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Interaction of Cupidin/Homer2 with two actin cytoskeletal regulators, Cdc42 small GTPase and Drebrin, in dendritic spines.

Shiraishi-Yamaguchi Y, Sato Y, Sakai R, Mizutani A, Knöpfel T, Mori N, Mikoshiba K, Furuichi T - BMC Neurosci (2009)

Bottom Line: Overexpression of CPDDelta191-230 decreased the number of synapses and reduced the amplitudes of miniature excitatory postsynaptic currents in hippocampal neurons.CPDDelta191-230 overexpression decreased Drebrin clustering in the dendritic spines of hippocampal neurons.These results indicate that Cupidin/Homer2 interacts with the dendritic spine actin regulators Cdc42 and Drebrin via its C-terminal and N-terminal domains, respectively, and that it may be involved in spine morphology and synaptic properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan. f1966@cc.nagasaki-u.ac.jp

ABSTRACT

Background: Homer is a postsynaptic scaffold protein that links various synaptic signaling proteins, including the type I metabotropic glutamate receptor subunits 1alpha and 5, the inositol 1,4,5-trisphosphate receptor, Shank and Cdc42 small GTPase. Overexpression of Homer induces changes in dendritic spine morphology in cultured hippocampal neurons. However, the molecular basis underpinning Homer-mediated spine morphogenesis remains unclear. In this study, we aimed to elucidate the structural and functional properties of the interaction between Cupidin/Homer2 and two actin-cytoskeletal regulators, Cdc42 small GTPase and Drebrin.

Results: Cupidin/Homer2 interacted with activated Cdc42 small GTPase via the Cdc42-binding domain that resides around amino acid residues 191-283, within the C-terminal coiled-coil domain. We generated a Cupidin deletion mutant lacking amino acids 191-230 (CPDDelta191-230), which showed decrease Cdc42-binding ability but maintained self-multimerization ability. Cupidin suppressed Cdc42-induced filopodia-like protrusion formation in HeLa cells, whereas CPDDelta191-230 failed to do so. In cultured hippocampal neurons, Cupidin was targeted to dendritic spines, whereas CPDDelta191-230 was distributed in dendritic shafts as well as spines. Overexpression of CPDDelta191-230 decreased the number of synapses and reduced the amplitudes of miniature excitatory postsynaptic currents in hippocampal neurons. Cupidin interacted with a dendritic spine F-actin-binding protein, Drebrin, which possesses two Homer ligand motifs, via the N-terminal EVH-1 domain. CPDDelta191-230 overexpression decreased Drebrin clustering in the dendritic spines of hippocampal neurons.

Conclusion: These results indicate that Cupidin/Homer2 interacts with the dendritic spine actin regulators Cdc42 and Drebrin via its C-terminal and N-terminal domains, respectively, and that it may be involved in spine morphology and synaptic properties.

