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Molecular basis of filamin A-FilGAP interaction and its impairment in congenital disorders associated with filamin A mutations.

Nakamura F, Heikkinen O, Pentikäinen OT, Osborn TM, Kasza KE, Weitz DA, Kupiainen O, Permi P, Kilpeläinen I, Ylänne J, Hartwig JH, Stossel TP - PLoS ONE (2009)

Bottom Line: We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction.FilGAP does not bind FLNa homologs FLNb or FLNc establishing the importance of this interaction to the human FLNa mutations.Disease-related FLNa mutations have demonstrable effects on FLNa function.

View Article: PubMed Central - PubMed

Affiliation: Translational Medicine Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. fnakamura@rics.bwh.harvard.edu

ABSTRACT

Background: Mutations in filamin A (FLNa), an essential cytoskeletal protein with multiple binding partners, cause developmental anomalies in humans.

Methodology/principal findings: We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction. A combination of NMR structural analysis and in silico modeling revealed the structural interface details between the C and D beta-strands of the IgFLNa23 and the C-terminal 32 residues of FilGAP. Mutagenesis of the predicted key interface residues confirmed the binding constraints between the two proteins. Specific loss-of-function FLNa constructs were generated and used to analyze the importance of the FLNa-FilGAP interaction in vivo. Point mutagenesis revealed that disruption of the FLNa-FilGAP interface perturbs cell spreading. FilGAP does not bind FLNa homologs FLNb or FLNc establishing the importance of this interaction to the human FLNa mutations. Tight complex formation requires dimerization of both partners and the correct alignment of the binding surfaces, which is promoted by a flexible hinge domain between repeats 23 and 24 of FLNa. FLNa mutations associated with human developmental anomalies disrupt the binding interaction and weaken the elasticity of FLNa/F-actin network under high mechanical stress.

Conclusions/significance: Mutational analysis informed by structure can generate reagents for probing specific cellular interactions of FLNa. Disease-related FLNa mutations have demonstrable effects on FLNa function.

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In vivo interaction of FLNa and FilGAP.(A) Immunoprecipitation of the FLNa/FilGAP complex expressed in FLNa-deficient M2 cells. Wild-type or mutant (M2474E) FLNa were co-expressed with wild-type or mutant (V734Y) HA-tagged FilGAP in M2 cells. FLNa was immunoprecipitated with FLNa-specific mAb and bound HA-FilGAP was detected by immunoblotting using HA-specific mAb. (B and C) Effects of wild-type and mutant FilGAP on cell spreading. M2 cells were transfected with EGFP-FLNa, wild-type or V734Y mutant HA-FilGAP. After 48 h, cells were trypsinized, plated on coverslips coated with fibronectin, and fixed at 15, 30, 60, and 120 min after plating. Cells were stained with mouse mAbs to HA for FilGAP (red, three lower right panels) or Texas-red phalloidin for F-actin (red, lower left panel). FLNa was detected with EGFP (green, upper panels). Stained cells at 120 min after plating are shown in (B). Scale bar, 50 ìm. The percentages of spread cells were plotted as the mean±SEM (n = 3) in (C).
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pone-0004928-g005: In vivo interaction of FLNa and FilGAP.(A) Immunoprecipitation of the FLNa/FilGAP complex expressed in FLNa-deficient M2 cells. Wild-type or mutant (M2474E) FLNa were co-expressed with wild-type or mutant (V734Y) HA-tagged FilGAP in M2 cells. FLNa was immunoprecipitated with FLNa-specific mAb and bound HA-FilGAP was detected by immunoblotting using HA-specific mAb. (B and C) Effects of wild-type and mutant FilGAP on cell spreading. M2 cells were transfected with EGFP-FLNa, wild-type or V734Y mutant HA-FilGAP. After 48 h, cells were trypsinized, plated on coverslips coated with fibronectin, and fixed at 15, 30, 60, and 120 min after plating. Cells were stained with mouse mAbs to HA for FilGAP (red, three lower right panels) or Texas-red phalloidin for F-actin (red, lower left panel). FLNa was detected with EGFP (green, upper panels). Stained cells at 120 min after plating are shown in (B). Scale bar, 50 ìm. The percentages of spread cells were plotted as the mean±SEM (n = 3) in (C).

Mentions: To study the effects of FLNa and FilGAP mutations in vivo, full length proteins were expressed in filamin-deficient M2 cells. Point mutations of FLNa(M2474E) and FilGAP(V734Y) confirm that these residues are critical for the binding partnering interaction in vivo (Figure 5A). Transfection of either wild-type or V734Y mutant hemaglutin (HA)-FilGAP in FLNa- M2 cells suppressed spreading on fibronectin-coated coverslips (Figure 5, B and C). Co-expression of EGFP-FLNa with wild-type FilGAP diminished the effect of FilGAP, but expression of the mutant FilGAP did not have this effect (Figure 5, B and C).


