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Different splice variants of filamin-B affect myogenesis, subcellular distribution, and determine binding to integrin [beta] subunits.

van der Flier A, Kuikman I, Kramer D, Geerts D, Kreft M, Takafuta T, Shapiro SS, Sonnenberg A - J. Cell Biol. (2002)

Bottom Line: When expressed in C2C12 cells, filamin-Bvar-1(DeltaH1) accelerates their differentiation into myotubes.Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region.These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins.

View Article: PubMed Central - PubMed

Affiliation: Netherlands Cancer Institute, Division of Cell Biology, 1066 CX Amsterdams, Netherlands.

ABSTRACT
Integrins connect the extracellular matrix with the cell interior, and transduce signals through interactions of their cytoplasmic tails with cytoskeletal and signaling proteins. Using the yeast two-hybrid system, we isolated a novel splice variant (filamin-Bvar-1) of the filamentous actin cross-linking protein, filamin-B, that interacts with the cytoplasmic domain of the integrin beta1A and beta1D subunits. RT-PCR analysis showed weak, but wide, expression of filamin-Bvar-1 and a similar splice variant of filamin-A (filamin-Avar-1) in human tissues. Furthermore, alternative splice variants of filamin-B and filamin-C, from which the flexible hinge-1 region is deleted (DeltaH1), were induced during in vitro differentiation of C2C12 mouse myoblasts. We show that both filamin-Avar-1 and filamin-Bvar-1 bind more strongly than their wild-type isoforms to different integrin beta subunits. The mere presence of the high-affinity binding site for beta1A is not sufficient for targeting the filamin-Bvar-1 construct to focal contacts. Interestingly, the simultaneous deletion of the H1 region is required for the localization of filamin-B at the tips of actin stress fibers. When expressed in C2C12 cells, filamin-Bvar-1(DeltaH1) accelerates their differentiation into myotubes. Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region. These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins.

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Binding of filamin isoforms and variants to GST–β1A and –β1D fusion proteins. (A) Detection of the expressed products from HA-tagged filamin-B cDNA constructs (filamin repeats 19–24) in lysates of COS-7 cells, transiently transfected with the indicated constructs. The upper band represents nonspecific reactivity of the antibody with an endogenous protein. (B) Expression of recombinant GST–β1A and –β1D fusion proteins. The GST fusion proteins containing the cytoplasmic domains of β1A and β1D were purified on glutathione-Sepharose 4B beads and analyzed by immunoblotting with antibodies against GST, β1A, and β1D subunits. (C) Pull-down assay with GST or GST–β1A and –β1D fusion proteins, immobilized on glutathione beads (A) containing HA-tagged fusion proteins as indicated. (Top) Immunoblot detection of bound filamin-Avar-1 and filamin-Bvar-1 to GST–β1A and very weak binding to GST–β1D. No binding was detected to filamin-A and filamin-B. (Bottom) Coomassie blue–stained polyvinylidene difluoride membrane showing the amount of GST fusion proteins used for each pull-down assay.
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fig3: Binding of filamin isoforms and variants to GST–β1A and –β1D fusion proteins. (A) Detection of the expressed products from HA-tagged filamin-B cDNA constructs (filamin repeats 19–24) in lysates of COS-7 cells, transiently transfected with the indicated constructs. The upper band represents nonspecific reactivity of the antibody with an endogenous protein. (B) Expression of recombinant GST–β1A and –β1D fusion proteins. The GST fusion proteins containing the cytoplasmic domains of β1A and β1D were purified on glutathione-Sepharose 4B beads and analyzed by immunoblotting with antibodies against GST, β1A, and β1D subunits. (C) Pull-down assay with GST or GST–β1A and –β1D fusion proteins, immobilized on glutathione beads (A) containing HA-tagged fusion proteins as indicated. (Top) Immunoblot detection of bound filamin-Avar-1 and filamin-Bvar-1 to GST–β1A and very weak binding to GST–β1D. No binding was detected to filamin-A and filamin-B. (Bottom) Coomassie blue–stained polyvinylidene difluoride membrane showing the amount of GST fusion proteins used for each pull-down assay.