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Cupidin-Drebrin interaction and colocalization in the spines of hippocampal neurons, and its inhibition by expressing a mutant with a deletion of the Cupidin Cdc42-binding domain. (A) Mouse Drebrin contains an actin depolymerization factor/cofilin-like domain (ADF) in the N-terminal region and two putative PPxxF Homer-binding motifs (592-PPPPATF-596 and 674-PPPVF-678) in the C-terminal region. Both Drebrin-A and Drebrin-E were co-immunoprecipitated with anti-Homer1, 2 and 3 antibodies (Homer-mix) and an anti-pan Homer antibody that recognizes all three Homer family members (right panel). Immunoprecipitates with control rabbit IgG showed no Drebrin bands. (B) The GST-fused mouse Drebrin A C-terminal region (Drebrin-C WT, aa 579-706 containing two Homer ligand motifs 592-PPATF-596 and 674-PPPVF-678) and its double mutant (Drebrin-C mutant with P593A and P675A) were bacterially expressed, electrophoresed and transferred to a nitrocellulose membrane followed by an overlay assay with GST-CPD protein extracts (see Methods). Drebrin-Cupidin binding was proven by Western blot analysis using anti-Cupidin (CPD C) antibody. Top panel, CCB stain; bottom panel, Western blot. (C) Immuno-fluorescent staining of endogenous Cupidin (green) and Drebrin (red) proteins colocalized in the dendritic spines of primary cultured rat hippocampal neurons. Bar: 5 μm. (D) GFP fluorescence (green) and Drebrin-immuno-fluorescence (red) images in the dendrites of hippocampal neurons overexpressing GFP, GFP-CPD or GFP-CPDΔ191–230 (= GFP-Δ191–230) by recombinant adenovirus infection. Bar: 5 μm. (E) The relative puncta number (top graph) and size (bottom graph) of Drebrin-positive puncta along dendrites overexpressing either GFP-CPD or GFP-CPDΔ191–230 (= GFP-Δ191–230) were quantified and normalized to those of dendrites overexpressing GFP alone, which was set to 1.0. *, p < 0.01.
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Figure 6: Cupidin-Drebrin interaction and colocalization in the spines of hippocampal neurons, and its inhibition by expressing a mutant with a deletion of the Cupidin Cdc42-binding domain. (A) Mouse Drebrin contains an actin depolymerization factor/cofilin-like domain (ADF) in the N-terminal region and two putative PPxxF Homer-binding motifs (592-PPPPATF-596 and 674-PPPVF-678) in the C-terminal region. Both Drebrin-A and Drebrin-E were co-immunoprecipitated with anti-Homer1, 2 and 3 antibodies (Homer-mix) and an anti-pan Homer antibody that recognizes all three Homer family members (right panel). Immunoprecipitates with control rabbit IgG showed no Drebrin bands. (B) The GST-fused mouse Drebrin A C-terminal region (Drebrin-C WT, aa 579-706 containing two Homer ligand motifs 592-PPATF-596 and 674-PPPVF-678) and its double mutant (Drebrin-C mutant with P593A and P675A) were bacterially expressed, electrophoresed and transferred to a nitrocellulose membrane followed by an overlay assay with GST-CPD protein extracts (see Methods). Drebrin-Cupidin binding was proven by Western blot analysis using anti-Cupidin (CPD C) antibody. Top panel, CCB stain; bottom panel, Western blot. (C) Immuno-fluorescent staining of endogenous Cupidin (green) and Drebrin (red) proteins colocalized in the dendritic spines of primary cultured rat hippocampal neurons. Bar: 5 μm. (D) GFP fluorescence (green) and Drebrin-immuno-fluorescence (red) images in the dendrites of hippocampal neurons overexpressing GFP, GFP-CPD or GFP-CPDΔ191–230 (= GFP-Δ191–230) by recombinant adenovirus infection. Bar: 5 μm. (E) The relative puncta number (top graph) and size (bottom graph) of Drebrin-positive puncta along dendrites overexpressing either GFP-CPD or GFP-CPDΔ191–230 (= GFP-Δ191–230) were quantified and normalized to those of dendrites overexpressing GFP alone, which was set to 1.0. *, p < 0.01.

Mentions: A homology search with the Homer ligand PPxxF motif identified two homologous sites (aa 592–596 and 674–678) in the C-terminal region of mouse Drebrin (Fig. 6A). Coimmunoprecipitation with anti-Homer antibodies (either anti-Homer 1, 2 and 3 antibody mixture or anti-pan-Homer antibody) revealed an association between Homer proteins and Drebrin proteins (two splicing variants A and E) in P8 mouse brains (Fig. 6A). Since the anti-Drebrin monoclonal antibody used recognizes the C-terminal region near the PPxxF motifs, the anti-Drebrin antibody might interfere with the Cupidin-Drebrin interaction in a reverse coimmunoprecipitation of Cupidin with anti-Drebrin antibody. Thus, we carried out an overlay assay in which Cupidin was tested for binding to a polypeptide containing two Drebrin PPxxF motifs (aa 579–706 of mouse Drebrin A) or its double mutant (P593A and P675A) blotted onto a membrane filter (Fig. 6B). Cupidin bound to the PPxxF-containing polypeptide, but not to the mutant polypeptide, again indicating that Cupidin interacts with the C-terminal PPxxF motifs of Drebrin.


