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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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Identification of three human filamin-B variants and analysis of the genomic organization of the FLN-B gene. (A) Tissue expression of filamin-B variants. PCR was performed on a human multiple tissue cDNA panel, using primers BV1 and BV2 designed to amplify repeats 19 and 20 of filamin-B. The 683-bp band, representing wild-type filamin-B, is present in all samples tested. The 560-bp product marked by one asterisk represents filamin-Bvar-1. The cardiac splice variants (filamin-Bvar-2, 830 bp, and filamin-Bvar-3, 753 bp) are indicated by two asterisks. (B) The partial cDNA sequences and deduced amino acid sequence of the human filamin-B variants. The translation of the two cardiac variants (Bvar-2 and Bvar-3), as well as wild-type and filamin-Bvar-1 is indicated in single letter code. Dots indicate the presence of nucleotides, whereas slashes indicate deletion of the corresponding nucleotides. The putative cAMP-kinase sequence is underlined. The nucleotide sequence data are available from GenBank/EMBL/DDBJ under accession no. AF353666. (C) Schematic diagram representing the genomic organization of the region of FLN-B encoding the repeats 19 and 20 and the composition of the cDNA splice variants. Arrows indicate positions of primers used for cloning. (D) Boundaries at the exon–intron junctions of the FLN-B gene segment. Sizes of exons and introns are indicated as are the consensus splice donor and acceptor sequences; GT/AG of each exon–intron border are underlined. Exons are numbered according to Chakarova et al. (2000). (Footnotes a and b) Nucleotides are numbered according to GenBank/EMBL/DDBJ accession nos. NM_001457 and AF353667 (Fig. 1 D), respectively.
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fig1: Identification of three human filamin-B variants and analysis of the genomic organization of the FLN-B gene. (A) Tissue expression of filamin-B variants. PCR was performed on a human multiple tissue cDNA panel, using primers BV1 and BV2 designed to amplify repeats 19 and 20 of filamin-B. The 683-bp band, representing wild-type filamin-B, is present in all samples tested. The 560-bp product marked by one asterisk represents filamin-Bvar-1. The cardiac splice variants (filamin-Bvar-2, 830 bp, and filamin-Bvar-3, 753 bp) are indicated by two asterisks. (B) The partial cDNA sequences and deduced amino acid sequence of the human filamin-B variants. The translation of the two cardiac variants (Bvar-2 and Bvar-3), as well as wild-type and filamin-Bvar-1 is indicated in single letter code. Dots indicate the presence of nucleotides, whereas slashes indicate deletion of the corresponding nucleotides. The putative cAMP-kinase sequence is underlined. The nucleotide sequence data are available from GenBank/EMBL/DDBJ under accession no. AF353666. (C) Schematic diagram representing the genomic organization of the region of FLN-B encoding the repeats 19 and 20 and the composition of the cDNA splice variants. Arrows indicate positions of primers used for cloning. (D) Boundaries at the exon–intron junctions of the FLN-B gene segment. Sizes of exons and introns are indicated as are the consensus splice donor and acceptor sequences; GT/AG of each exon–intron border are underlined. Exons are numbered according to Chakarova et al. (2000). (Footnotes a and b) Nucleotides are numbered according to GenBank/EMBL/DDBJ accession nos. NM_001457 and AF353667 (Fig. 1 D), respectively.

Mentions: To explore the expression pattern of the transcript for filamin-Bvar-1, cDNAs of multiple human tissues were analyzed in a PCR reaction, using primers that flank repeats 19 and 20 of filamin-B. Fig. 1 A shows that the cDNA encoding the previously reported filamin-B wild-type sequence was amplified from all tissues tested (683-bp product). In addition, a smaller PCR product of 560 bp, corresponding to the filamin-Bvar-1 specific part, was detectable in heart, lung, and skeletal muscle. Nested PCR analysis revealed a weak expression of filamin-Bvar-1 in all tissues tested (unpublished data). A similar splice variant lacking the corresponding region (amino acids 2127–2167) in human filamin-A, filamin-Avar-1, was also detected by (nested) PCR analysis (unpublished data). Two additional filamin-B–specific PCR products of 830 (filamin-Bvar-2) and 753 bp (filamin-Bvar-3) were detected in cardiac tissue (Fig. 1 A). Cloning and sequencing of these PCR fragments revealed that they represent two partially overlapping cardiac filamin-B cDNAs (Fig. 1 B). Intriguingly, in filamin-Bvar-2, the insertion of a 147-bp sequence results in a truncated protein with a unique COOH-terminal sequence of 24 amino acids. The insertion in filamin-Bvar-3 of only the first 70 of the 147 bp inserted in filamin-Bvar-2 leads to a frameshift, and as a result, four more amino acids are encoded COOH-terminally in this protein. Hence, both cardiac-specific filamin-B transcripts encode truncated filamin-B proteins that lack the four COOH-terminal repeats, including the 24th dimerization domain.


