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The neuronal scaffold protein Shank3 mediates signaling and biological function of the receptor tyrosine kinase Ret in epithelial cells.

Schuetz G, Rosário M, Grimm J, Boeckers TM, Gundelfinger ED, Birchmeier W - J. Cell Biol. (2004)

Bottom Line: The PDZ domain-containing Shank3 protein was found to represent a novel interaction partner of the receptor tyrosine kinase Ret, which binds specifically to a PDZ-binding motif present in the Ret9 but not in the Ret51 isoform.Shank3 protein mediates sustained Erk-MAPK and PI3K signaling, which is crucial for tubule formation, through recruitment of the adaptor protein Grb2.These results demonstrate that the Shank3 adaptor protein can mediate cellular signaling, and provide a molecular mechanism for the biological divergence between the Ret9 and Ret51 isoform.

View Article: PubMed Central - PubMed

Affiliation: MaxDelbrück-Center for Molecular Medicine, Berlin, Germany.

ABSTRACT
Shank proteins, initially also described as ProSAP proteins, are scaffolding adaptors that have been previously shown to integrate neurotransmitter receptors into the cortical cytoskeleton at postsynaptic densities. We show here that Shank proteins are also crucial in receptor tyrosine kinase signaling. The PDZ domain-containing Shank3 protein was found to represent a novel interaction partner of the receptor tyrosine kinase Ret, which binds specifically to a PDZ-binding motif present in the Ret9 but not in the Ret51 isoform. Furthermore, we show that Ret9 but not Ret51 induces epithelial cells to form branched tubular structures in three-dimensional cultures in a Shank3-dependent manner. Ret9 but not Ret51 has been previously shown to be required for kidney development. Shank3 protein mediates sustained Erk-MAPK and PI3K signaling, which is crucial for tubule formation, through recruitment of the adaptor protein Grb2. These results demonstrate that the Shank3 adaptor protein can mediate cellular signaling, and provide a molecular mechanism for the biological divergence between the Ret9 and Ret51 isoform.

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Ret9 interacts with Shank3 in vivo. (a and b) Immunofluorescence for Shank3 of embryonic kidney sections. Frozen sections of E16.5 mouse embryos were stained for Shank3 in green and DNA in blue. The dashed lines in b outline epithelial tubules. (c) Coimmunoprecipitation of Ret9 with Shank3 from lysates of mouse kidneys. Immunoprecipitations were subjected to SDS-PAGE and Western blotting. The Western blot was developed using antibodies against Ret9 and Shank3 as indicated. Controls were an immunoprecipitation with a control antibody, whole cell lysate from mouse kidneys, and whole cell lysate from Neuro-2A cells (mouse neuroblastoma 2A), which are rich in Ret9 and Shank3. Bars: 50 μm.
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fig3: Ret9 interacts with Shank3 in vivo. (a and b) Immunofluorescence for Shank3 of embryonic kidney sections. Frozen sections of E16.5 mouse embryos were stained for Shank3 in green and DNA in blue. The dashed lines in b outline epithelial tubules. (c) Coimmunoprecipitation of Ret9 with Shank3 from lysates of mouse kidneys. Immunoprecipitations were subjected to SDS-PAGE and Western blotting. The Western blot was developed using antibodies against Ret9 and Shank3 as indicated. Controls were an immunoprecipitation with a control antibody, whole cell lysate from mouse kidneys, and whole cell lysate from Neuro-2A cells (mouse neuroblastoma 2A), which are rich in Ret9 and Shank3. Bars: 50 μm.

Mentions: Both Ret and Shank2 have been reported to localize to the renal tubular system (Pachnis et al., 1993; Redecker et al., 2001). Using immunohistochemistry with a specific antibody, we could show that Shank3 localizes to basolateral cell membranes in epithelial tubules of the developing kidney in E16.5 mouse embryos (Fig. 3, a and b). No expression of Shank was observed in surrounding mesenchymal cells. Strong staining was also observed at apical sites of renal tubules, which may indicate a further scaffolding function of Shank proteins. We could also demonstrate interaction of endogenous Ret9 with Shank3 in lysates of mouse kidneys by coimmunoprecipitation (Fig. 3 c). Taken together, our data indicate that Ret and Shank3 interact in the kidney in vivo, and that Shank3 is expressed at sites of Ret function.