Mentions: To confirm the interactions between the different filamin splice variants and the β1A and β1D subunits observed in yeast, we expressed the regions containing repeats 19–24 of filamin-Avar-1 or filamin-Bvar-1, or the corresponding regions of wild-type filamin-A(19–24) or filamin-B(19–24), in COS-7 cells. The proteins were tagged at their NH2 terminus with hemagglutinin A (HA) and expression of equivalent amounts of proteins in COS-7 cells was confirmed by immunoblotting with anti-HA antibody (Fig. 3 A). Binding of the filamin constructs to β1A and β1D was tested in a pull-down assay using glutathione-S-transferase (GST) fusion proteins containing the cytoplasmic domains of these integrin subunits, immobilized on glutathione-Sepharose beads (Fig. 3 B). As shown in Fig. 3 C, GST–β1A bound to filamin-Avar-1(19–24) and filamin-Bvar-1(19–24), but not to the corresponding fragments of the wild-type filamin isoforms (filamin-A, 19–24, and filamin-B, 19–24). Binding of GST–β1D to the different filamin-A and filamin-B constructs was either undetectable or very weak. The interaction between β1A and filamin-B(19–24) detected in yeast could not be confirmed in the pull-down assay, probably because it is too weak. Interestingly, we found that a truncated filamin-B construct that lacks the first 14 amino acids of repeat 19 (filamin-B, 2009–2602) could be efficiently precipitated with GST–β1A (unpublished data). Thus, it appears that not only the deletion of COOH-terminal residues of repeat 19, as in the variant-1 protein, but also the deletion of NH2-terminal residues of repeat 19 results in stronger binding of filamin-B to β1A. Together, these data suggest that repeat 19 contains an inhibitory element for binding of filamin-B to β subunits.


Different splice variants of filamin-B affect myogenesis, subcellular distribution, and determine binding to integrin [beta] subunits.

van der Flier A, Kuikman I, Kramer D, Geerts D, Kreft M, Takafuta T, Shapiro SS, Sonnenberg A - J. Cell Biol. (2002)

Binding of filamin isoforms and variants to GST–β1A and –β1D fusion proteins. (A) Detection of the expressed products from HA-tagged filamin-B cDNA constructs (filamin repeats 19–24) in lysates of COS-7 cells, transiently transfected with the indicated constructs. The upper band represents nonspecific reactivity of the antibody with an endogenous protein. (B) Expression of recombinant GST–β1A and –β1D fusion proteins. The GST fusion proteins containing the cytoplasmic domains of β1A and β1D were purified on glutathione-Sepharose 4B beads and analyzed by immunoblotting with antibodies against GST, β1A, and β1D subunits. (C) Pull-down assay with GST or GST–β1A and –β1D fusion proteins, immobilized on glutathione beads (A) containing HA-tagged fusion proteins as indicated. (Top) Immunoblot detection of bound filamin-Avar-1 and filamin-Bvar-1 to GST–β1A and very weak binding to GST–β1D. No binding was detected to filamin-A and filamin-B. (Bottom) Coomassie blue–stained polyvinylidene difluoride membrane showing the amount of GST fusion proteins used for each pull-down assay.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199218&req=5

