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Supervillin (p205): A novel membrane-associated, F-actin-binding protein in the villin/gelsolin superfamily.

Pestonjamasp KN, Pope RK, Wulfkuhle JD, Luna EJ - J. Cell Biol. (1997)

Bottom Line: The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin.The NH2 terminus is novel, but contains four potential nuclear targeting signals.Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

View Article: PubMed Central - PubMed

Affiliation: Worcester Foundation for Biomedical Research, University of Massachusetts Medical Center, Shrewsbury, Massachusetts 01545, USA.

ABSTRACT
Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE-purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell-cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions "rings." At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH2 terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

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p205 cosediments  with endogenous, phalloidin-stabilized actin (A), and actin  coimmunoprecipitates with  p205 (B). (A) Neutrophil  plasma membranes (lane 1)  were treated with either fluorescein-phalloidin (lanes 2  and 3) or unlabeled phalloidin (lane 4), solubilized in  TEB, and then incubated  with nonspecific IgG (lane  2) or antifluorescein IgG  (lanes 3 and 4) bound to protein A–agarose. p205 was visualized by staining with affinity-purified pepA IgG  and by F-actin blot overlays (not shown). (B) IgG and actin in  immunoprecipitates generated with either nonspecific rabbit IgG  (lane 1) or affinity-purified, anti-pepA antibody (lane 2) after  three washes with RIPA buffer. The relative amounts of actin  cited in the text were normalized by reference to the amounts of  IgG visualized by labeling with radiolabeled secondary IgG.
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Figure 6: p205 cosediments with endogenous, phalloidin-stabilized actin (A), and actin coimmunoprecipitates with p205 (B). (A) Neutrophil plasma membranes (lane 1) were treated with either fluorescein-phalloidin (lanes 2 and 3) or unlabeled phalloidin (lane 4), solubilized in TEB, and then incubated with nonspecific IgG (lane 2) or antifluorescein IgG (lanes 3 and 4) bound to protein A–agarose. p205 was visualized by staining with affinity-purified pepA IgG and by F-actin blot overlays (not shown). (B) IgG and actin in immunoprecipitates generated with either nonspecific rabbit IgG (lane 1) or affinity-purified, anti-pepA antibody (lane 2) after three washes with RIPA buffer. The relative amounts of actin cited in the text were normalized by reference to the amounts of IgG visualized by labeling with radiolabeled secondary IgG.

Mentions: More evidence for the association of p205 with actin in situ was obtained by demonstrating that p205 and F-actin cosediment in reciprocal immunoprecipitation assays (Fig. 6, A and B). Because antiactin antibodies were less than optimal in these assays, we developed a procedure in which high affinity polyclonal antibodies against fluorescein (53, 83) were used to pellet actin filaments stabilized with fluorescein-labeled phalloidin. Neutrophil plasma membranes were incubated with fluorescein-phalloidin, and then phalloidin-bound actin filaments were precipitated from detergent-solubilized extracts with antifluorescein IgG (see Materials and Methods). Essentially all of the p205 in the initial extract (Fig. 6 A, lane 1) coprecipitated with F-actin bound to fluorescein-phalloidin (Fig. 6 A, lane 3), whereas little or no p205 was found in control experiments with nonspecific IgG (Fig. 6 A, lane 2) or with unlabeled phalloidin (Fig. 6 A, lane 4).


Supervillin (p205): A novel membrane-associated, F-actin-binding protein in the villin/gelsolin superfamily.

Pestonjamasp KN, Pope RK, Wulfkuhle JD, Luna EJ - J. Cell Biol. (1997)

p205 cosediments  with endogenous, phalloidin-stabilized actin (A), and actin  coimmunoprecipitates with  p205 (B). (A) Neutrophil  plasma membranes (lane 1)  were treated with either fluorescein-phalloidin (lanes 2  and 3) or unlabeled phalloidin (lane 4), solubilized in  TEB, and then incubated  with nonspecific IgG (lane  2) or antifluorescein IgG  (lanes 3 and 4) bound to protein A–agarose. p205 was visualized by staining with affinity-purified pepA IgG  and by F-actin blot overlays (not shown). (B) IgG and actin in  immunoprecipitates generated with either nonspecific rabbit IgG  (lane 1) or affinity-purified, anti-pepA antibody (lane 2) after  three washes with RIPA buffer. The relative amounts of actin  cited in the text were normalized by reference to the amounts of  IgG visualized by labeling with radiolabeled secondary IgG.
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Figure 6: p205 cosediments with endogenous, phalloidin-stabilized actin (A), and actin coimmunoprecipitates with p205 (B). (A) Neutrophil plasma membranes (lane 1) were treated with either fluorescein-phalloidin (lanes 2 and 3) or unlabeled phalloidin (lane 4), solubilized in TEB, and then incubated with nonspecific IgG (lane 2) or antifluorescein IgG (lanes 3 and 4) bound to protein A–agarose. p205 was visualized by staining with affinity-purified pepA IgG and by F-actin blot overlays (not shown). (B) IgG and actin in immunoprecipitates generated with either nonspecific rabbit IgG (lane 1) or affinity-purified, anti-pepA antibody (lane 2) after three washes with RIPA buffer. The relative amounts of actin cited in the text were normalized by reference to the amounts of IgG visualized by labeling with radiolabeled secondary IgG.
Mentions: More evidence for the association of p205 with actin in situ was obtained by demonstrating that p205 and F-actin cosediment in reciprocal immunoprecipitation assays (Fig. 6, A and B). Because antiactin antibodies were less than optimal in these assays, we developed a procedure in which high affinity polyclonal antibodies against fluorescein (53, 83) were used to pellet actin filaments stabilized with fluorescein-labeled phalloidin. Neutrophil plasma membranes were incubated with fluorescein-phalloidin, and then phalloidin-bound actin filaments were precipitated from detergent-solubilized extracts with antifluorescein IgG (see Materials and Methods). Essentially all of the p205 in the initial extract (Fig. 6 A, lane 1) coprecipitated with F-actin bound to fluorescein-phalloidin (Fig. 6 A, lane 3), whereas little or no p205 was found in control experiments with nonspecific IgG (Fig. 6 A, lane 2) or with unlabeled phalloidin (Fig. 6 A, lane 4).

Bottom Line: The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin.The NH2 terminus is novel, but contains four potential nuclear targeting signals.Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

View Article: PubMed Central - PubMed

Affiliation: Worcester Foundation for Biomedical Research, University of Massachusetts Medical Center, Shrewsbury, Massachusetts 01545, USA.

ABSTRACT
Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE-purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell-cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions "rings." At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH2 terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

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Related in: MedlinePlus