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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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PDZ class-switch experiment. (a) Schematic representation of the PDZ class-switch mutants in Ret9 and Shank3. (b) Coimmunoprecipitation of Ret9 and Shank3 and their mutants. Flag-tagged Shank3–PDZ and Myc-tagged Ret9 were coexpressed in HEK293 cells, and Shank3–PDZ was precipitated using Flag–M2 Sepharose followed by SDS-PAGE and Western blotting with anti-Myc or anti-Flag tag antibodies. The Ret PDZ-binding class and the Shank3 PDZ domain class are indicated.
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fig2: PDZ class-switch experiment. (a) Schematic representation of the PDZ class-switch mutants in Ret9 and Shank3. (b) Coimmunoprecipitation of Ret9 and Shank3 and their mutants. Flag-tagged Shank3–PDZ and Myc-tagged Ret9 were coexpressed in HEK293 cells, and Shank3–PDZ was precipitated using Flag–M2 Sepharose followed by SDS-PAGE and Western blotting with anti-Myc or anti-Flag tag antibodies. The Ret PDZ-binding class and the Shank3 PDZ domain class are indicated.

Mentions: To confirm that the Shank–Ret9 interaction is direct, we performed a PDZ class switch experiment (Kaech et al., 1998) by introducing compensatory mutations in Shank3 and Ret9. PDZ domains can be subdivided by their ability to bind to different receptor tails (Songyang et al., 1997; Vaccaro and Dente, 2002). Shank3 harbors a type I PDZ domain characterized by the presence of a histidine residue at position 716 (Fig. 2 a). Ret9 contains a type I PDZ-binding motif characterized by a threonine residue at position 1070. Mutation of the critical histidine residue to valine converts the Shank3 PDZ domain to a class II domain (Shank3–PDZ HV), which no longer binds to Ret9 (Fig. 2 b). Introduction of a compensatory mutation in Ret9 (threonine 1070 to tyrosine, Ret9 TY) converts the PDZ-binding motif into a type II–binding motif and reconstitutes the Shank3–Ret9 interaction (Fig. 2 b). These data demonstrate that Ret9 and Shank3 interact in a PDZ domain–mediated fashion, and that this interaction is direct.


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)

PDZ class-switch experiment. (a) Schematic representation of the PDZ class-switch mutants in Ret9 and Shank3. (b) Coimmunoprecipitation of Ret9 and Shank3 and their mutants. Flag-tagged Shank3–PDZ and Myc-tagged Ret9 were coexpressed in HEK293 cells, and Shank3–PDZ was precipitated using Flag–M2 Sepharose followed by SDS-PAGE and Western blotting with anti-Myc or anti-Flag tag antibodies. The Ret PDZ-binding class and the Shank3 PDZ domain class are indicated.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: PDZ class-switch experiment. (a) Schematic representation of the PDZ class-switch mutants in Ret9 and Shank3. (b) Coimmunoprecipitation of Ret9 and Shank3 and their mutants. Flag-tagged Shank3–PDZ and Myc-tagged Ret9 were coexpressed in HEK293 cells, and Shank3–PDZ was precipitated using Flag–M2 Sepharose followed by SDS-PAGE and Western blotting with anti-Myc or anti-Flag tag antibodies. The Ret PDZ-binding class and the Shank3 PDZ domain class are indicated.
Mentions: To confirm that the Shank–Ret9 interaction is direct, we performed a PDZ class switch experiment (Kaech et al., 1998) by introducing compensatory mutations in Shank3 and Ret9. PDZ domains can be subdivided by their ability to bind to different receptor tails (Songyang et al., 1997; Vaccaro and Dente, 2002). Shank3 harbors a type I PDZ domain characterized by the presence of a histidine residue at position 716 (Fig. 2 a). Ret9 contains a type I PDZ-binding motif characterized by a threonine residue at position 1070. Mutation of the critical histidine residue to valine converts the Shank3 PDZ domain to a class II domain (Shank3–PDZ HV), which no longer binds to Ret9 (Fig. 2 b). Introduction of a compensatory mutation in Ret9 (threonine 1070 to tyrosine, Ret9 TY) converts the PDZ-binding motif into a type II–binding motif and reconstitutes the Shank3–Ret9 interaction (Fig. 2 b). These data demonstrate that Ret9 and Shank3 interact in a PDZ domain–mediated fashion, and that this interaction is direct.

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