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

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.

Figure 1.

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.

Figure 2.

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.

Figure 3.

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.

Figure 4.

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.

Figure 5.

The Shank3 proline-rich domain is crucial for Ret-induced tubule formation. MDCK cell lines expressing fusion proteins of the full-length Ret9 receptor with either the NH₂ terminus of Shank3 (Ret–Shank3 NT; a–c) or the proline-rich domain of Shank3 (Ret9–Shank3–Pro1; 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.

Figure 6.

The Ret9 PDZ-binding motif mediates sustained Erk–MAPK and PI3K signaling. MDCK cells, expressing Ret9 (a and e), Ret9 FA (b and f), Ret51 (c and g), or control cells without Ret (d and h) were stimulated with GDNF and sGFRα1 and examined for Erk–MAPK and Akt phosphorylation by SDS-PAGE. Phosphorylated Erk1/2 (P-Erk1/2) or Akt (P-Akt) proteins, total Erk2 and total Akt were detected by Western blotting with specific antibodies. The specific bands for phosphorylated and unphosphorylated proteins are indicated by arrows. Western blots were quantified and activation levels over time are shown. Error bars show standard deviation from three independent experiments.

Figure 7.

Shank3 interacts with Grb2. (a) Schematic representation of the used Ret9 and Shank3 mutants. The consensus Grb2-binding motif in Shank3 is underlined. (b) Interaction of Shank3 with endogenous Grb2. HEK293 cells were cotransfected with Flag-tagged Shank3–Pro2 or Shank3–Pro2 containing the Y1006F mutant and Ret9, Ret9FA, or Ret9Y1062F. Immunoprecipitation of Shank3 using Flag–M2 Sepharose beads was performed followed by SDS-PAGE and Western blotting. Western blots of immunoprecipitations and total lysates were developed with antibodies against Grb2, Flag, phospho-tyrosine (PY), and Ret9 as indicated.

Figure 8.

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.