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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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The GFP tag on the COOH-terminal part of filamin-B does not interfere with dimerization in vivo. CHO cells were transfected with either the NH2-terminal HA- or the COOH-terminal GFP-tagged FLN-B(19–24) or with the NH2-terminal HA-tagged FLN-Bvar-1(19–23) construct lacking the last repeat. 2 d after transfection, filamin dimers in either cell lysates (lysate) or intact cells (cells) were stabilized by chemical cross-linking at two different concentrations of DSP for 1 h, and dimerization of the epitope-tagged filamins was analyzed by immunoblotting under nonreducing conditions (NR) using anti-HA and anti-GFP antibodies. The specificity of the cross-linkage was checked by including NH2-terminal HA-tagged FLN-Bvar-1(19–23), in which the truncation of COOH-terminal repeat 24 abolished dimerization. In addition, cross-linked samples were separated under reducing (R), DSP disrupting conditions resulting in the presence of filamin-B monomers. Closed arrowheads indicate HA-tagged products, and open arrowheads indicate GFP-tagged fusion proteins. A single arrow indicates a filamin monomer, and an arrow doublet indicates dimers.
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fig6: The GFP tag on the COOH-terminal part of filamin-B does not interfere with dimerization in vivo. CHO cells were transfected with either the NH2-terminal HA- or the COOH-terminal GFP-tagged FLN-B(19–24) or with the NH2-terminal HA-tagged FLN-Bvar-1(19–23) construct lacking the last repeat. 2 d after transfection, filamin dimers in either cell lysates (lysate) or intact cells (cells) were stabilized by chemical cross-linking at two different concentrations of DSP for 1 h, and dimerization of the epitope-tagged filamins was analyzed by immunoblotting under nonreducing conditions (NR) using anti-HA and anti-GFP antibodies. The specificity of the cross-linkage was checked by including NH2-terminal HA-tagged FLN-Bvar-1(19–23), in which the truncation of COOH-terminal repeat 24 abolished dimerization. In addition, cross-linked samples were separated under reducing (R), DSP disrupting conditions resulting in the presence of filamin-B monomers. Closed arrowheads indicate HA-tagged products, and open arrowheads indicate GFP-tagged fusion proteins. A single arrow indicates a filamin monomer, and an arrow doublet indicates dimers.

Mentions: Before initiating studies to define the cellular localization of the different splice variants of filamin-B, we examined whether the addition of GFP at the COOH terminus of filamin influences the ability of this protein to form dimers. To this end, a filamin-B(19–24) construct with GFP at the COOH-terminal end, and a control construct, tagged with HA at the NH2-terminal end, were transiently expressed in CHO cells. After 2 d, cell lysates and intact cells were treated with the chemical cross-linking reagent dithiobis-succinimidyl propionate (DSP) at two concentrations. As shown in Fig. 6 , the addition of increasing amounts of cross-linker led to a shift from monomeric to dimeric tagged filamins, as visualized by immunoblotting with antibodies against HA or GFP. The similar dimerization capacity of HA- and GFP-tagged filamin-B(19–24) indicates that the GFP tag had no effect on dimerization. The same samples under reducing conditions, which disrupt the disulfide bond, only contained filamin monomers. Specificity of the cross-linking reaction was checked using the NH2-terminal HA-tagged filamin-Bvar-1(19–23) construct, which did not form dimers due to truncation of the COOH-terminal repeat 24, which is required for dimerization.


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)

The GFP tag on the COOH-terminal part of filamin-B does not interfere with dimerization in vivo. CHO cells were transfected with either the NH2-terminal HA- or the COOH-terminal GFP-tagged FLN-B(19–24) or with the NH2-terminal HA-tagged FLN-Bvar-1(19–23) construct lacking the last repeat. 2 d after transfection, filamin dimers in either cell lysates (lysate) or intact cells (cells) were stabilized by chemical cross-linking at two different concentrations of DSP for 1 h, and dimerization of the epitope-tagged filamins was analyzed by immunoblotting under nonreducing conditions (NR) using anti-HA and anti-GFP antibodies. The specificity of the cross-linkage was checked by including NH2-terminal HA-tagged FLN-Bvar-1(19–23), in which the truncation of COOH-terminal repeat 24 abolished dimerization. In addition, cross-linked samples were separated under reducing (R), DSP disrupting conditions resulting in the presence of filamin-B monomers. Closed arrowheads indicate HA-tagged products, and open arrowheads indicate GFP-tagged fusion proteins. A single arrow indicates a filamin monomer, and an arrow doublet indicates dimers.
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Related In: Results  -  Collection

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fig6: The GFP tag on the COOH-terminal part of filamin-B does not interfere with dimerization in vivo. CHO cells were transfected with either the NH2-terminal HA- or the COOH-terminal GFP-tagged FLN-B(19–24) or with the NH2-terminal HA-tagged FLN-Bvar-1(19–23) construct lacking the last repeat. 2 d after transfection, filamin dimers in either cell lysates (lysate) or intact cells (cells) were stabilized by chemical cross-linking at two different concentrations of DSP for 1 h, and dimerization of the epitope-tagged filamins was analyzed by immunoblotting under nonreducing conditions (NR) using anti-HA and anti-GFP antibodies. The specificity of the cross-linkage was checked by including NH2-terminal HA-tagged FLN-Bvar-1(19–23), in which the truncation of COOH-terminal repeat 24 abolished dimerization. In addition, cross-linked samples were separated under reducing (R), DSP disrupting conditions resulting in the presence of filamin-B monomers. Closed arrowheads indicate HA-tagged products, and open arrowheads indicate GFP-tagged fusion proteins. A single arrow indicates a filamin monomer, and an arrow doublet indicates dimers.
Mentions: Before initiating studies to define the cellular localization of the different splice variants of filamin-B, we examined whether the addition of GFP at the COOH terminus of filamin influences the ability of this protein to form dimers. To this end, a filamin-B(19–24) construct with GFP at the COOH-terminal end, and a control construct, tagged with HA at the NH2-terminal end, were transiently expressed in CHO cells. After 2 d, cell lysates and intact cells were treated with the chemical cross-linking reagent dithiobis-succinimidyl propionate (DSP) at two concentrations. As shown in Fig. 6 , the addition of increasing amounts of cross-linker led to a shift from monomeric to dimeric tagged filamins, as visualized by immunoblotting with antibodies against HA or GFP. The similar dimerization capacity of HA- and GFP-tagged filamin-B(19–24) indicates that the GFP tag had no effect on dimerization. The same samples under reducing conditions, which disrupt the disulfide bond, only contained filamin monomers. Specificity of the cross-linking reaction was checked using the NH2-terminal HA-tagged filamin-Bvar-1(19–23) construct, which did not form dimers due to truncation of the COOH-terminal repeat 24, which is required for dimerization.

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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