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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 the Shank3 PDZ domain. (a) Schematic representation of Ret9, Ret51, and of COOH-terminal mutants. Cad, cadherin domain; Tm, transmembrane domain; Tk, tyrosine kinase domain; Ret9 Δ4, deletion of the PDZ-binding motif; Ret9 FA point mutation in the PDZ-binding motif; Ret51-9, Ret9 PDZ-binding motif attached to Ret51. (b) Domain structure of Shank3 and Shank3 deletion mutants. Ank, ankyrin repeats; SH3, src homology 3 domain; Pro, proline-rich region; SAM, sterile alpha motif. (c) Interaction of Ret9 and Shank3 in a yeast two-hybrid analysis. Binding of the GAL4-binding domain–Shank3 PDZ domain fusion protein (or of the GAL4-binding domain alone) to GAL4-activation domain fusion proteins of the cytoplasmic domains of Ret9, Ret51, Ret9 Δ4, and Ret9 FA was tested. Growth of yeast on selective medium is shown. (d) Interaction of Ret9 and Shank3 in mammalian cells. HEK293 cells were cotransfected with the indicated Ret mutants and Flag-tagged Shank3–PDZ. Coimmunoprecipitation with Flag–M2 Sepharose was performed followed by SDS-PAGE and Western blotting with the indicated antibodies.
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fig1: Ret9 interacts with the Shank3 PDZ domain. (a) Schematic representation of Ret9, Ret51, and of COOH-terminal mutants. Cad, cadherin domain; Tm, transmembrane domain; Tk, tyrosine kinase domain; Ret9 Δ4, deletion of the PDZ-binding motif; Ret9 FA point mutation in the PDZ-binding motif; Ret51-9, Ret9 PDZ-binding motif attached to Ret51. (b) Domain structure of Shank3 and Shank3 deletion mutants. Ank, ankyrin repeats; SH3, src homology 3 domain; Pro, proline-rich region; SAM, sterile alpha motif. (c) Interaction of Ret9 and Shank3 in a yeast two-hybrid analysis. Binding of the GAL4-binding domain–Shank3 PDZ domain fusion protein (or of the GAL4-binding domain alone) to GAL4-activation domain fusion proteins of the cytoplasmic domains of Ret9, Ret51, Ret9 Δ4, and Ret9 FA was tested. Growth of yeast on selective medium is shown. (d) Interaction of Ret9 and Shank3 in mammalian cells. HEK293 cells were cotransfected with the indicated Ret mutants and Flag-tagged Shank3–PDZ. Coimmunoprecipitation with Flag–M2 Sepharose was performed followed by SDS-PAGE and Western blotting with the indicated antibodies.

Mentions: The protooncogene Ret is expressed in two splice variants, Ret9 and Ret51, which differ only in carboxyl-terminal residues. 9 additional amino acids are present in Ret9; 51 entirely different amino acids in Ret51 (Fig. 1 a). To identify signaling effectors downstream of the functionally essential Ret9 isoform, we performed yeast two-hybrid screens with the entire cytoplasmic domain of Ret9 as a bait against a mouse embryonic cDNA library (Weidner et al., 1996). From these screens, we isolated cDNA clones encoding the PDZ domain of Shank3, e.g., amino acids 588–741 (Fig. 1 b). Shank proteins are scaffolding adaptors, which contain several domains such as ankyrin repeats, SH3, PDZ, and SAM domains, and multiple proline-rich regions (Boeckers et al., 1999; Naisbitt et al., 1999; Tu et al., 1999; Yao et al., 1999). The Shank3 PDZ domain binds exclusively to Ret9 and not to Ret51, as shown by yeast two-hybrid analysis (Fig. 1 c). Upon sequence analysis, we observed that Ret9, but not Ret51, harbors a putative PDZ-binding motif (FTRF) at the very COOH terminus (Fig. 1 a). Deletion of this motif (Ret9 Δ4) or mutation of the crucial COOH-terminal phenylalanine to alanine (Ret9 FA; Borg et al., 2000) prevents interaction with Shank3 (Fig. 1, a and c), demonstrating that Shank3 interacts with Ret9 through the newly identified PDZ-binding motif.


