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Synbindin, A novel syndecan-2-binding protein in neuronal dendritic spines.

Ethell IM, Hagihara K, Miura Y, Irie F, Yamaguchi Y - J. Cell Biol. (2000)

Bottom Line: This effect requires the COOH-terminal EFYA sequence of syndecan-2, suggesting that cytoplasmic molecules interacting with this sequence play a critical role in spine morphogenesis.Our results show that synbindin is a physiological syndecan-2 ligand on dendritic spines.We suggest that syndecan-2 induces spine formation by recruiting intracellular vesicles toward postsynaptic sites through the interaction with synbindin.

View Article: PubMed Central - PubMed

Affiliation: The Burnham Institute, La Jolla, California 92037, USA.

ABSTRACT
Dendritic spines are small protrusions on the surface of dendrites that receive the vast majority of excitatory synapses. We previously showed that the cell-surface heparan sulfate proteoglycan syndecan-2 induces spine formation upon transfection into hippocampal neurons. This effect requires the COOH-terminal EFYA sequence of syndecan-2, suggesting that cytoplasmic molecules interacting with this sequence play a critical role in spine morphogenesis. Here, we report a novel protein that binds to the EFYA motif of syndecan-2. This protein, named synbindin, is expressed by neurons in a pattern similar to that of syndecan-2, and colocalizes with syndecan-2 in the spines of cultured hippocampal neurons. In transfected hippocampal neurons, synbindin undergoes syndecan-2-dependent clustering. Synbindin is structurally related to yeast proteins known to be involved in vesicle transport. Immunoelectron microscopy localized synbindin on postsynaptic membranes and intracellular vesicles within dendrites, suggesting a role in postsynaptic membrane trafficking. Synbindin coimmunoprecipitates with syndecan-2 from synaptic membrane fractions. Our results show that synbindin is a physiological syndecan-2 ligand on dendritic spines. We suggest that syndecan-2 induces spine formation by recruiting intracellular vesicles toward postsynaptic sites through the interaction with synbindin.