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)

Identification of three human filamin-B variants and analysis of the genomic organization of the FLN-B gene. (A) Tissue expression of filamin-B variants. PCR was performed on a human multiple tissue cDNA panel, using primers BV1 and BV2 designed to amplify repeats 19 and 20 of filamin-B. The 683-bp band, representing wild-type filamin-B, is present in all samples tested. The 560-bp product marked by one asterisk represents filamin-Bvar-1. The cardiac splice variants (filamin-Bvar-2, 830 bp, and filamin-Bvar-3, 753 bp) are indicated by two asterisks. (B) The partial cDNA sequences and deduced amino acid sequence of the human filamin-B variants. The translation of the two cardiac variants (Bvar-2 and Bvar-3), as well as wild-type and filamin-Bvar-1 is indicated in single letter code. Dots indicate the presence of nucleotides, whereas slashes indicate deletion of the corresponding nucleotides. The putative cAMP-kinase sequence is underlined. The nucleotide sequence data are available from GenBank/EMBL/DDBJ under accession no. AF353666. (C) Schematic diagram representing the genomic organization of the region of FLN-B encoding the repeats 19 and 20 and the composition of the cDNA splice variants. Arrows indicate positions of primers used for cloning. (D) Boundaries at the exon–intron junctions of the FLN-B gene segment. Sizes of exons and introns are indicated as are the consensus splice donor and acceptor sequences; GT/AG of each exon–intron border are underlined. Exons are numbered according to Chakarova et al. (2000). (Footnotes a and b) Nucleotides are numbered according to GenBank/EMBL/DDBJ accession nos. NM_001457 and AF353667 (Fig. 1 D), respectively.
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fig1: Identification of three human filamin-B variants and analysis of the genomic organization of the FLN-B gene. (A) Tissue expression of filamin-B variants. PCR was performed on a human multiple tissue cDNA panel, using primers BV1 and BV2 designed to amplify repeats 19 and 20 of filamin-B. The 683-bp band, representing wild-type filamin-B, is present in all samples tested. The 560-bp product marked by one asterisk represents filamin-Bvar-1. The cardiac splice variants (filamin-Bvar-2, 830 bp, and filamin-Bvar-3, 753 bp) are indicated by two asterisks. (B) The partial cDNA sequences and deduced amino acid sequence of the human filamin-B variants. The translation of the two cardiac variants (Bvar-2 and Bvar-3), as well as wild-type and filamin-Bvar-1 is indicated in single letter code. Dots indicate the presence of nucleotides, whereas slashes indicate deletion of the corresponding nucleotides. The putative cAMP-kinase sequence is underlined. The nucleotide sequence data are available from GenBank/EMBL/DDBJ under accession no. AF353666. (C) Schematic diagram representing the genomic organization of the region of FLN-B encoding the repeats 19 and 20 and the composition of the cDNA splice variants. Arrows indicate positions of primers used for cloning. (D) Boundaries at the exon–intron junctions of the FLN-B gene segment. Sizes of exons and introns are indicated as are the consensus splice donor and acceptor sequences; GT/AG of each exon–intron border are underlined. Exons are numbered according to Chakarova et al. (2000). (Footnotes a and b) Nucleotides are numbered according to GenBank/EMBL/DDBJ accession nos. NM_001457 and AF353667 (Fig. 1 D), respectively.
Mentions: To explore the expression pattern of the transcript for filamin-Bvar-1, cDNAs of multiple human tissues were analyzed in a PCR reaction, using primers that flank repeats 19 and 20 of filamin-B. Fig. 1 A shows that the cDNA encoding the previously reported filamin-B wild-type sequence was amplified from all tissues tested (683-bp product). In addition, a smaller PCR product of 560 bp, corresponding to the filamin-Bvar-1 specific part, was detectable in heart, lung, and skeletal muscle. Nested PCR analysis revealed a weak expression of filamin-Bvar-1 in all tissues tested (unpublished data). A similar splice variant lacking the corresponding region (amino acids 2127–2167) in human filamin-A, filamin-Avar-1, was also detected by (nested) PCR analysis (unpublished data). Two additional filamin-B–specific PCR products of 830 (filamin-Bvar-2) and 753 bp (filamin-Bvar-3) were detected in cardiac tissue (Fig. 1 A). Cloning and sequencing of these PCR fragments revealed that they represent two partially overlapping cardiac filamin-B cDNAs (Fig. 1 B). Intriguingly, in filamin-Bvar-2, the insertion of a 147-bp sequence results in a truncated protein with a unique COOH-terminal sequence of 24 amino acids. The insertion in filamin-Bvar-3 of only the first 70 of the 147 bp inserted in filamin-Bvar-2 leads to a frameshift, and as a result, four more amino acids are encoded COOH-terminally in this protein. Hence, both cardiac-specific filamin-B transcripts encode truncated filamin-B proteins that lack the four COOH-terminal repeats, including the 24th dimerization domain.

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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