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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 branching tubulogenesis of GFRα1- and Ret/GFRα1-expressing cells require c-Src kinase. GFRα1- and Ret/GFRα1-expressing and wild-type MDCK cells were infected with adenovirus constructs containing DN c-Src, activated c-Src, or adeno-GFP. (A) GDNF-induced Met activation depends on c-Src kinase. 1 d after the adenovirus infection, GFRα1- and Ret/GFRα1-expressing MDCK cells were induced with GDNF (50 ng/ml) and wild-type MDCK also with HGF (50 ng/ml). Aliquots of total cell lysates were immunoblotted with anti-Y418 Src, and the rest of lysates were immunoprecipitated with anti-Met antibodies and immunoblotted using antiphosphotyrosine antibodies. The results are representative of two independent experiments. (B) After infection cells were put in collagen gel culture, GFRα1- and Ret/GFRα1-expressing cells were grown with or without GDNF (50 ng/ml), wild-type MDCK with or without HGF (50 ng/ml). After 3 d, the cells were fixed and counted as described in Materials and methods. The results are representative of three independent experiments.
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fig7: GDNF-induced branching tubulogenesis of GFRα1- and Ret/GFRα1-expressing cells require c-Src kinase. GFRα1- and Ret/GFRα1-expressing and wild-type MDCK cells were infected with adenovirus constructs containing DN c-Src, activated c-Src, or adeno-GFP. (A) GDNF-induced Met activation depends on c-Src kinase. 1 d after the adenovirus infection, GFRα1- and Ret/GFRα1-expressing MDCK cells were induced with GDNF (50 ng/ml) and wild-type MDCK also with HGF (50 ng/ml). Aliquots of total cell lysates were immunoblotted with anti-Y418 Src, and the rest of lysates were immunoprecipitated with anti-Met antibodies and immunoblotted using antiphosphotyrosine antibodies. The results are representative of two independent experiments. (B) After infection cells were put in collagen gel culture, GFRα1- and Ret/GFRα1-expressing cells were grown with or without GDNF (50 ng/ml), wild-type MDCK with or without HGF (50 ng/ml). After 3 d, the cells were fixed and counted as described in Materials and methods. The results are representative of three independent experiments.

Mentions: We used adenoviruses to introduce DN c-Src or activated c-Src to GFRα1- and Ret/GFRα1-expressing and wild-type MDCK cells. Both constructs contained gfp under a separate promoter, which enabled us to monitor the infection efficiency. It was close to 100% in all experiments. Adenoviruses with gfp alone were used as a control. In accordance with the results with PP2, DN c-Src efficiently blocked Src phosphorylation and Met activation induced by GDNF but not that induced by HGF (Fig. 7 A). Expression of activated c-Src resulted in GDNF-independent phosphorylation of Met, which could not be further increased by GDNF. In contrast, HGF enhanced Met phosphorylation in wild-type MDCK-expressing activated c-Src (Fig. 7 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)

GDNF-induced branching tubulogenesis of GFRα1- and Ret/GFRα1-expressing cells require c-Src kinase. GFRα1- and Ret/GFRα1-expressing and wild-type MDCK cells were infected with adenovirus constructs containing DN c-Src, activated c-Src, or adeno-GFP. (A) GDNF-induced Met activation depends on c-Src kinase. 1 d after the adenovirus infection, GFRα1- and Ret/GFRα1-expressing MDCK cells were induced with GDNF (50 ng/ml) and wild-type MDCK also with HGF (50 ng/ml). Aliquots of total cell lysates were immunoblotted with anti-Y418 Src, and the rest of lysates were immunoprecipitated with anti-Met antibodies and immunoblotted using antiphosphotyrosine antibodies. The results are representative of two independent experiments. (B) After infection cells were put in collagen gel culture, GFRα1- and Ret/GFRα1-expressing cells were grown with or without GDNF (50 ng/ml), wild-type MDCK with or without HGF (50 ng/ml). After 3 d, the cells were fixed and counted as described in Materials and methods. The results are representative of three independent experiments.
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Related In: Results  -  Collection

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fig7: GDNF-induced branching tubulogenesis of GFRα1- and Ret/GFRα1-expressing cells require c-Src kinase. GFRα1- and Ret/GFRα1-expressing and wild-type MDCK cells were infected with adenovirus constructs containing DN c-Src, activated c-Src, or adeno-GFP. (A) GDNF-induced Met activation depends on c-Src kinase. 1 d after the adenovirus infection, GFRα1- and Ret/GFRα1-expressing MDCK cells were induced with GDNF (50 ng/ml) and wild-type MDCK also with HGF (50 ng/ml). Aliquots of total cell lysates were immunoblotted with anti-Y418 Src, and the rest of lysates were immunoprecipitated with anti-Met antibodies and immunoblotted using antiphosphotyrosine antibodies. The results are representative of two independent experiments. (B) After infection cells were put in collagen gel culture, GFRα1- and Ret/GFRα1-expressing cells were grown with or without GDNF (50 ng/ml), wild-type MDCK with or without HGF (50 ng/ml). After 3 d, the cells were fixed and counted as described in Materials and methods. The results are representative of three independent experiments.
Mentions: We used adenoviruses to introduce DN c-Src or activated c-Src to GFRα1- and Ret/GFRα1-expressing and wild-type MDCK cells. Both constructs contained gfp under a separate promoter, which enabled us to monitor the infection efficiency. It was close to 100% in all experiments. Adenoviruses with gfp alone were used as a control. In accordance with the results with PP2, DN c-Src efficiently blocked Src phosphorylation and Met activation induced by GDNF but not that induced by HGF (Fig. 7 A). Expression of activated c-Src resulted in GDNF-independent phosphorylation of Met, which could not be further increased by GDNF. In contrast, HGF enhanced Met phosphorylation in wild-type MDCK-expressing activated c-Src (Fig. 7 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