Limits...
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.

Show MeSH

Related in: MedlinePlus

Determination of the activated Cdc42-binding region of Cupidin and generation of deletion mutants lacking binding activities. (A-D) Top, the domain structure of Cupidinα/Homer2a (CPDα) and its mutants used in the ligand overlay assay. The Cdc42 binding activities are summarized on the right, presented as normalized values corresponding to CPD (A) or CPD191–283 (B, C, and D) set to 1.00. Asterisks indicate values over 0.70, which was arbitrarily considered to represent positive values. CPDα is composed of 343 amino-acid residues. The EVH1 domain in the N-terminal 110 residues, the coiled-coil domain (CC) predicted within the amino acid stretch 173–317, and two Leu zipper motifs A (LZA, 238–296 aa) and B (LZB, 311–339 aa) are indicated. The deletion constructs consist of the regions shown by solid thick bars; Middle, autoradiograms resulting from the [35S]-GTPγS-bound Cdc42 ligand overlay assay; Bottom, Coomassie Brilliant Blue (CBB)-stained gel corresponding to the ligand overlay assay, in which all GST-fused CPD-relating proteins migrated as top bands by SDS-PAGE. (A) First trial to determine the Cdc42-binding region using rough deletion of CPD C. (B) Second trial to narrow down the Cdc42-binding region by making mutants of CPD 191–283. (C) Third trial to narrow down the Cdc42-binding region by using shortened fragments of CPD 191–283. (D) Final trial to determine the deletion mutants of Cupidin lacking Cdc42-binding ability.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2666743&req=5

Figure 1: Determination of the activated Cdc42-binding region of Cupidin and generation of deletion mutants lacking binding activities. (A-D) Top, the domain structure of Cupidinα/Homer2a (CPDα) and its mutants used in the ligand overlay assay. The Cdc42 binding activities are summarized on the right, presented as normalized values corresponding to CPD (A) or CPD191–283 (B, C, and D) set to 1.00. Asterisks indicate values over 0.70, which was arbitrarily considered to represent positive values. CPDα is composed of 343 amino-acid residues. The EVH1 domain in the N-terminal 110 residues, the coiled-coil domain (CC) predicted within the amino acid stretch 173–317, and two Leu zipper motifs A (LZA, 238–296 aa) and B (LZB, 311–339 aa) are indicated. The deletion constructs consist of the regions shown by solid thick bars; Middle, autoradiograms resulting from the [35S]-GTPγS-bound Cdc42 ligand overlay assay; Bottom, Coomassie Brilliant Blue (CBB)-stained gel corresponding to the ligand overlay assay, in which all GST-fused CPD-relating proteins migrated as top bands by SDS-PAGE. (A) First trial to determine the Cdc42-binding region using rough deletion of CPD C. (B) Second trial to narrow down the Cdc42-binding region by making mutants of CPD 191–283. (C) Third trial to narrow down the Cdc42-binding region by using shortened fragments of CPD 191–283. (D) Final trial to determine the deletion mutants of Cupidin lacking Cdc42-binding ability.

Mentions: Cupidin/Homer2 is comprised of an N-terminal EVH1 domain, a C-terminal coiled-coil (CC) motif and two Leu zipper motifs A and B (LZA and LZB, respectively) (Fig. 1). Our previous study indicated that Cupidin interacts with Cdc42 small GTPase in a GTP-dependent manner via the C-terminal region [6]. To narrow down the region responsible for Cdc42 binding activity, we generated a series of glutathione S-transferase (GST) fusion constructs containing various regions of Cupidin (CPD), as shown in Fig. 1. These GST fusion proteins were blotted onto membrane filters and probed by a ligand overlay assay with GST-fused Cdc42 in the presence of [35S]-GTPγS. As described previously [6], GST-CPD and GST-CPD C (C-terminal amino acids (aa) 111–343), but not GST-CPD N (N-terminal aa 1–110), were radioactively labeled (Fig. 1A). No specific radioactive labeling of GST-CPD C was observed in the presence of [35S]-GDPβS and Cdc42, as previously reported [6].


