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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-B variants in human tissues, and characterization of the binding of full-length filamin-B variants to integrin cytoplasmic domains. (A) Schematic presentation of COOH-terminal GFP-tagged filamin-B variant constructs. The internal deletions of filamin-B are indicated by a single line. (B) Detection by PCR amplification of filamin-Bvar-1 and filamin-Bvar-1(ΔH1) transcripts. PCR was performed on cDNAs from different human tissues, using primers BV12 and BV13 (A) designed to specifically amplify filamin-Bvar-1(±ΔH1) cDNAs. cDNAs encoding filamin-B variants with and without the var-1 region were used as positive and negative controls, respectively. As a further negative control, cDNA for filamin-A was used. A PCR product for filamin-Bvar-1 was amplified from most tissues. Heart, spleen, and thymus contained transcripts for filamin-Bvar-1(ΔH1). A third PCR product (indicated by an asterisk) that migrated slightly faster than the filamin-Bvar-1 variant and is present in heart, kidney, liver, lung, and colon, corresponds to filamin-Bvar-1(H1s). The authenticity of the PCR products has been confirmed by sequencing. (C) Expression of GFP-tagged filamin-B variants in C2C12 myoblasts. Stably transduced C2C12 cells were obtained as described in the Materials and methods. Equal amounts of total cells lysed in boiling sample buffer were analyzed by immunoblotting. Filamin-B–GFP was detected by mouse anti-GFP antibody. The upper bands represent the full-length filamin–GFP fusion proteins whereas some fainter lower bands represent COOH-terminal proteolytic products. (D) Pull-down assay of full-length filamin-B variants with GST or GST–β1A and –β1D fusion proteins immobilized on glutathione beads containing GFP-tagged fusion proteins as indicated. Immunoblotting showed binding of filamin-Bvar-1 and filamin-Bvar-1(ΔH1) to GST–β1A, weak binding to GST–β1D, whereas filamin-B and filamin-B(ΔH1) did not bind.
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fig7: Expression of filamin-B variants in human tissues, and characterization of the binding of full-length filamin-B variants to integrin cytoplasmic domains. (A) Schematic presentation of COOH-terminal GFP-tagged filamin-B variant constructs. The internal deletions of filamin-B are indicated by a single line. (B) Detection by PCR amplification of filamin-Bvar-1 and filamin-Bvar-1(ΔH1) transcripts. PCR was performed on cDNAs from different human tissues, using primers BV12 and BV13 (A) designed to specifically amplify filamin-Bvar-1(±ΔH1) cDNAs. cDNAs encoding filamin-B variants with and without the var-1 region were used as positive and negative controls, respectively. As a further negative control, cDNA for filamin-A was used. A PCR product for filamin-Bvar-1 was amplified from most tissues. Heart, spleen, and thymus contained transcripts for filamin-Bvar-1(ΔH1). A third PCR product (indicated by an asterisk) that migrated slightly faster than the filamin-Bvar-1 variant and is present in heart, kidney, liver, lung, and colon, corresponds to filamin-Bvar-1(H1s). The authenticity of the PCR products has been confirmed by sequencing. (C) Expression of GFP-tagged filamin-B variants in C2C12 myoblasts. Stably transduced C2C12 cells were obtained as described in the Materials and methods. Equal amounts of total cells lysed in boiling sample buffer were analyzed by immunoblotting. Filamin-B–GFP was detected by mouse anti-GFP antibody. The upper bands represent the full-length filamin–GFP fusion proteins whereas some fainter lower bands represent COOH-terminal proteolytic products. (D) Pull-down assay of full-length filamin-B variants with GST or GST–β1A and –β1D fusion proteins immobilized on glutathione beads containing GFP-tagged fusion proteins as indicated. Immunoblotting showed binding of filamin-Bvar-1 and filamin-Bvar-1(ΔH1) to GST–β1A, weak binding to GST–β1D, whereas filamin-B and filamin-B(ΔH1) did not bind.

Mentions: After excluding potential disadvantageous effects of the GFP tag on the dimerization of filamin, we generated full-length cDNAs encoding four different filamin-B splice variants and tagged them with GFP (Fig. 7 A). These include the previously reported filamin-B and filamin-B lacking H1 (filamin-B[ΔH1]), as well as the novel identified filamin-B lacking the 41 residues between repeats 19 and 20 (filamin-Bvar-1), and filamin-Bvar-1 without H1 (filamin-Bvar-1[ΔH1]). As shown in Fig. 7 B, the latter two filamin-B variants are expressed in a variety of tissues and cell types, including heart, lung, testis, spleen, thymus, and leukocytes. Several cell types were retrovirally transduced, and after fluorescence-activated cell sorting for filamin-GFP–expressing cells, the stable expression of the different filamin fusion proteins was verified by immunoblotting. In all cell lines, full-length GFP-tagged filamin-B variants migrating at ∼300 kD could be detected (shown for C2C12, Fig. 7 C). Filamin-B expression levels were consistently lower than those of the other variants, which were comparable to each other. Occasionally, smaller protein degradation products were detected, which varied in size and quantity, depending on the transduced cell line and the filamin variant (Fig. 7 C). GST pull-down assays confirmed that deletion of the variant-1 region from full-length filamin-B increases binding to β1 integrins (Fig. 7 D). Only the two filamin-B constructs, filamin-Bvar-1 and filamin-Bvar-1 (ΔH1), in which the variant-1 region had been deleted, but not filamin-B or filamin-B(ΔH1), were precipitated by GST–β1A (Fig. 7 D). Binding of filamin-Bvar-1 and filamin-Bvar-1(ΔH1) to β1D again proved to be weak and could only be demonstrated after long exposures of the film. None of the filamin-B constructs interacted with GST.