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Characterization of the synbindin–syndecan-2 interaction by pull-down, coimmunoprecipitation, ligand overlay, and two-hybrid assays. (A) GST pull-down assay. Lysates from human 293 cells transfected with Myc-tagged syndecan-2 were pulled down with GST-synbindin fusion protein (a and b) or nonfused GST (c) and immunoblotted with anti–syndecan-2 mAb 6G12 (a) or anti-Myc polyclonal antibody A14 (b and c). In d, cell lysates were directly immunoblotted with anti–syndecan-2 mAb without pull-down. In each panel, three different lysates were tested: (lane 1) lysates from 293 cells transfected with a control vector; (lane 2) lysates from 293 cells transfected with Myc-tagged syndecan-2, which were undigested; and (lane 3) lysates from 293 cells transfected with Myc-tagged syndecan-2, which were digested with heparitinase. The arrowhead indicates the 34-kD syndecan-2 core protein. (B) Coimmunoprecipitation of synbindin and syndecan-2. The 293 cells were transfected with FLAG-syndecan-2 alone (lane 1), FLAG-syndecan-2 and Myc-synbindin (lane 2), or the FLAG-syndecan-2ΔEFYA deletion mutant and Myc-synbindin (lane 3). The cell lysates were immunoprecipitated with anti-Myc polyclonal antibody and immunoblotted with anti-FLAG antibody (c). The arrowhead indicates the syndecan-2 core protein. Note that the intact syndecan-2 was coimmunoprecipitated with synbindin (c, lane 2), whereas the syndecan-2ΔEFYA deletion mutant was not (c, lane 3). In a and b, the lysates were directly immunoblotted with anti-Myc antibody (a) and anti-FLAG antibody (b), respectively, to show that similar amounts of proteins were expressed. (C) GST-synbindin overlay assay. His-tagged recombinant proteins of intact syndecan-2 cytoplasmic domain (SDC2) and ΔEFYA cytoplasmic domain (SDC2ΔEFYA, right lane) were resolved on a 10–20% tricine gel, blotted, and overlaid with GST-synbindin (bottom panel) as described in Materials and Methods. (top panel) Ponceau S staining of the blot. (D) Two-hybrid assays to analyze the syndecan-2–binding site in synbindin. Four synbindin fragments (shown in the bottom panel) were tested with two syndecan-2 baits: one representing the intact syndecan-2 cytoplasmic domain (SDC2) and the other representing the ΔEFYA cytoplasmic domain (SDC2ΔEFYA). The interactions were scored by His prototrophy (left) and β-galactosidase activity (right).
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Figure 2: Characterization of the synbindin–syndecan-2 interaction by pull-down, coimmunoprecipitation, ligand overlay, and two-hybrid assays. (A) GST pull-down assay. Lysates from human 293 cells transfected with Myc-tagged syndecan-2 were pulled down with GST-synbindin fusion protein (a and b) or nonfused GST (c) and immunoblotted with anti–syndecan-2 mAb 6G12 (a) or anti-Myc polyclonal antibody A14 (b and c). In d, cell lysates were directly immunoblotted with anti–syndecan-2 mAb without pull-down. In each panel, three different lysates were tested: (lane 1) lysates from 293 cells transfected with a control vector; (lane 2) lysates from 293 cells transfected with Myc-tagged syndecan-2, which were undigested; and (lane 3) lysates from 293 cells transfected with Myc-tagged syndecan-2, which were digested with heparitinase. The arrowhead indicates the 34-kD syndecan-2 core protein. (B) Coimmunoprecipitation of synbindin and syndecan-2. The 293 cells were transfected with FLAG-syndecan-2 alone (lane 1), FLAG-syndecan-2 and Myc-synbindin (lane 2), or the FLAG-syndecan-2ΔEFYA deletion mutant and Myc-synbindin (lane 3). The cell lysates were immunoprecipitated with anti-Myc polyclonal antibody and immunoblotted with anti-FLAG antibody (c). The arrowhead indicates the syndecan-2 core protein. Note that the intact syndecan-2 was coimmunoprecipitated with synbindin (c, lane 2), whereas the syndecan-2ΔEFYA deletion mutant was not (c, lane 3). In a and b, the lysates were directly immunoblotted with anti-Myc antibody (a) and anti-FLAG antibody (b), respectively, to show that similar amounts of proteins were expressed. (C) GST-synbindin overlay assay. His-tagged recombinant proteins of intact syndecan-2 cytoplasmic domain (SDC2) and ΔEFYA cytoplasmic domain (SDC2ΔEFYA, right lane) were resolved on a 10–20% tricine gel, blotted, and overlaid with GST-synbindin (bottom panel) as described in Materials and Methods. (top panel) Ponceau S staining of the blot. (D) Two-hybrid assays to analyze the syndecan-2–binding site in synbindin. Four synbindin fragments (shown in the bottom panel) were tested with two syndecan-2 baits: one representing the intact syndecan-2 cytoplasmic domain (SDC2) and the other representing the ΔEFYA cytoplasmic domain (SDC2ΔEFYA). The interactions were scored by His prototrophy (left) and β-galactosidase activity (right).

Mentions: Yeast two-hybrid screens were performed using the L40 yeast strain, where the expression of both the reporter genes HIS3 and LacZ are driven by minimal GAL1 promoters fused to LexA-binding sites. A bait consisting of the entire cytoplasmic domain of the syndecan-2 fused in-frame to the LexA DNA–binding domain was constructed by PCR with the BTM116 vector. An embryonic mouse cDNA library constructed into the NotI site of pVP16 containing the Leu2 activation domain (Vojtek and Hollenberg 1995; a gift from Dr. Erkki Ruoslahti, The Burnham Institute) was screened with the syndecan-2 bait. Positive clones were selected by His prototrophy and assayed for β-galactosidase activity. Double-positive clones were isolated and characterized by sequencing. A double-positive clone (clone 28), which encodes a putative cytoplasmic protein with similarities to several PDZ domain–containing proteins (see Results), was further investigated as described in this paper. We named this protein synbindin. The specificity of the interaction between synbindin and the cytoplasmic domain of syndecan-2 was analyzed by two-hybrid assays (see Fig. 1 A). For this, we generated (by PCR) the following additional baits of the syndecan cytoplasmic domains: (1) a syndecan-2 deletion mutant lacking the COOH-terminal EFYA sequence (syndecan-2ΔEFYA); (2) a syndecan-4 deletion mutant lacking the COOH-terminal EFYA sequence (syndecan-4ΔEFYA); and (3) a bait with reverse sequence of syndecan-2 cytoplasmic domain. To determine the syndecan-2–binding site in synbindin, we generated by PCR four Leu2 fusion constructs representing the NH2-terminal half of synbindin (N-Sbd), the PDZ-related domain (P-Sbd), the COOH-terminal half (C-Sbd), and the PDZ-related domain plus the COOH-terminal half (P/C-Sbd) (see Fig. 2 D). Two-hybrid assays were performed as described above using HIS3 and LacZ as reporter genes.


