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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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Grb2 binding to Shank3 is required for tubule formation. MDCK cells expressing either Ret9–Shank3–Pro2 (a–c) or Ret–Shank3–Pro2 containing the Y1006F mutation (d–f) were examined for tubulogenesis in three-dimensional collagen matrices. After stimulation with GDNF–sGFRα1 or HGF/SF, cell cultures were fixed and photographed. Bar in f: 200 μm (applies to a–f). (g) Expression of the indicated cDNA constructs shown by RT-PCR.
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fig8: Grb2 binding to Shank3 is required for tubule formation. MDCK cells expressing either Ret9–Shank3–Pro2 (a–c) or Ret–Shank3–Pro2 containing the Y1006F mutation (d–f) were examined for tubulogenesis in three-dimensional collagen matrices. After stimulation with GDNF–sGFRα1 or HGF/SF, cell cultures were fixed and photographed. Bar in f: 200 μm (applies to a–f). (g) Expression of the indicated cDNA constructs shown by RT-PCR.

Mentions: The PDZ-binding motif of Ret9 is also required for Shank3 binding. To analyze whether Shank proteins indeed mediate tube formation, we fused distinct fragments of Shank3 to Ret9 and examined the hybrids in the biological assays. It should be noted that fusing any sequence to the COOH terminus of Ret9 abrogates PDZ binding (Songyang et al., 1997) and tubule formation (unpublished results). However, by attachment of the proline-rich domain of Shank3 to Ret9 (Ret9–Shank3–Pro1), we could reconstitute tube formation (Fig. 5, d and e). A fusion construct of Ret with a shorter fragment of the proline-rich region of Shank3 (Shank3-Pro2, aa 632–1057; Fig. 1 b) did also induce tubule formation (see Fig. 8, a and b). Fusion of other domains of Shank3 to Ret9, e.g., the ankyrin repeat and the SH3 domains, are not sufficient to induce tubulogenesis (Ret9–Shank3–NT; Fig. 5, a and b). Expression of Ret9–Shank3–NT and Ret9–Shank3–Pro1 is shown by RT-PCR (Fig. 5 g). Again, all cell lines were responsive to HGF/SF (Fig. 5, c and f). We could not obtain expression of a fusion construct between Ret and full-size Shank3. These results indicate that Shank3 can indeed act downstream of Ret9 to stimulate the formation of epithelial tubes. This activity is mediated by the proline-rich domain of Shank3.


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)

Grb2 binding to Shank3 is required for tubule formation. MDCK cells expressing either Ret9–Shank3–Pro2 (a–c) or Ret–Shank3–Pro2 containing the Y1006F mutation (d–f) were examined for tubulogenesis in three-dimensional collagen matrices. After stimulation with GDNF–sGFRα1 or HGF/SF, cell cultures were fixed and photographed. Bar in f: 200 μm (applies to a–f). (g) Expression of the indicated cDNA constructs shown by RT-PCR.
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Related In: Results  -  Collection

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fig8: Grb2 binding to Shank3 is required for tubule formation. MDCK cells expressing either Ret9–Shank3–Pro2 (a–c) or Ret–Shank3–Pro2 containing the Y1006F mutation (d–f) were examined for tubulogenesis in three-dimensional collagen matrices. After stimulation with GDNF–sGFRα1 or HGF/SF, cell cultures were fixed and photographed. Bar in f: 200 μm (applies to a–f). (g) Expression of the indicated cDNA constructs shown by RT-PCR.
Mentions: The PDZ-binding motif of Ret9 is also required for Shank3 binding. To analyze whether Shank proteins indeed mediate tube formation, we fused distinct fragments of Shank3 to Ret9 and examined the hybrids in the biological assays. It should be noted that fusing any sequence to the COOH terminus of Ret9 abrogates PDZ binding (Songyang et al., 1997) and tubule formation (unpublished results). However, by attachment of the proline-rich domain of Shank3 to Ret9 (Ret9–Shank3–Pro1), we could reconstitute tube formation (Fig. 5, d and e). A fusion construct of Ret with a shorter fragment of the proline-rich region of Shank3 (Shank3-Pro2, aa 632–1057; Fig. 1 b) did also induce tubule formation (see Fig. 8, a and b). Fusion of other domains of Shank3 to Ret9, e.g., the ankyrin repeat and the SH3 domains, are not sufficient to induce tubulogenesis (Ret9–Shank3–NT; Fig. 5, a and b). Expression of Ret9–Shank3–NT and Ret9–Shank3–Pro1 is shown by RT-PCR (Fig. 5 g). Again, all cell lines were responsive to HGF/SF (Fig. 5, c and f). We could not obtain expression of a fusion construct between Ret and full-size Shank3. These results indicate that Shank3 can indeed act downstream of Ret9 to stimulate the formation of epithelial tubes. This activity is mediated by the proline-rich domain of Shank3.

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