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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 does not complex with Met. Binding of 125I-GDNF to COS7 cells transfected with gfrα1 and 125I-HGF to wild-type COS7 followed by cross-linking with EDC together with sulfo-NHS. Immunoprecipitates with anti-Met antibodies (IP:Met) were analyzed by SDS-PAGE under reducing conditions. In total lysates (TL), different complexes of 125I-GDNF (monomers or dimers) and the dimers of GFRα1 are marked with a square bracket. 125I-HGF α subunit and proHGF are marked by arrows. 125I-HGF–Met complexes are indicated by arrowheads. The results are representative of five independent experiments.
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fig5: GFRα1 does not complex with Met. Binding of 125I-GDNF to COS7 cells transfected with gfrα1 and 125I-HGF to wild-type COS7 followed by cross-linking with EDC together with sulfo-NHS. Immunoprecipitates with anti-Met antibodies (IP:Met) were analyzed by SDS-PAGE under reducing conditions. In total lysates (TL), different complexes of 125I-GDNF (monomers or dimers) and the dimers of GFRα1 are marked with a square bracket. 125I-HGF α subunit and proHGF are marked by arrows. 125I-HGF–Met complexes are indicated by arrowheads. The results are representative of five independent experiments.

Mentions: In a series of cross-linking immunoprecipitation experiments, we tested whether GDNF activates Met directly or indirectly. Binding of 125I-GDNF to GFRα1-expressing MDCK, SHEP, or COS7 cells and NIH 3T3 cells transiently transfected with gfrα1 was followed by chemical cross-linking and immunoprecipitation with anti-Met antibodies. No high molecular weight complexes were revealed, and in total lysates the bands represent different complexes of 125I-GDNF (monomers or dimers) and the dimers of GFRα1 (Fig. 5) . Different cross-linkers, such as EDC with sulfo-NHS, BS3, DSS, and DSP, were tested, and the result remained the same (Fig. 5 and unpublished data). A direct association of the GDNF receptor complex and Met was not detected in Ret/GFRα1-expressing MDCK cells either (unpublished data).


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 does not complex with Met. Binding of 125I-GDNF to COS7 cells transfected with gfrα1 and 125I-HGF to wild-type COS7 followed by cross-linking with EDC together with sulfo-NHS. Immunoprecipitates with anti-Met antibodies (IP:Met) were analyzed by SDS-PAGE under reducing conditions. In total lysates (TL), different complexes of 125I-GDNF (monomers or dimers) and the dimers of GFRα1 are marked with a square bracket. 125I-HGF α subunit and proHGF are marked by arrows. 125I-HGF–Met complexes are indicated by arrowheads. The results are representative of five independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172872&req=5

fig5: GFRα1 does not complex with Met. Binding of 125I-GDNF to COS7 cells transfected with gfrα1 and 125I-HGF to wild-type COS7 followed by cross-linking with EDC together with sulfo-NHS. Immunoprecipitates with anti-Met antibodies (IP:Met) were analyzed by SDS-PAGE under reducing conditions. In total lysates (TL), different complexes of 125I-GDNF (monomers or dimers) and the dimers of GFRα1 are marked with a square bracket. 125I-HGF α subunit and proHGF are marked by arrows. 125I-HGF–Met complexes are indicated by arrowheads. The results are representative of five independent experiments.
Mentions: In a series of cross-linking immunoprecipitation experiments, we tested whether GDNF activates Met directly or indirectly. Binding of 125I-GDNF to GFRα1-expressing MDCK, SHEP, or COS7 cells and NIH 3T3 cells transiently transfected with gfrα1 was followed by chemical cross-linking and immunoprecipitation with anti-Met antibodies. No high molecular weight complexes were revealed, and in total lysates the bands represent different complexes of 125I-GDNF (monomers or dimers) and the dimers of GFRα1 (Fig. 5) . Different cross-linkers, such as EDC with sulfo-NHS, BS3, DSS, and DSP, were tested, and the result remained the same (Fig. 5 and unpublished data). A direct association of the GDNF receptor complex and Met was not detected in Ret/GFRα1-expressing MDCK cells either (unpublished data).

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