Synbindin, A novel syndecan-2-binding protein in neuronal dendritic spines.

Ethell IM, Hagihara K, Miura Y, Irie F, Yamaguchi Y - J. Cell Biol. (2000)

Characterization of the synbindin–syndecan-2 interaction by pull-down, coimmunoprecipitation, ligand overlay, and two-hybrid assays. (A) GST pull-down assay. Lysates from human 293 cells transfected with Myc-tagged syndecan-2 were pulled down with GST-synbindin fusion protein (a and b) or nonfused GST (c) and immunoblotted with anti–syndecan-2 mAb 6G12 (a) or anti-Myc polyclonal antibody A14 (b and c). In d, cell lysates were directly immunoblotted with anti–syndecan-2 mAb without pull-down. In each panel, three different lysates were tested: (lane 1) lysates from 293 cells transfected with a control vector; (lane 2) lysates from 293 cells transfected with Myc-tagged syndecan-2, which were undigested; and (lane 3) lysates from 293 cells transfected with Myc-tagged syndecan-2, which were digested with heparitinase. The arrowhead indicates the 34-kD syndecan-2 core protein. (B) Coimmunoprecipitation of synbindin and syndecan-2. The 293 cells were transfected with FLAG-syndecan-2 alone (lane 1), FLAG-syndecan-2 and Myc-synbindin (lane 2), or the FLAG-syndecan-2ΔEFYA deletion mutant and Myc-synbindin (lane 3). The cell lysates were immunoprecipitated with anti-Myc polyclonal antibody and immunoblotted with anti-FLAG antibody (c). The arrowhead indicates the syndecan-2 core protein. Note that the intact syndecan-2 was coimmunoprecipitated with synbindin (c, lane 2), whereas the syndecan-2ΔEFYA deletion mutant was not (c, lane 3). In a and b, the lysates were directly immunoblotted with anti-Myc antibody (a) and anti-FLAG antibody (b), respectively, to show that similar amounts of proteins were expressed. (C) GST-synbindin overlay assay. His-tagged recombinant proteins of intact syndecan-2 cytoplasmic domain (SDC2) and ΔEFYA cytoplasmic domain (SDC2ΔEFYA, right lane) were resolved on a 10–20% tricine gel, blotted, and overlaid with GST-synbindin (bottom panel) as described in Materials and Methods. (top panel) Ponceau S staining of the blot. (D) Two-hybrid assays to analyze the syndecan-2–binding site in synbindin. Four synbindin fragments (shown in the bottom panel) were tested with two syndecan-2 baits: one representing the intact syndecan-2 cytoplasmic domain (SDC2) and the other representing the ΔEFYA cytoplasmic domain (SDC2ΔEFYA). The interactions were scored by His prototrophy (left) and β-galactosidase activity (right).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2189810&req=5

