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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-induced activation of Met requires Src kinase. (A) Dose-dependent Src kinase activation by GDNF in GFRα1- and Ret/GFRα1-expressing MDCK cells. The activation of Src-type kinases was observed after 15 min. The concentrations of GDNF are marked. The bottom panel shows mock-transfected MDCK cells induced with 50 ng/ml of HGF and 100 ng/ml of GDNF. The numbers below the lane indicate the fold of increase in phosphorylation of Tyr418 of Src. The bottom panels show the reprobing of the same filter with anti-Src antibodies. The results are representative of three independent experiments. (B) SHEP cells were grown with GDNF (10 ng/ml and 5 pg/ml) or HGF (10 ng/ml) in the presence of PP2 (1 and 10 μM). To exclude a possible cytotoxic effect of the solvent, DMSO was added to the controls. The bottom panel shows the reprobing of the same filter with anti-Met antibodies. Bottom picture demonstrates wild-type MDCK cells induced with 50 ng/ml of HGF and 100 ng/ml of GDNF. Numbers below the lane indicate the fold of induction of Met tyrosine kinase. The results are representative of three independent experiments.
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fig6: GDNF-induced activation of Met requires Src kinase. (A) Dose-dependent Src kinase activation by GDNF in GFRα1- and Ret/GFRα1-expressing MDCK cells. The activation of Src-type kinases was observed after 15 min. The concentrations of GDNF are marked. The bottom panel shows mock-transfected MDCK cells induced with 50 ng/ml of HGF and 100 ng/ml of GDNF. The numbers below the lane indicate the fold of increase in phosphorylation of Tyr418 of Src. The bottom panels show the reprobing of the same filter with anti-Src antibodies. The results are representative of three independent experiments. (B) SHEP cells were grown with GDNF (10 ng/ml and 5 pg/ml) or HGF (10 ng/ml) in the presence of PP2 (1 and 10 μM). To exclude a possible cytotoxic effect of the solvent, DMSO was added to the controls. The bottom panel shows the reprobing of the same filter with anti-Met antibodies. Bottom picture demonstrates wild-type MDCK cells induced with 50 ng/ml of HGF and 100 ng/ml of GDNF. Numbers below the lane indicate the fold of induction of Met tyrosine kinase. The results are representative of three independent experiments.

Mentions: Already 0.1 pg/ml of GDNF saturated Src phosphorylation at Tyr418, demonstrating Src activation in both GFRα1- and Ret/GFRα1-expressing MDCK cells (Fig. 6 A). 1 μM concentration of the Src-type kinase inhibitor PP2 inhibited GDNF-induced Met activation in both GFRα1- and Ret/GFRα1-expressing MDCK cells but not in the HGF-induced Met phosphorylation in these cells (unpublished data). Accordingly, PP2 strongly reduced the GDNF-dependent phosphorylation of Met in SHEP cells endogenously expressing GFRα1 but not Ret (Fig. 6 B) and did not affect HGF-induced Met phosphorylation (Fig. 6 B).


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-induced activation of Met requires Src kinase. (A) Dose-dependent Src kinase activation by GDNF in GFRα1- and Ret/GFRα1-expressing MDCK cells. The activation of Src-type kinases was observed after 15 min. The concentrations of GDNF are marked. The bottom panel shows mock-transfected MDCK cells induced with 50 ng/ml of HGF and 100 ng/ml of GDNF. The numbers below the lane indicate the fold of increase in phosphorylation of Tyr418 of Src. The bottom panels show the reprobing of the same filter with anti-Src antibodies. The results are representative of three independent experiments. (B) SHEP cells were grown with GDNF (10 ng/ml and 5 pg/ml) or HGF (10 ng/ml) in the presence of PP2 (1 and 10 μM). To exclude a possible cytotoxic effect of the solvent, DMSO was added to the controls. The bottom panel shows the reprobing of the same filter with anti-Met antibodies. Bottom picture demonstrates wild-type MDCK cells induced with 50 ng/ml of HGF and 100 ng/ml of GDNF. Numbers below the lane indicate the fold of induction of Met tyrosine kinase. The results are representative of three independent experiments.
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Related In: Results  -  Collection

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fig6: GDNF-induced activation of Met requires Src kinase. (A) Dose-dependent Src kinase activation by GDNF in GFRα1- and Ret/GFRα1-expressing MDCK cells. The activation of Src-type kinases was observed after 15 min. The concentrations of GDNF are marked. The bottom panel shows mock-transfected MDCK cells induced with 50 ng/ml of HGF and 100 ng/ml of GDNF. The numbers below the lane indicate the fold of increase in phosphorylation of Tyr418 of Src. The bottom panels show the reprobing of the same filter with anti-Src antibodies. The results are representative of three independent experiments. (B) SHEP cells were grown with GDNF (10 ng/ml and 5 pg/ml) or HGF (10 ng/ml) in the presence of PP2 (1 and 10 μM). To exclude a possible cytotoxic effect of the solvent, DMSO was added to the controls. The bottom panel shows the reprobing of the same filter with anti-Met antibodies. Bottom picture demonstrates wild-type MDCK cells induced with 50 ng/ml of HGF and 100 ng/ml of GDNF. Numbers below the lane indicate the fold of induction of Met tyrosine kinase. The results are representative of three independent experiments.
Mentions: Already 0.1 pg/ml of GDNF saturated Src phosphorylation at Tyr418, demonstrating Src activation in both GFRα1- and Ret/GFRα1-expressing MDCK cells (Fig. 6 A). 1 μM concentration of the Src-type kinase inhibitor PP2 inhibited GDNF-induced Met activation in both GFRα1- and Ret/GFRα1-expressing MDCK cells but not in the HGF-induced Met phosphorylation in these cells (unpublished data). Accordingly, PP2 strongly reduced the GDNF-dependent phosphorylation of Met in SHEP cells endogenously expressing GFRα1 but not Ret (Fig. 6 B) and did not affect HGF-induced Met phosphorylation (Fig. 6 B).

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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