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-B variants in human tissues, and characterization of the binding of full-length filamin-B variants to integrin cytoplasmic domains. (A) Schematic presentation of COOH-terminal GFP-tagged filamin-B variant constructs. The internal deletions of filamin-B are indicated by a single line. (B) Detection by PCR amplification of filamin-Bvar-1 and filamin-Bvar-1(ΔH1) transcripts. PCR was performed on cDNAs from different human tissues, using primers BV12 and BV13 (A) designed to specifically amplify filamin-Bvar-1(±ΔH1) cDNAs. cDNAs encoding filamin-B variants with and without the var-1 region were used as positive and negative controls, respectively. As a further negative control, cDNA for filamin-A was used. A PCR product for filamin-Bvar-1 was amplified from most tissues. Heart, spleen, and thymus contained transcripts for filamin-Bvar-1(ΔH1). A third PCR product (indicated by an asterisk) that migrated slightly faster than the filamin-Bvar-1 variant and is present in heart, kidney, liver, lung, and colon, corresponds to filamin-Bvar-1(H1s). The authenticity of the PCR products has been confirmed by sequencing. (C) Expression of GFP-tagged filamin-B variants in C2C12 myoblasts. Stably transduced C2C12 cells were obtained as described in the Materials and methods. Equal amounts of total cells lysed in boiling sample buffer were analyzed by immunoblotting. Filamin-B–GFP was detected by mouse anti-GFP antibody. The upper bands represent the full-length filamin–GFP fusion proteins whereas some fainter lower bands represent COOH-terminal proteolytic products. (D) Pull-down assay of full-length filamin-B variants with GST or GST–β1A and –β1D fusion proteins immobilized on glutathione beads containing GFP-tagged fusion proteins as indicated. Immunoblotting showed binding of filamin-Bvar-1 and filamin-Bvar-1(ΔH1) to GST–β1A, weak binding to GST–β1D, whereas filamin-B and filamin-B(ΔH1) did not bind.
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fig7: Expression of filamin-B variants in human tissues, and characterization of the binding of full-length filamin-B variants to integrin cytoplasmic domains. (A) Schematic presentation of COOH-terminal GFP-tagged filamin-B variant constructs. The internal deletions of filamin-B are indicated by a single line. (B) Detection by PCR amplification of filamin-Bvar-1 and filamin-Bvar-1(ΔH1) transcripts. PCR was performed on cDNAs from different human tissues, using primers BV12 and BV13 (A) designed to specifically amplify filamin-Bvar-1(±ΔH1) cDNAs. cDNAs encoding filamin-B variants with and without the var-1 region were used as positive and negative controls, respectively. As a further negative control, cDNA for filamin-A was used. A PCR product for filamin-Bvar-1 was amplified from most tissues. Heart, spleen, and thymus contained transcripts for filamin-Bvar-1(ΔH1). A third PCR product (indicated by an asterisk) that migrated slightly faster than the filamin-Bvar-1 variant and is present in heart, kidney, liver, lung, and colon, corresponds to filamin-Bvar-1(H1s). The authenticity of the PCR products has been confirmed by sequencing. (C) Expression of GFP-tagged filamin-B variants in C2C12 myoblasts. Stably transduced C2C12 cells were obtained as described in the Materials and methods. Equal amounts of total cells lysed in boiling sample buffer were analyzed by immunoblotting. Filamin-B–GFP was detected by mouse anti-GFP antibody. The upper bands represent the full-length filamin–GFP fusion proteins whereas some fainter lower bands represent COOH-terminal proteolytic products. (D) Pull-down assay of full-length filamin-B variants with GST or GST–β1A and –β1D fusion proteins immobilized on glutathione beads containing GFP-tagged fusion proteins as indicated. Immunoblotting showed binding of filamin-Bvar-1 and filamin-Bvar-1(ΔH1) to GST–β1A, weak binding to GST–β1D, whereas filamin-B and filamin-B(ΔH1) did not bind.
Mentions: After excluding potential disadvantageous effects of the GFP tag on the dimerization of filamin, we generated full-length cDNAs encoding four different filamin-B splice variants and tagged them with GFP (Fig. 7 A). These include the previously reported filamin-B and filamin-B lacking H1 (filamin-B[ΔH1]), as well as the novel identified filamin-B lacking the 41 residues between repeats 19 and 20 (filamin-Bvar-1), and filamin-Bvar-1 without H1 (filamin-Bvar-1[ΔH1]). As shown in Fig. 7 B, the latter two filamin-B variants are expressed in a variety of tissues and cell types, including heart, lung, testis, spleen, thymus, and leukocytes. Several cell types were retrovirally transduced, and after fluorescence-activated cell sorting for filamin-GFP–expressing cells, the stable expression of the different filamin fusion proteins was verified by immunoblotting. In all cell lines, full-length GFP-tagged filamin-B variants migrating at ∼300 kD could be detected (shown for C2C12, Fig. 7 C). Filamin-B expression levels were consistently lower than those of the other variants, which were comparable to each other. Occasionally, smaller protein degradation products were detected, which varied in size and quantity, depending on the transduced cell line and the filamin variant (Fig. 7 C). GST pull-down assays confirmed that deletion of the variant-1 region from full-length filamin-B increases binding to β1 integrins (Fig. 7 D). Only the two filamin-B constructs, filamin-Bvar-1 and filamin-Bvar-1 (ΔH1), in which the variant-1 region had been deleted, but not filamin-B or filamin-B(ΔH1), were precipitated by GST–β1A (Fig. 7 D). Binding of filamin-Bvar-1 and filamin-Bvar-1(ΔH1) to β1D again proved to be weak and could only be demonstrated after long exposures of the film. None of the filamin-B constructs interacted with GST.

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