The neuronal scaffold protein Shank3 mediates signaling and biological function of the receptor tyrosine kinase Ret in epithelial cells.

Schuetz G, Rosário M, Grimm J, Boeckers TM, Gundelfinger ED, Birchmeier W - J. Cell Biol. (2004)

Ret9 interacts with Shank3 in vivo. (a and b) Immunofluorescence for Shank3 of embryonic kidney sections. Frozen sections of E16.5 mouse embryos were stained for Shank3 in green and DNA in blue. The dashed lines in b outline epithelial tubules. (c) Coimmunoprecipitation of Ret9 with Shank3 from lysates of mouse kidneys. Immunoprecipitations were subjected to SDS-PAGE and Western blotting. The Western blot was developed using antibodies against Ret9 and Shank3 as indicated. Controls were an immunoprecipitation with a control antibody, whole cell lysate from mouse kidneys, and whole cell lysate from Neuro-2A cells (mouse neuroblastoma 2A), which are rich in Ret9 and Shank3. Bars: 50 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172453&req=5

fig3: Ret9 interacts with Shank3 in vivo. (a and b) Immunofluorescence for Shank3 of embryonic kidney sections. Frozen sections of E16.5 mouse embryos were stained for Shank3 in green and DNA in blue. The dashed lines in b outline epithelial tubules. (c) Coimmunoprecipitation of Ret9 with Shank3 from lysates of mouse kidneys. Immunoprecipitations were subjected to SDS-PAGE and Western blotting. The Western blot was developed using antibodies against Ret9 and Shank3 as indicated. Controls were an immunoprecipitation with a control antibody, whole cell lysate from mouse kidneys, and whole cell lysate from Neuro-2A cells (mouse neuroblastoma 2A), which are rich in Ret9 and Shank3. Bars: 50 μm.
Mentions: Both Ret and Shank2 have been reported to localize to the renal tubular system (Pachnis et al., 1993; Redecker et al., 2001). Using immunohistochemistry with a specific antibody, we could show that Shank3 localizes to basolateral cell membranes in epithelial tubules of the developing kidney in E16.5 mouse embryos (Fig. 3, a and b). No expression of Shank was observed in surrounding mesenchymal cells. Strong staining was also observed at apical sites of renal tubules, which may indicate a further scaffolding function of Shank proteins. We could also demonstrate interaction of endogenous Ret9 with Shank3 in lysates of mouse kidneys by coimmunoprecipitation (Fig. 3 c). Taken together, our data indicate that Ret and Shank3 interact in the kidney in vivo, and that Shank3 is expressed at sites of Ret function.

Bottom Line: The PDZ domain-containing Shank3 protein was found to represent a novel interaction partner of the receptor tyrosine kinase Ret, which binds specifically to a PDZ-binding motif present in the Ret9 but not in the Ret51 isoform.Shank3 protein mediates sustained Erk-MAPK and PI3K signaling, which is crucial for tubule formation, through recruitment of the adaptor protein Grb2.These results demonstrate that the Shank3 adaptor protein can mediate cellular signaling, and provide a molecular mechanism for the biological divergence between the Ret9 and Ret51 isoform.

View Article: PubMed Central - PubMed

Affiliation: MaxDelbrück-Center for Molecular Medicine, Berlin, Germany.

ABSTRACT
Shank proteins, initially also described as ProSAP proteins, are scaffolding adaptors that have been previously shown to integrate neurotransmitter receptors into the cortical cytoskeleton at postsynaptic densities. We show here that Shank proteins are also crucial in receptor tyrosine kinase signaling. The PDZ domain-containing Shank3 protein was found to represent a novel interaction partner of the receptor tyrosine kinase Ret, which binds specifically to a PDZ-binding motif present in the Ret9 but not in the Ret51 isoform. Furthermore, we show that Ret9 but not Ret51 induces epithelial cells to form branched tubular structures in three-dimensional cultures in a Shank3-dependent manner. Ret9 but not Ret51 has been previously shown to be required for kidney development. Shank3 protein mediates sustained Erk-MAPK and PI3K signaling, which is crucial for tubule formation, through recruitment of the adaptor protein Grb2. These results demonstrate that the Shank3 adaptor protein can mediate cellular signaling, and provide a molecular mechanism for the biological divergence between the Ret9 and Ret51 isoform.

Show MeSH
Related in: MedlinePlus