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 the Shank3 PDZ domain. (a) Schematic representation of Ret9, Ret51, and of COOH-terminal mutants. Cad, cadherin domain; Tm, transmembrane domain; Tk, tyrosine kinase domain; Ret9 Δ4, deletion of the PDZ-binding motif; Ret9 FA point mutation in the PDZ-binding motif; Ret51-9, Ret9 PDZ-binding motif attached to Ret51. (b) Domain structure of Shank3 and Shank3 deletion mutants. Ank, ankyrin repeats; SH3, src homology 3 domain; Pro, proline-rich region; SAM, sterile alpha motif. (c) Interaction of Ret9 and Shank3 in a yeast two-hybrid analysis. Binding of the GAL4-binding domain–Shank3 PDZ domain fusion protein (or of the GAL4-binding domain alone) to GAL4-activation domain fusion proteins of the cytoplasmic domains of Ret9, Ret51, Ret9 Δ4, and Ret9 FA was tested. Growth of yeast on selective medium is shown. (d) Interaction of Ret9 and Shank3 in mammalian cells. HEK293 cells were cotransfected with the indicated Ret mutants and Flag-tagged Shank3–PDZ. Coimmunoprecipitation with Flag–M2 Sepharose was performed followed by SDS-PAGE and Western blotting with the indicated antibodies.
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Related In: Results  -  Collection

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fig1: Ret9 interacts with the Shank3 PDZ domain. (a) Schematic representation of Ret9, Ret51, and of COOH-terminal mutants. Cad, cadherin domain; Tm, transmembrane domain; Tk, tyrosine kinase domain; Ret9 Δ4, deletion of the PDZ-binding motif; Ret9 FA point mutation in the PDZ-binding motif; Ret51-9, Ret9 PDZ-binding motif attached to Ret51. (b) Domain structure of Shank3 and Shank3 deletion mutants. Ank, ankyrin repeats; SH3, src homology 3 domain; Pro, proline-rich region; SAM, sterile alpha motif. (c) Interaction of Ret9 and Shank3 in a yeast two-hybrid analysis. Binding of the GAL4-binding domain–Shank3 PDZ domain fusion protein (or of the GAL4-binding domain alone) to GAL4-activation domain fusion proteins of the cytoplasmic domains of Ret9, Ret51, Ret9 Δ4, and Ret9 FA was tested. Growth of yeast on selective medium is shown. (d) Interaction of Ret9 and Shank3 in mammalian cells. HEK293 cells were cotransfected with the indicated Ret mutants and Flag-tagged Shank3–PDZ. Coimmunoprecipitation with Flag–M2 Sepharose was performed followed by SDS-PAGE and Western blotting with the indicated antibodies.
Mentions: The protooncogene Ret is expressed in two splice variants, Ret9 and Ret51, which differ only in carboxyl-terminal residues. 9 additional amino acids are present in Ret9; 51 entirely different amino acids in Ret51 (Fig. 1 a). To identify signaling effectors downstream of the functionally essential Ret9 isoform, we performed yeast two-hybrid screens with the entire cytoplasmic domain of Ret9 as a bait against a mouse embryonic cDNA library (Weidner et al., 1996). From these screens, we isolated cDNA clones encoding the PDZ domain of Shank3, e.g., amino acids 588–741 (Fig. 1 b). Shank proteins are scaffolding adaptors, which contain several domains such as ankyrin repeats, SH3, PDZ, and SAM domains, and multiple proline-rich regions (Boeckers et al., 1999; Naisbitt et al., 1999; Tu et al., 1999; Yao et al., 1999). The Shank3 PDZ domain binds exclusively to Ret9 and not to Ret51, as shown by yeast two-hybrid analysis (Fig. 1 c). Upon sequence analysis, we observed that Ret9, but not Ret51, harbors a putative PDZ-binding motif (FTRF) at the very COOH terminus (Fig. 1 a). Deletion of this motif (Ret9 Δ4) or mutation of the crucial COOH-terminal phenylalanine to alanine (Ret9 FA; Borg et al., 2000) prevents interaction with Shank3 (Fig. 1, a and c), demonstrating that Shank3 interacts with Ret9 through the newly identified PDZ-binding motif.

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