Figure 2: Characterization of the synbindin–syndecan-2 interaction by pull-down, coimmunoprecipitation, ligand overlay, and two-hybrid assays. (A) GST pull-down assay. Lysates from human 293 cells transfected with Myc-tagged syndecan-2 were pulled down with GST-synbindin fusion protein (a and b) or nonfused GST (c) and immunoblotted with anti–syndecan-2 mAb 6G12 (a) or anti-Myc polyclonal antibody A14 (b and c). In d, cell lysates were directly immunoblotted with anti–syndecan-2 mAb without pull-down. In each panel, three different lysates were tested: (lane 1) lysates from 293 cells transfected with a control vector; (lane 2) lysates from 293 cells transfected with Myc-tagged syndecan-2, which were undigested; and (lane 3) lysates from 293 cells transfected with Myc-tagged syndecan-2, which were digested with heparitinase. The arrowhead indicates the 34-kD syndecan-2 core protein. (B) Coimmunoprecipitation of synbindin and syndecan-2. The 293 cells were transfected with FLAG-syndecan-2 alone (lane 1), FLAG-syndecan-2 and Myc-synbindin (lane 2), or the FLAG-syndecan-2ΔEFYA deletion mutant and Myc-synbindin (lane 3). The cell lysates were immunoprecipitated with anti-Myc polyclonal antibody and immunoblotted with anti-FLAG antibody (c). The arrowhead indicates the syndecan-2 core protein. Note that the intact syndecan-2 was coimmunoprecipitated with synbindin (c, lane 2), whereas the syndecan-2ΔEFYA deletion mutant was not (c, lane 3). In a and b, the lysates were directly immunoblotted with anti-Myc antibody (a) and anti-FLAG antibody (b), respectively, to show that similar amounts of proteins were expressed. (C) GST-synbindin overlay assay. His-tagged recombinant proteins of intact syndecan-2 cytoplasmic domain (SDC2) and ΔEFYA cytoplasmic domain (SDC2ΔEFYA, right lane) were resolved on a 10–20% tricine gel, blotted, and overlaid with GST-synbindin (bottom panel) as described in Materials and Methods. (top panel) Ponceau S staining of the blot. (D) Two-hybrid assays to analyze the syndecan-2–binding site in synbindin. Four synbindin fragments (shown in the bottom panel) were tested with two syndecan-2 baits: one representing the intact syndecan-2 cytoplasmic domain (SDC2) and the other representing the ΔEFYA cytoplasmic domain (SDC2ΔEFYA). The interactions were scored by His prototrophy (left) and β-galactosidase activity (right).
Mentions: Yeast two-hybrid screens were performed using the L40 yeast strain, where the expression of both the reporter genes HIS3 and LacZ are driven by minimal GAL1 promoters fused to LexA-binding sites. A bait consisting of the entire cytoplasmic domain of the syndecan-2 fused in-frame to the LexA DNA–binding domain was constructed by PCR with the BTM116 vector. An embryonic mouse cDNA library constructed into the NotI site of pVP16 containing the Leu2 activation domain (Vojtek and Hollenberg 1995; a gift from Dr. Erkki Ruoslahti, The Burnham Institute) was screened with the syndecan-2 bait. Positive clones were selected by His prototrophy and assayed for β-galactosidase activity. Double-positive clones were isolated and characterized by sequencing. A double-positive clone (clone 28), which encodes a putative cytoplasmic protein with similarities to several PDZ domain–containing proteins (see Results), was further investigated as described in this paper. We named this protein synbindin. The specificity of the interaction between synbindin and the cytoplasmic domain of syndecan-2 was analyzed by two-hybrid assays (see Fig. 1 A). For this, we generated (by PCR) the following additional baits of the syndecan cytoplasmic domains: (1) a syndecan-2 deletion mutant lacking the COOH-terminal EFYA sequence (syndecan-2ΔEFYA); (2) a syndecan-4 deletion mutant lacking the COOH-terminal EFYA sequence (syndecan-4ΔEFYA); and (3) a bait with reverse sequence of syndecan-2 cytoplasmic domain. To determine the syndecan-2–binding site in synbindin, we generated by PCR four Leu2 fusion constructs representing the NH2-terminal half of synbindin (N-Sbd), the PDZ-related domain (P-Sbd), the COOH-terminal half (C-Sbd), and the PDZ-related domain plus the COOH-terminal half (P/C-Sbd) (see Fig. 2 D). Two-hybrid assays were performed as described above using HIS3 and LacZ as reporter genes.

Bottom Line: This effect requires the COOH-terminal EFYA sequence of syndecan-2, suggesting that cytoplasmic molecules interacting with this sequence play a critical role in spine morphogenesis.Our results show that synbindin is a physiological syndecan-2 ligand on dendritic spines.We suggest that syndecan-2 induces spine formation by recruiting intracellular vesicles toward postsynaptic sites through the interaction with synbindin.

View Article: PubMed Central - PubMed

Affiliation: The Burnham Institute, La Jolla, California 92037, USA.

ABSTRACT
Dendritic spines are small protrusions on the surface of dendrites that receive the vast majority of excitatory synapses. We previously showed that the cell-surface heparan sulfate proteoglycan syndecan-2 induces spine formation upon transfection into hippocampal neurons. This effect requires the COOH-terminal EFYA sequence of syndecan-2, suggesting that cytoplasmic molecules interacting with this sequence play a critical role in spine morphogenesis. Here, we report a novel protein that binds to the EFYA motif of syndecan-2. This protein, named synbindin, is expressed by neurons in a pattern similar to that of syndecan-2, and colocalizes with syndecan-2 in the spines of cultured hippocampal neurons. In transfected hippocampal neurons, synbindin undergoes syndecan-2-dependent clustering. Synbindin is structurally related to yeast proteins known to be involved in vesicle transport. Immunoelectron microscopy localized synbindin on postsynaptic membranes and intracellular vesicles within dendrites, suggesting a role in postsynaptic membrane trafficking. Synbindin coimmunoprecipitates with syndecan-2 from synaptic membrane fractions. Our results show that synbindin is a physiological syndecan-2 ligand on dendritic spines. We suggest that syndecan-2 induces spine formation by recruiting intracellular vesicles toward postsynaptic sites through the interaction with synbindin.

Show MeSH
Related in: MedlinePlus