representative of three independent experiments.
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Related In: Results  -  Collection

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fig4: GDNF induces phosphorylation of Met. (A and B) Dose-dependent phosphorylation of Met by GDNF in GFRα1- and Ret/GFRα1-expressing MDCK cells. Met was activated in 15 min after GDNF application. The bottom panels show the reprobing of the same filter with anti-Met antibodies. The numbers below the lanes indicate the fold of induction of Met tyrosine kinase. (C) Phosphorylation of Met in mock-transfected MDCK cells. Concentrations of GDNF and HGF are given in ng/ml. 30 μg of total proteins were incubated with 10 μl of immobilized phosphotyrosine mAbs, and immunocomplexes were washed and analyzed as described in Materials and methods. (D) Dose-dependent activation of Ret by GDNF in Ret/GFRα1-expressing MDCK cells. The bottom panel shows the reprobing of the same filter with anti-Ret antibodies. The numbers below the lanes indicate the fold of induction of Ret tyrosine kinase. IP, immunoprecipitation; WB, Western blotting; P-tyr, phosphotyrosine. The results are representative of three independent experiments.
Mentions: Since both GDNF and HGF promoted branching of MDCK cells and Met is the only receptor known to promote tubule formation in these cells (Santos et al., 1993), we suggested that GDNF may induce Met phosphorylation. In 15 min, GDNF indeed evoked Met phosphorylation in GFRα1- and Ret/GFRα1-expressing MDCK cells but not in wild-type MDCK cells. Saturation was reached at 0.1 pg/ml (Fig. 4, A and B) . The same concentration of GDNF also induced rapid Met phosphorylation in human neuroblastoma SHEP cells (unpublished data), which express GFRα1 but no Ret (Poteryaev et al., 1999). GDNF activated Met in GFRα1- and Ret/GFRα1-expressing MDCK cells already in 15 min (Fig. 4, A and B), and the activation lasted at least 2 h (unpublished data). In the mock-transfected MDCK cells, only HGF phosphorylated Met (Fig. 4 C). In Ret/GFRα1-expressing MDCK cells, Ret was phosphorylated already at 0.1 pg/ml of GDNF, and saturation was reached at 10 ng/ml (Fig. 4 D).

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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