fig3: Binding of filamin isoforms and variants to GST–β1A and –β1D fusion proteins. (A) Detection of the expressed products from HA-tagged filamin-B cDNA constructs (filamin repeats 19–24) in lysates of COS-7 cells, transiently transfected with the indicated constructs. The upper band represents nonspecific reactivity of the antibody with an endogenous protein. (B) Expression of recombinant GST–β1A and –β1D fusion proteins. The GST fusion proteins containing the cytoplasmic domains of β1A and β1D were purified on glutathione-Sepharose 4B beads and analyzed by immunoblotting with antibodies against GST, β1A, and β1D subunits. (C) Pull-down assay with GST or GST–β1A and –β1D fusion proteins, immobilized on glutathione beads (A) containing HA-tagged fusion proteins as indicated. (Top) Immunoblot detection of bound filamin-Avar-1 and filamin-Bvar-1 to GST–β1A and very weak binding to GST–β1D. No binding was detected to filamin-A and filamin-B. (Bottom) Coomassie blue–stained polyvinylidene difluoride membrane showing the amount of GST fusion proteins used for each pull-down assay.
Mentions: To confirm the interactions between the different filamin splice variants and the β1A and β1D subunits observed in yeast, we expressed the regions containing repeats 19–24 of filamin-Avar-1 or filamin-Bvar-1, or the corresponding regions of wild-type filamin-A(19–24) or filamin-B(19–24), in COS-7 cells. The proteins were tagged at their NH2 terminus with hemagglutinin A (HA) and expression of equivalent amounts of proteins in COS-7 cells was confirmed by immunoblotting with anti-HA antibody (Fig. 3 A). Binding of the filamin constructs to β1A and β1D was tested in a pull-down assay using glutathione-S-transferase (GST) fusion proteins containing the cytoplasmic domains of these integrin subunits, immobilized on glutathione-Sepharose beads (Fig. 3 B). As shown in Fig. 3 C, GST–β1A bound to filamin-Avar-1(19–24) and filamin-Bvar-1(19–24), but not to the corresponding fragments of the wild-type filamin isoforms (filamin-A, 19–24, and filamin-B, 19–24). Binding of GST–β1D to the different filamin-A and filamin-B constructs was either undetectable or very weak. The interaction between β1A and filamin-B(19–24) detected in yeast could not be confirmed in the pull-down assay, probably because it is too weak. Interestingly, we found that a truncated filamin-B construct that lacks the first 14 amino acids of repeat 19 (filamin-B, 2009–2602) could be efficiently precipitated with GST–β1A (unpublished data). Thus, it appears that not only the deletion of COOH-terminal residues of repeat 19, as in the variant-1 protein, but also the deletion of NH2-terminal residues of repeat 19 results in stronger binding of filamin-B to β1A. Together, these data suggest that repeat 19 contains an inhibitory element for binding of filamin-B to β subunits.

Bottom Line: When expressed in C2C12 cells, filamin-Bvar-1(DeltaH1) accelerates their differentiation into myotubes.Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region.These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins.

View Article: PubMed Central - PubMed

Affiliation: Netherlands Cancer Institute, Division of Cell Biology, 1066 CX Amsterdams, Netherlands.

ABSTRACT
Integrins connect the extracellular matrix with the cell interior, and transduce signals through interactions of their cytoplasmic tails with cytoskeletal and signaling proteins. Using the yeast two-hybrid system, we isolated a novel splice variant (filamin-Bvar-1) of the filamentous actin cross-linking protein, filamin-B, that interacts with the cytoplasmic domain of the integrin beta1A and beta1D subunits. RT-PCR analysis showed weak, but wide, expression of filamin-Bvar-1 and a similar splice variant of filamin-A (filamin-Avar-1) in human tissues. Furthermore, alternative splice variants of filamin-B and filamin-C, from which the flexible hinge-1 region is deleted (DeltaH1), were induced during in vitro differentiation of C2C12 mouse myoblasts. We show that both filamin-Avar-1 and filamin-Bvar-1 bind more strongly than their wild-type isoforms to different integrin beta subunits. The mere presence of the high-affinity binding site for beta1A is not sufficient for targeting the filamin-Bvar-1 construct to focal contacts. Interestingly, the simultaneous deletion of the H1 region is required for the localization of filamin-B at the tips of actin stress fibers. When expressed in C2C12 cells, filamin-Bvar-1(DeltaH1) accelerates their differentiation into myotubes. Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region. These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins.

Show MeSH