Interaction of Cupidin/Homer2 with two actin cytoskeletal regulators, Cdc42 small GTPase and Drebrin, in dendritic spines.

Shiraishi-Yamaguchi Y, Sato Y, Sakai R, Mizutani A, Knöpfel T, Mori N, Mikoshiba K, Furuichi T - BMC Neurosci (2009)

Cupidin-Drebrin interaction and colocalization in the spines of hippocampal neurons, and its inhibition by expressing a mutant with a deletion of the Cupidin Cdc42-binding domain. (A) Mouse Drebrin contains an actin depolymerization factor/cofilin-like domain (ADF) in the N-terminal region and two putative PPxxF Homer-binding motifs (592-PPPPATF-596 and 674-PPPVF-678) in the C-terminal region. Both Drebrin-A and Drebrin-E were co-immunoprecipitated with anti-Homer1, 2 and 3 antibodies (Homer-mix) and an anti-pan Homer antibody that recognizes all three Homer family members (right panel). Immunoprecipitates with control rabbit IgG showed no Drebrin bands. (B) The GST-fused mouse Drebrin A C-terminal region (Drebrin-C WT, aa 579-706 containing two Homer ligand motifs 592-PPATF-596 and 674-PPPVF-678) and its double mutant (Drebrin-C mutant with P593A and P675A) were bacterially expressed, electrophoresed and transferred to a nitrocellulose membrane followed by an overlay assay with GST-CPD protein extracts (see Methods). Drebrin-Cupidin binding was proven by Western blot analysis using anti-Cupidin (CPD C) antibody. Top panel, CCB stain; bottom panel, Western blot. (C) Immuno-fluorescent staining of endogenous Cupidin (green) and Drebrin (red) proteins colocalized in the dendritic spines of primary cultured rat hippocampal neurons. Bar: 5 μm. (D) GFP fluorescence (green) and Drebrin-immuno-fluorescence (red) images in the dendrites of hippocampal neurons overexpressing GFP, GFP-CPD or GFP-CPDΔ191–230 (= GFP-Δ191–230) by recombinant adenovirus infection. Bar: 5 μm. (E) The relative puncta number (top graph) and size (bottom graph) of Drebrin-positive puncta along dendrites overexpressing either GFP-CPD or GFP-CPDΔ191–230 (= GFP-Δ191–230) were quantified and normalized to those of dendrites overexpressing GFP alone, which was set to 1.0. *, p < 0.01.
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Figure 6: Cupidin-Drebrin interaction and colocalization in the spines of hippocampal neurons, and its inhibition by expressing a mutant with a deletion of the Cupidin Cdc42-binding domain. (A) Mouse Drebrin contains an actin depolymerization factor/cofilin-like domain (ADF) in the N-terminal region and two putative PPxxF Homer-binding motifs (592-PPPPATF-596 and 674-PPPVF-678) in the C-terminal region. Both Drebrin-A and Drebrin-E were co-immunoprecipitated with anti-Homer1, 2 and 3 antibodies (Homer-mix) and an anti-pan Homer antibody that recognizes all three Homer family members (right panel). Immunoprecipitates with control rabbit IgG showed no Drebrin bands. (B) The GST-fused mouse Drebrin A C-terminal region (Drebrin-C WT, aa 579-706 containing two Homer ligand motifs 592-PPATF-596 and 674-PPPVF-678) and its double mutant (Drebrin-C mutant with P593A and P675A) were bacterially expressed, electrophoresed and transferred to a nitrocellulose membrane followed by an overlay assay with GST-CPD protein extracts (see Methods). Drebrin-Cupidin binding was proven by Western blot analysis using anti-Cupidin (CPD C) antibody. Top panel, CCB stain; bottom panel, Western blot. (C) Immuno-fluorescent staining of endogenous Cupidin (green) and Drebrin (red) proteins colocalized in the dendritic spines of primary cultured rat hippocampal neurons. Bar: 5 μm. (D) GFP fluorescence (green) and Drebrin-immuno-fluorescence (red) images in the dendrites of hippocampal neurons overexpressing GFP, GFP-CPD or GFP-CPDΔ191–230 (= GFP-Δ191–230) by recombinant adenovirus infection. Bar: 5 μm. (E) The relative puncta number (top graph) and size (bottom graph) of Drebrin-positive puncta along dendrites overexpressing either GFP-CPD or GFP-CPDΔ191–230 (= GFP-Δ191–230) were quantified and normalized to those of dendrites overexpressing GFP alone, which was set to 1.0. *, p < 0.01.
Mentions: A homology search with the Homer ligand PPxxF motif identified two homologous sites (aa 592–596 and 674–678) in the C-terminal region of mouse Drebrin (Fig. 6A). Coimmunoprecipitation with anti-Homer antibodies (either anti-Homer 1, 2 and 3 antibody mixture or anti-pan-Homer antibody) revealed an association between Homer proteins and Drebrin proteins (two splicing variants A and E) in P8 mouse brains (Fig. 6A). Since the anti-Drebrin monoclonal antibody used recognizes the C-terminal region near the PPxxF motifs, the anti-Drebrin antibody might interfere with the Cupidin-Drebrin interaction in a reverse coimmunoprecipitation of Cupidin with anti-Drebrin antibody. Thus, we carried out an overlay assay in which Cupidin was tested for binding to a polypeptide containing two Drebrin PPxxF motifs (aa 579–706 of mouse Drebrin A) or its double mutant (P593A and P675A) blotted onto a membrane filter (Fig. 6B). Cupidin bound to the PPxxF-containing polypeptide, but not to the mutant polypeptide, again indicating that Cupidin interacts with the C-terminal PPxxF motifs of Drebrin.