Molecular basis of filamin A-FilGAP interaction and its impairment in congenital disorders associated with filamin A mutations.

Nakamura F, Heikkinen O, Pentikäinen OT, Osborn TM, Kasza KE, Weitz DA, Kupiainen O, Permi P, Kilpeläinen I, Ylänne J, Hartwig JH, Stossel TP - PLoS ONE (2009)

In vivo interaction of FLNa and FilGAP.(A) Immunoprecipitation of the FLNa/FilGAP complex expressed in FLNa-deficient M2 cells. Wild-type or mutant (M2474E) FLNa were co-expressed with wild-type or mutant (V734Y) HA-tagged FilGAP in M2 cells. FLNa was immunoprecipitated with FLNa-specific mAb and bound HA-FilGAP was detected by immunoblotting using HA-specific mAb. (B and C) Effects of wild-type and mutant FilGAP on cell spreading. M2 cells were transfected with EGFP-FLNa, wild-type or V734Y mutant HA-FilGAP. After 48 h, cells were trypsinized, plated on coverslips coated with fibronectin, and fixed at 15, 30, 60, and 120 min after plating. Cells were stained with mouse mAbs to HA for FilGAP (red, three lower right panels) or Texas-red phalloidin for F-actin (red, lower left panel). FLNa was detected with EGFP (green, upper panels). Stained cells at 120 min after plating are shown in (B). Scale bar, 50 ìm. The percentages of spread cells were plotted as the mean±SEM (n = 3) in (C).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654154&req=5

pone-0004928-g005: In vivo interaction of FLNa and FilGAP.(A) Immunoprecipitation of the FLNa/FilGAP complex expressed in FLNa-deficient M2 cells. Wild-type or mutant (M2474E) FLNa were co-expressed with wild-type or mutant (V734Y) HA-tagged FilGAP in M2 cells. FLNa was immunoprecipitated with FLNa-specific mAb and bound HA-FilGAP was detected by immunoblotting using HA-specific mAb. (B and C) Effects of wild-type and mutant FilGAP on cell spreading. M2 cells were transfected with EGFP-FLNa, wild-type or V734Y mutant HA-FilGAP. After 48 h, cells were trypsinized, plated on coverslips coated with fibronectin, and fixed at 15, 30, 60, and 120 min after plating. Cells were stained with mouse mAbs to HA for FilGAP (red, three lower right panels) or Texas-red phalloidin for F-actin (red, lower left panel). FLNa was detected with EGFP (green, upper panels). Stained cells at 120 min after plating are shown in (B). Scale bar, 50 ìm. The percentages of spread cells were plotted as the mean±SEM (n = 3) in (C).
Mentions: To study the effects of FLNa and FilGAP mutations in vivo, full length proteins were expressed in filamin-deficient M2 cells. Point mutations of FLNa(M2474E) and FilGAP(V734Y) confirm that these residues are critical for the binding partnering interaction in vivo (Figure 5A). Transfection of either wild-type or V734Y mutant hemaglutin (HA)-FilGAP in FLNa- M2 cells suppressed spreading on fibronectin-coated coverslips (Figure 5, B and C). Co-expression of EGFP-FLNa with wild-type FilGAP diminished the effect of FilGAP, but expression of the mutant FilGAP did not have this effect (Figure 5, B and C).

Bottom Line: We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction.FilGAP does not bind FLNa homologs FLNb or FLNc establishing the importance of this interaction to the human FLNa mutations.Disease-related FLNa mutations have demonstrable effects on FLNa function.

View Article: PubMed Central - PubMed

Affiliation: Translational Medicine Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. fnakamura@rics.bwh.harvard.edu

ABSTRACT

Background: Mutations in filamin A (FLNa), an essential cytoskeletal protein with multiple binding partners, cause developmental anomalies in humans.

Methodology/principal findings: We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction. A combination of NMR structural analysis and in silico modeling revealed the structural interface details between the C and D beta-strands of the IgFLNa23 and the C-terminal 32 residues of FilGAP. Mutagenesis of the predicted key interface residues confirmed the binding constraints between the two proteins. Specific loss-of-function FLNa constructs were generated and used to analyze the importance of the FLNa-FilGAP interaction in vivo. Point mutagenesis revealed that disruption of the FLNa-FilGAP interface perturbs cell spreading. FilGAP does not bind FLNa homologs FLNb or FLNc establishing the importance of this interaction to the human FLNa mutations. Tight complex formation requires dimerization of both partners and the correct alignment of the binding surfaces, which is promoted by a flexible hinge domain between repeats 23 and 24 of FLNa. FLNa mutations associated with human developmental anomalies disrupt the binding interaction and weaken the elasticity of FLNa/F-actin network under high mechanical stress.

Conclusions/significance: Mutational analysis informed by structure can generate reagents for probing specific cellular interactions of FLNa. Disease-related FLNa mutations have demonstrable effects on FLNa function.

Show MeSH
Related in: MedlinePlus