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)

Determination of the activated Cdc42-binding region of Cupidin and generation of deletion mutants lacking binding activities. (A-D) Top, the domain structure of Cupidinα/Homer2a (CPDα) and its mutants used in the ligand overlay assay. The Cdc42 binding activities are summarized on the right, presented as normalized values corresponding to CPD (A) or CPD191–283 (B, C, and D) set to 1.00. Asterisks indicate values over 0.70, which was arbitrarily considered to represent positive values. CPDα is composed of 343 amino-acid residues. The EVH1 domain in the N-terminal 110 residues, the coiled-coil domain (CC) predicted within the amino acid stretch 173–317, and two Leu zipper motifs A (LZA, 238–296 aa) and B (LZB, 311–339 aa) are indicated. The deletion constructs consist of the regions shown by solid thick bars; Middle, autoradiograms resulting from the [35S]-GTPγS-bound Cdc42 ligand overlay assay; Bottom, Coomassie Brilliant Blue (CBB)-stained gel corresponding to the ligand overlay assay, in which all GST-fused CPD-relating proteins migrated as top bands by SDS-PAGE. (A) First trial to determine the Cdc42-binding region using rough deletion of CPD C. (B) Second trial to narrow down the Cdc42-binding region by making mutants of CPD 191–283. (C) Third trial to narrow down the Cdc42-binding region by using shortened fragments of CPD 191–283. (D) Final trial to determine the deletion mutants of Cupidin lacking Cdc42-binding ability.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666743&req=5

Figure 1: Determination of the activated Cdc42-binding region of Cupidin and generation of deletion mutants lacking binding activities. (A-D) Top, the domain structure of Cupidinα/Homer2a (CPDα) and its mutants used in the ligand overlay assay. The Cdc42 binding activities are summarized on the right, presented as normalized values corresponding to CPD (A) or CPD191–283 (B, C, and D) set to 1.00. Asterisks indicate values over 0.70, which was arbitrarily considered to represent positive values. CPDα is composed of 343 amino-acid residues. The EVH1 domain in the N-terminal 110 residues, the coiled-coil domain (CC) predicted within the amino acid stretch 173–317, and two Leu zipper motifs A (LZA, 238–296 aa) and B (LZB, 311–339 aa) are indicated. The deletion constructs consist of the regions shown by solid thick bars; Middle, autoradiograms resulting from the [35S]-GTPγS-bound Cdc42 ligand overlay assay; Bottom, Coomassie Brilliant Blue (CBB)-stained gel corresponding to the ligand overlay assay, in which all GST-fused CPD-relating proteins migrated as top bands by SDS-PAGE. (A) First trial to determine the Cdc42-binding region using rough deletion of CPD C. (B) Second trial to narrow down the Cdc42-binding region by making mutants of CPD 191–283. (C) Third trial to narrow down the Cdc42-binding region by using shortened fragments of CPD 191–283. (D) Final trial to determine the deletion mutants of Cupidin lacking Cdc42-binding ability.
Mentions: Cupidin/Homer2 is comprised of an N-terminal EVH1 domain, a C-terminal coiled-coil (CC) motif and two Leu zipper motifs A and B (LZA and LZB, respectively) (Fig. 1). Our previous study indicated that Cupidin interacts with Cdc42 small GTPase in a GTP-dependent manner via the C-terminal region [6]. To narrow down the region responsible for Cdc42 binding activity, we generated a series of glutathione S-transferase (GST) fusion constructs containing various regions of Cupidin (CPD), as shown in Fig. 1. These GST fusion proteins were blotted onto membrane filters and probed by a ligand overlay assay with GST-fused Cdc42 in the presence of [35S]-GTPγS. As described previously [6], GST-CPD and GST-CPD C (C-terminal amino acids (aa) 111–343), but not GST-CPD N (N-terminal aa 1–110), were radioactively labeled (Fig. 1A). No specific radioactive labeling of GST-CPD C was observed in the presence of [35S]-GDPβS and Cdc42, as previously reported [6].

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