Bottom Line: Overexpression of CPDDelta191-230 decreased the number of synapses and reduced the amplitudes of miniature excitatory postsynaptic currents in hippocampal neurons.CPDDelta191-230 overexpression decreased Drebrin clustering in the dendritic spines of hippocampal neurons.These results indicate that Cupidin/Homer2 interacts with the dendritic spine actin regulators Cdc42 and Drebrin via its C-terminal and N-terminal domains, respectively, and that it may be involved in spine morphology and synaptic properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan. f1966@cc.nagasaki-u.ac.jp

ABSTRACT

Background: Homer is a postsynaptic scaffold protein that links various synaptic signaling proteins, including the type I metabotropic glutamate receptor subunits 1alpha and 5, the inositol 1,4,5-trisphosphate receptor, Shank and Cdc42 small GTPase. Overexpression of Homer induces changes in dendritic spine morphology in cultured hippocampal neurons. However, the molecular basis underpinning Homer-mediated spine morphogenesis remains unclear. In this study, we aimed to elucidate the structural and functional properties of the interaction between Cupidin/Homer2 and two actin-cytoskeletal regulators, Cdc42 small GTPase and Drebrin.

Results: Cupidin/Homer2 interacted with activated Cdc42 small GTPase via the Cdc42-binding domain that resides around amino acid residues 191-283, within the C-terminal coiled-coil domain. We generated a Cupidin deletion mutant lacking amino acids 191-230 (CPDDelta191-230), which showed decrease Cdc42-binding ability but maintained self-multimerization ability. Cupidin suppressed Cdc42-induced filopodia-like protrusion formation in HeLa cells, whereas CPDDelta191-230 failed to do so. In cultured hippocampal neurons, Cupidin was targeted to dendritic spines, whereas CPDDelta191-230 was distributed in dendritic shafts as well as spines. Overexpression of CPDDelta191-230 decreased the number of synapses and reduced the amplitudes of miniature excitatory postsynaptic currents in hippocampal neurons. Cupidin interacted with a dendritic spine F-actin-binding protein, Drebrin, which possesses two Homer ligand motifs, via the N-terminal EVH-1 domain. CPDDelta191-230 overexpression decreased Drebrin clustering in the dendritic spines of hippocampal neurons.

Conclusion: These results indicate that Cupidin/Homer2 interacts with the dendritic spine actin regulators Cdc42 and Drebrin via its C-terminal and N-terminal domains, respectively, and that it may be involved in spine morphology and synaptic properties.

Show MeSH
Related in: MedlinePlus