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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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Characterization of the interaction between mutants of the β1A and β1D cytoplasmic domain with filamin-A and filamin-B splice variants by yeast two-hybrid analysis. Cotransformation of yeast host strain PJ69–4A with the listed combinations of β integrin cytoplasmic domains (in pAS) and filamin (in pACT). Interaction was scored as in Fig. 2. The numbers represent the amino acid positions in β1A and β1D. The conserved NPXY motifs are underlined and the amino acids in β1D that were swapped in the different chimeras are indicated by shading. The common membrane proximal region, which is shared between β1A and β1D, is boxed.
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fig4: Characterization of the interaction between mutants of the β1A and β1D cytoplasmic domain with filamin-A and filamin-B splice variants by yeast two-hybrid analysis. Cotransformation of yeast host strain PJ69–4A with the listed combinations of β integrin cytoplasmic domains (in pAS) and filamin (in pACT). Interaction was scored as in Fig. 2. The numbers represent the amino acid positions in β1A and β1D. The conserved NPXY motifs are underlined and the amino acids in β1D that were swapped in the different chimeras are indicated by shading. The common membrane proximal region, which is shared between β1A and β1D, is boxed.

Mentions: Next, we determined the binding sites for filamin-B and the splice variants of filamin-A and filamin-B on the cytoplasmic domains of the β1A and β1D subunits (Fig. 4) . Swapping the COOH-terminal residues of β1A and β1D in β1A/D796 (EGK to GRKAGL) and β1A/D792 (NPKYEGK to NPNYGRAGL) had no effect on their binding to filamin-B(19–24), filamin-Bvar-1(19–24), and filamin-A(20–24). A swap involving the membrane-proximal region of β1D, where the G778 and A786 residues in β1A are replaced by the corresponding Q778 and P786 residues from β1D, leads to complete abolition of the interaction with filamin-B(19–24) and filamin-A(20*–24). Binding of filamin-Bvar-1 (19–24) could still be detected, although it was weaker. These results indicate that the specificity of the binding of filamin-B and filamin-Avar-1 with β1A depends on the two residues, G778 and A786, that have been substituted in β1D by Q778 and P786. Although a role for Q778 cannot be excluded, we propose that P786, by its ability to create a bend or a rigid kink in polypeptide chains, changes the conformation of the β1D cytoplasmic domain in such a way that it can no longer bind to filamin-B or filamin-Avar-1.


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)

Characterization of the interaction between mutants of the β1A and β1D cytoplasmic domain with filamin-A and filamin-B splice variants by yeast two-hybrid analysis. Cotransformation of yeast host strain PJ69–4A with the listed combinations of β integrin cytoplasmic domains (in pAS) and filamin (in pACT). Interaction was scored as in Fig. 2. The numbers represent the amino acid positions in β1A and β1D. The conserved NPXY motifs are underlined and the amino acids in β1D that were swapped in the different chimeras are indicated by shading. The common membrane proximal region, which is shared between β1A and β1D, is boxed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199218&req=5

fig4: Characterization of the interaction between mutants of the β1A and β1D cytoplasmic domain with filamin-A and filamin-B splice variants by yeast two-hybrid analysis. Cotransformation of yeast host strain PJ69–4A with the listed combinations of β integrin cytoplasmic domains (in pAS) and filamin (in pACT). Interaction was scored as in Fig. 2. The numbers represent the amino acid positions in β1A and β1D. The conserved NPXY motifs are underlined and the amino acids in β1D that were swapped in the different chimeras are indicated by shading. The common membrane proximal region, which is shared between β1A and β1D, is boxed.
Mentions: Next, we determined the binding sites for filamin-B and the splice variants of filamin-A and filamin-B on the cytoplasmic domains of the β1A and β1D subunits (Fig. 4) . Swapping the COOH-terminal residues of β1A and β1D in β1A/D796 (EGK to GRKAGL) and β1A/D792 (NPKYEGK to NPNYGRAGL) had no effect on their binding to filamin-B(19–24), filamin-Bvar-1(19–24), and filamin-A(20–24). A swap involving the membrane-proximal region of β1D, where the G778 and A786 residues in β1A are replaced by the corresponding Q778 and P786 residues from β1D, leads to complete abolition of the interaction with filamin-B(19–24) and filamin-A(20*–24). Binding of filamin-Bvar-1 (19–24) could still be detected, although it was weaker. These results indicate that the specificity of the binding of filamin-B and filamin-Avar-1 with β1A depends on the two residues, G778 and A786, that have been substituted in β1D by Q778 and P786. Although a role for Q778 cannot be excluded, we propose that P786, by its ability to create a bend or a rigid kink in polypeptide chains, changes the conformation of the β1D cytoplasmic domain in such a way that it can no longer bind to filamin-B or filamin-Avar-1.

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
Related in: MedlinePlus