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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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Expression of filamin isoforms and variants during in vitro myogenesis. (A) Schematic representation of the organization of the filamin domains and the regions analyzed by RT-PCR. (B) RT-PCR analysis of alternative splicing of the H1 region of filamin-A, filamin-B, and filamin-C in mouse C2C12 myoblasts and murine tissues, as indicated. Splicing is analyzed at the indicated time points after induction of differentiation. The H1 region of filamin-A is not spliced, whereas in filamin-B and filamin-C the H1 region is removed during myogenesis. Filamin-B(H1s) is detected in C2C12 cells and adult tissues. The switching from the β1A to the β1D variant during C2C12 differentiation is shown. (C) Immunoblot of the protein expression levels of filamin-B β1D, and MHC during myogenesis. Filamin-B expression decreases, while the myogenic differentiation markers β1D and MHC are both induced. (D) Amino acid alignment of the human and murine H1 and variable-1 region. The partial murine filamin sequences are available from GenBank/EMBL/DDBJ under accession nos. AF353668–353670.
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fig5: Expression of filamin isoforms and variants during in vitro myogenesis. (A) Schematic representation of the organization of the filamin domains and the regions analyzed by RT-PCR. (B) RT-PCR analysis of alternative splicing of the H1 region of filamin-A, filamin-B, and filamin-C in mouse C2C12 myoblasts and murine tissues, as indicated. Splicing is analyzed at the indicated time points after induction of differentiation. The H1 region of filamin-A is not spliced, whereas in filamin-B and filamin-C the H1 region is removed during myogenesis. Filamin-B(H1s) is detected in C2C12 cells and adult tissues. The switching from the β1A to the β1D variant during C2C12 differentiation is shown. (C) Immunoblot of the protein expression levels of filamin-B β1D, and MHC during myogenesis. Filamin-B expression decreases, while the myogenic differentiation markers β1D and MHC are both induced. (D) Amino acid alignment of the human and murine H1 and variable-1 region. The partial murine filamin sequences are available from GenBank/EMBL/DDBJ under accession nos. AF353668–353670.

Mentions: During differentiation of mouse C2C12 myoblasts into myotubes, the expression of β1D is induced, whereas that of β1A is downregulated (Belkin et al., 1997; van der Flier et al., 1997). We investigated whether this switch is paralleled by changes in the expression of filamin isoforms and/or their variants. Total RNA was isolated at different time points of myogenic differentiation, and the expression of filamin isoforms was analyzed by RT-PCR using appropriate primers. We studied the splicing of the region encoding the 41 amino acids in filamin-A and filamin-B, as well that of the H1 region of filamin-A, filamin-B, and filamin-C (Fig. 5 A). The latter were included because variants of human filamin-B and filamin-C, lacking the H1 region, have been described previously (Xie et al., 1998; Xu et al., 1998). Fig. 5, B–D, shows that C2C12 myoblasts express all three murine filamin isoforms. In addition, whereas the H1 region is present in filamin-A throughout differentiation, this region is absent from the filamin-B and filamin-C isoforms. This deletion appears to precede the switch from β1A to β1D that occurs during myogenic differentiation. Interestingly, we detected in C2C12 cells and among several murine and human cDNAs (unpublished data) a third filamin-B transcript that encodes a variant with a shorter H1 (H1s) region (Fig. 5 D). This transcript arises as a result of the usage of intrinsic splice-donor and acceptor sites that are present in the murine and human filamin-B genes (position 5280 and 5312, respectively; GenBank/EMBL/DDBJ accession no. NM_001457). We did not detect the filamin-Avar-1 and filamin-Bvar-1 transcripts.


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)

Expression of filamin isoforms and variants during in vitro myogenesis. (A) Schematic representation of the organization of the filamin domains and the regions analyzed by RT-PCR. (B) RT-PCR analysis of alternative splicing of the H1 region of filamin-A, filamin-B, and filamin-C in mouse C2C12 myoblasts and murine tissues, as indicated. Splicing is analyzed at the indicated time points after induction of differentiation. The H1 region of filamin-A is not spliced, whereas in filamin-B and filamin-C the H1 region is removed during myogenesis. Filamin-B(H1s) is detected in C2C12 cells and adult tissues. The switching from the β1A to the β1D variant during C2C12 differentiation is shown. (C) Immunoblot of the protein expression levels of filamin-B β1D, and MHC during myogenesis. Filamin-B expression decreases, while the myogenic differentiation markers β1D and MHC are both induced. (D) Amino acid alignment of the human and murine H1 and variable-1 region. The partial murine filamin sequences are available from GenBank/EMBL/DDBJ under accession nos. AF353668–353670.
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Related In: Results  -  Collection

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fig5: Expression of filamin isoforms and variants during in vitro myogenesis. (A) Schematic representation of the organization of the filamin domains and the regions analyzed by RT-PCR. (B) RT-PCR analysis of alternative splicing of the H1 region of filamin-A, filamin-B, and filamin-C in mouse C2C12 myoblasts and murine tissues, as indicated. Splicing is analyzed at the indicated time points after induction of differentiation. The H1 region of filamin-A is not spliced, whereas in filamin-B and filamin-C the H1 region is removed during myogenesis. Filamin-B(H1s) is detected in C2C12 cells and adult tissues. The switching from the β1A to the β1D variant during C2C12 differentiation is shown. (C) Immunoblot of the protein expression levels of filamin-B β1D, and MHC during myogenesis. Filamin-B expression decreases, while the myogenic differentiation markers β1D and MHC are both induced. (D) Amino acid alignment of the human and murine H1 and variable-1 region. The partial murine filamin sequences are available from GenBank/EMBL/DDBJ under accession nos. AF353668–353670.
Mentions: During differentiation of mouse C2C12 myoblasts into myotubes, the expression of β1D is induced, whereas that of β1A is downregulated (Belkin et al., 1997; van der Flier et al., 1997). We investigated whether this switch is paralleled by changes in the expression of filamin isoforms and/or their variants. Total RNA was isolated at different time points of myogenic differentiation, and the expression of filamin isoforms was analyzed by RT-PCR using appropriate primers. We studied the splicing of the region encoding the 41 amino acids in filamin-A and filamin-B, as well that of the H1 region of filamin-A, filamin-B, and filamin-C (Fig. 5 A). The latter were included because variants of human filamin-B and filamin-C, lacking the H1 region, have been described previously (Xie et al., 1998; Xu et al., 1998). Fig. 5, B–D, shows that C2C12 myoblasts express all three murine filamin isoforms. In addition, whereas the H1 region is present in filamin-A throughout differentiation, this region is absent from the filamin-B and filamin-C isoforms. This deletion appears to precede the switch from β1A to β1D that occurs during myogenic differentiation. Interestingly, we detected in C2C12 cells and among several murine and human cDNAs (unpublished data) a third filamin-B transcript that encodes a variant with a shorter H1 (H1s) region (Fig. 5 D). This transcript arises as a result of the usage of intrinsic splice-donor and acceptor sites that are present in the murine and human filamin-B genes (position 5280 and 5312, respectively; GenBank/EMBL/DDBJ accession no. NM_001457). We did not detect the filamin-Avar-1 and filamin-Bvar-1 transcripts.

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