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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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Induction of tubular structures by Ret depends on the PDZ-binding motif. MDCK cell lines expressing Ret9 (a–c), Ret51 (d–f), Ret9 FA (g–i), Ret51-9 (j–l), or control cells without Ret (m–o) were seeded as single cell suspensions into three-dimensional collagen matrices and stimulated with GDNF–sGFRα1 or HGF/SF. Photographs were taken after fixation. Bar in o: 200 μm (applies to a–o). (p) Expression of the Ret cDNA constructs in the various MDCK cell lines is shown by RT-PCR. (q) Expression of endogenous Shank3 in mouse brain and MDCK cells is shown by RT–PCR.
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fig4: Induction of tubular structures by Ret depends on the PDZ-binding motif. MDCK cell lines expressing Ret9 (a–c), Ret51 (d–f), Ret9 FA (g–i), Ret51-9 (j–l), or control cells without Ret (m–o) were seeded as single cell suspensions into three-dimensional collagen matrices and stimulated with GDNF–sGFRα1 or HGF/SF. Photographs were taken after fixation. Bar in o: 200 μm (applies to a–o). (p) Expression of the Ret cDNA constructs in the various MDCK cell lines is shown by RT-PCR. (q) Expression of endogenous Shank3 in mouse brain and MDCK cells is shown by RT–PCR.

Mentions: In vivo experiments have indicated that only Ret9 is crucial for kidney development (Srinivas et al., 1999; de Graaff et al., 2001). Here we have used an in vitro tubulogenesis assay with MDCK kidney epithelial cells to mimic the formation of kidney tubules (Montesano et al., 1991; Santos and Nigam, 1993; Weidner et al., 1993). MDCK cells seeded in a three-dimensional collagen matrix form cysts, consisting of a single polarized epithelial cell layer enclosing a fluid-filled lumen. Stimulation with growth factors such as hepatocyte growth factor/scatter factor (HGF/SF) induces the reorganization and growth of these cysts into branched, hollow tubes, which invade the surrounding matrix. We have generated MDCK cell lines that express various forms of Ret and stimulated these with the Ret ligand GDNF and the essential soluble coreceptor GFRα1 (sGFRα1; Paratcha et al., 2001). We found that cells expressing Ret9, but not Ret51, respond to ligand stimulation and form tubules (Fig. 4, a, b, d, and e). Remarkably, mutation of the PDZ-binding motif in Ret9 (Ret9 FA) prevented tubule formation (Fig. 4, g and h). In contrast, cells expressing the Ret51-9 protein, where the Ret9 PDZ-binding motif is attached, are capable of undergoing tubule formation (Fig. 4, j and k). Parental MDCK cells, which do not express endogenous Ret, do not respond to stimulation with GDNF–sGFRα1 (Fig. 4, m and n). As a further control, addition of HGF/SF leads to tubulogenesis in all cell lines (Fig 4, HGF/SF column). The expression of the various Ret constructs and of endogenous Shank3 in the MDCK cell lines is shown by RT-PCR (Fig. 4, p and q). These data demonstrate that signaling by the Ret9 isoform is sufficient to induce epithelial tube formation, and that this activity depends on an intact PDZ-binding motif on Ret.


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)

Induction of tubular structures by Ret depends on the PDZ-binding motif. MDCK cell lines expressing Ret9 (a–c), Ret51 (d–f), Ret9 FA (g–i), Ret51-9 (j–l), or control cells without Ret (m–o) were seeded as single cell suspensions into three-dimensional collagen matrices and stimulated with GDNF–sGFRα1 or HGF/SF. Photographs were taken after fixation. Bar in o: 200 μm (applies to a–o). (p) Expression of the Ret cDNA constructs in the various MDCK cell lines is shown by RT-PCR. (q) Expression of endogenous Shank3 in mouse brain and MDCK cells is shown by RT–PCR.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172453&req=5

fig4: Induction of tubular structures by Ret depends on the PDZ-binding motif. MDCK cell lines expressing Ret9 (a–c), Ret51 (d–f), Ret9 FA (g–i), Ret51-9 (j–l), or control cells without Ret (m–o) were seeded as single cell suspensions into three-dimensional collagen matrices and stimulated with GDNF–sGFRα1 or HGF/SF. Photographs were taken after fixation. Bar in o: 200 μm (applies to a–o). (p) Expression of the Ret cDNA constructs in the various MDCK cell lines is shown by RT-PCR. (q) Expression of endogenous Shank3 in mouse brain and MDCK cells is shown by RT–PCR.
Mentions: In vivo experiments have indicated that only Ret9 is crucial for kidney development (Srinivas et al., 1999; de Graaff et al., 2001). Here we have used an in vitro tubulogenesis assay with MDCK kidney epithelial cells to mimic the formation of kidney tubules (Montesano et al., 1991; Santos and Nigam, 1993; Weidner et al., 1993). MDCK cells seeded in a three-dimensional collagen matrix form cysts, consisting of a single polarized epithelial cell layer enclosing a fluid-filled lumen. Stimulation with growth factors such as hepatocyte growth factor/scatter factor (HGF/SF) induces the reorganization and growth of these cysts into branched, hollow tubes, which invade the surrounding matrix. We have generated MDCK cell lines that express various forms of Ret and stimulated these with the Ret ligand GDNF and the essential soluble coreceptor GFRα1 (sGFRα1; Paratcha et al., 2001). We found that cells expressing Ret9, but not Ret51, respond to ligand stimulation and form tubules (Fig. 4, a, b, d, and e). Remarkably, mutation of the PDZ-binding motif in Ret9 (Ret9 FA) prevented tubule formation (Fig. 4, g and h). In contrast, cells expressing the Ret51-9 protein, where the Ret9 PDZ-binding motif is attached, are capable of undergoing tubule formation (Fig. 4, j and k). Parental MDCK cells, which do not express endogenous Ret, do not respond to stimulation with GDNF–sGFRα1 (Fig. 4, m and n). As a further control, addition of HGF/SF leads to tubulogenesis in all cell lines (Fig 4, HGF/SF column). The expression of the various Ret constructs and of endogenous Shank3 in the MDCK cell lines is shown by RT-PCR (Fig. 4, p and q). These data demonstrate that signaling by the Ret9 isoform is sufficient to induce epithelial tube formation, and that this activity depends on an intact PDZ-binding motif on Ret.

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