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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 of filamin isoforms and variants with the cytoplasmic domains of different integrin β subunits in yeast. (A) Cotransformation of yeast host strain PJ69–4A with the listed combinations of β integrin cytoplasmic domains (pAS2.1) and filamin-A and filamin-B deletion constructs (pACT). The NH2- and COOH-terminal positions of the different filamin constructs are indicated. Numbers in parentheses refer to the repeats present in the different constructs. The partially truncated repeat 20 is indicated by an asterisk. Interactions were scored (+) when the plating efficiencies on selective SC-LTHA plates were greater than 80% of those on nonselective SC-LT plates at 5 d of growth, (±) when they were greater than 80% at 10 d of growth, and (−) when no colonies were detected after 10 d of growth. (B) β-Galactosidase activity of the indicated cotransformants was measured by a liquid culture assay with O-nitrophenyl B-d-galactopyranoside as substrate. Controls were used: negative interaction control, empty vectors pAS/pACT; positive interaction control, p53/pSV-40 large T and the complete Gal4 domain pCL1/pAS (171 ± 6 β-galactosidase units; unpublished data). Data are shown as mean ± SD (n = 3).
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fig2: Characterization of the interaction of filamin isoforms and variants with the cytoplasmic domains of different integrin β subunits in yeast. (A) Cotransformation of yeast host strain PJ69–4A with the listed combinations of β integrin cytoplasmic domains (pAS2.1) and filamin-A and filamin-B deletion constructs (pACT). The NH2- and COOH-terminal positions of the different filamin constructs are indicated. Numbers in parentheses refer to the repeats present in the different constructs. The partially truncated repeat 20 is indicated by an asterisk. Interactions were scored (+) when the plating efficiencies on selective SC-LTHA plates were greater than 80% of those on nonselective SC-LT plates at 5 d of growth, (±) when they were greater than 80% at 10 d of growth, and (−) when no colonies were detected after 10 d of growth. (B) β-Galactosidase activity of the indicated cotransformants was measured by a liquid culture assay with O-nitrophenyl B-d-galactopyranoside as substrate. Controls were used: negative interaction control, empty vectors pAS/pACT; positive interaction control, p53/pSV-40 large T and the complete Gal4 domain pCL1/pAS (171 ± 6 β-galactosidase units; unpublished data). Data are shown as mean ± SD (n = 3).

Mentions: We next tested the interaction of the COOH-terminal domain, i.e., repeats 19–24 of filamin-B and filamin-Bvar-1 with different β subunits, using the yeast two-hybrid system. In addition, we tested the homologous regions of filamin-A and filamin-Avar-1. The results (Fig. 2 A) show that the COOH-terminal domain of wild-type filamin-B(19–24), containing repeats 19–24, interacts only with β1A. In contrast, an equivalent construct encoding filamin-Bvar-1(19–24), which lacks amino acids 2082–2122 that span repeats 19 and 20, and an NH2-terminal deletion mutant of filamin-B, truncated at amino acid 2123 (filamin-B, 20*–24), bind not only to β1A but also to the β1D, β3, and β6 subunits. Similar results were obtained with proteins from the original isolated filamin-Bvar-1 clones that contain amino acids 2027–2602 (unpublished data). Quantitative β-galactosidase activity assays indicated that filamin-Bvar-1(19–24) bound two to three times more strongly to β1A than did wild-type filamin-B(19–24) (Fig. 2 B). The binding of filamin-Bvar-1(19–24) to β3 was weaker and comparable to that of wild-type filamin-B(19–24) to β1A, and that of filamin-Bvar-1(19–24) to β1D was of intermediate strength.


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 of filamin isoforms and variants with the cytoplasmic domains of different integrin β subunits in yeast. (A) Cotransformation of yeast host strain PJ69–4A with the listed combinations of β integrin cytoplasmic domains (pAS2.1) and filamin-A and filamin-B deletion constructs (pACT). The NH2- and COOH-terminal positions of the different filamin constructs are indicated. Numbers in parentheses refer to the repeats present in the different constructs. The partially truncated repeat 20 is indicated by an asterisk. Interactions were scored (+) when the plating efficiencies on selective SC-LTHA plates were greater than 80% of those on nonselective SC-LT plates at 5 d of growth, (±) when they were greater than 80% at 10 d of growth, and (−) when no colonies were detected after 10 d of growth. (B) β-Galactosidase activity of the indicated cotransformants was measured by a liquid culture assay with O-nitrophenyl B-d-galactopyranoside as substrate. Controls were used: negative interaction control, empty vectors pAS/pACT; positive interaction control, p53/pSV-40 large T and the complete Gal4 domain pCL1/pAS (171 ± 6 β-galactosidase units; unpublished data). Data are shown as mean ± SD (n = 3).
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Related In: Results  -  Collection

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

fig2: Characterization of the interaction of filamin isoforms and variants with the cytoplasmic domains of different integrin β subunits in yeast. (A) Cotransformation of yeast host strain PJ69–4A with the listed combinations of β integrin cytoplasmic domains (pAS2.1) and filamin-A and filamin-B deletion constructs (pACT). The NH2- and COOH-terminal positions of the different filamin constructs are indicated. Numbers in parentheses refer to the repeats present in the different constructs. The partially truncated repeat 20 is indicated by an asterisk. Interactions were scored (+) when the plating efficiencies on selective SC-LTHA plates were greater than 80% of those on nonselective SC-LT plates at 5 d of growth, (±) when they were greater than 80% at 10 d of growth, and (−) when no colonies were detected after 10 d of growth. (B) β-Galactosidase activity of the indicated cotransformants was measured by a liquid culture assay with O-nitrophenyl B-d-galactopyranoside as substrate. Controls were used: negative interaction control, empty vectors pAS/pACT; positive interaction control, p53/pSV-40 large T and the complete Gal4 domain pCL1/pAS (171 ± 6 β-galactosidase units; unpublished data). Data are shown as mean ± SD (n = 3).
Mentions: We next tested the interaction of the COOH-terminal domain, i.e., repeats 19–24 of filamin-B and filamin-Bvar-1 with different β subunits, using the yeast two-hybrid system. In addition, we tested the homologous regions of filamin-A and filamin-Avar-1. The results (Fig. 2 A) show that the COOH-terminal domain of wild-type filamin-B(19–24), containing repeats 19–24, interacts only with β1A. In contrast, an equivalent construct encoding filamin-Bvar-1(19–24), which lacks amino acids 2082–2122 that span repeats 19 and 20, and an NH2-terminal deletion mutant of filamin-B, truncated at amino acid 2123 (filamin-B, 20*–24), bind not only to β1A but also to the β1D, β3, and β6 subunits. Similar results were obtained with proteins from the original isolated filamin-Bvar-1 clones that contain amino acids 2027–2602 (unpublished data). Quantitative β-galactosidase activity assays indicated that filamin-Bvar-1(19–24) bound two to three times more strongly to β1A than did wild-type filamin-B(19–24) (Fig. 2 B). The binding of filamin-Bvar-1(19–24) to β3 was weaker and comparable to that of wild-type filamin-B(19–24) to β1A, and that of filamin-Bvar-1(19–24) to β1D was of intermediate strength.

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