A novel filamin-B splice variant interacts with the β1D integrin subunit in yeast
To identify proteins that interact with the cytoplasmic domain of β1D, specific for cardiac and skeletal muscle, we screened a human skeletal muscle cDNA library for interacting clones, using the β1D cytoplasmic domain as bait in a yeast two-hybrid screen. Approximately 6 × 106 clones were screened. From 67 positive clones, two identical clones encoding the 5 COOH-terminal repeats of filamin-B (amino acids 2027–2602) were isolated. Strikingly, in both filamin-B clones, 41 amino acids were deleted from the region between filamin repeats 19 and 20 (residues 2082–2122). This filamin variant was named filamin-Bvar-1. In a yeast two-hybrid screen for clones interacting with β1A, using a human keratinocyte cDNA library (29 × 106 clones), we isolated, from 16 positive clones, 4 identical filamin-Bvar-1 clones. The other isolated clones encoded either false positives or will be described elsewhere. Additionally, a filamin-Bvar-1 clone encoding EST was published under GenBank/EMBL/DDBJ accession no. W40525 (pancreatic islets). These results show that filamin-Bvar-1 binds to the cytoplasmic domains of both β1A and β1D, and the expression of filamin-Bvar-1 is not restricted to skeletal muscle.
Expression and genomic determination of novel filamin-B splice variants
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. 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 Nested PCR analysis revealed a weak expression of filamin-Bvar-1 in all tissues tested (unpublished data).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region (amino acids 2127–2167) in human filamin-A, filamin-Avar-1, was also detected by (nested) PCR analysis (unpublished data).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 ( A). Cloning and sequencing of these PCR fragments revealed that they represent two partially overlapping cardiac filamin-B cDNAs ( 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.
Figure 1. 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 (more ...)
Genomic PCR mapping with primers designed from exons 39 and 41, encoding the filamin repeats 19 and 20 (), revealed that the stretch of 41 amino acids, deleted in filamin-Bvar-1, is encoded by a single exon of 123 bp. This exon is preceded by a 102-bp intron and followed by an intron of ~2.5 kb. Furthermore, we found that the cDNA sequence of filamin-Bvar-2, specific for heart, is encoded by a 147-bp exon, exon 40B/C. Alternative RNA splicing at the internal splice site, located in exon 40B/C, produces the filamin-Bvar-3 transcript.
Filamin isoforms and their variants determine specificity for association with β subunits
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 ( 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) ( 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.
Figure 2. 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 (more ...)
In contrast to filamin-B(19–24), the corresponding filamin-A(19–24) construct did not bind to β1A or any other β subunit. However, filamin-Avar-1(19–24) and the shorter filamin-A(20*–24) construct (amino acids 2168–2647) interacted strongly with β1A and weakly with β3 and β6, but neither of them interacted with β1D. None of the aforementioned filamin constructs interacted with the β2 cytoplasmic domain (unpublished data). Taken together, these data show that the affinity of filamin-B for β1A is strongly increased when the stretch of 41 amino acids is deleted from it. Similarly, the removal of the same region from filamin-A induces binding to β1A. Furthermore, both variants become capable of binding to other integrin β subunits.
Analysis of a series of filamin-B constructs truncated at the NH2 or COOH terminus indicates that repeat 21 is necessary, but not sufficient, for interaction with β1A ( A). It is possible that the presence of repeat 24 facilitates the dimerization of filamin, and thus of repeat 21, thereby greatly increasing the strength of the binding to β1A. The interaction of filamin-Bvar-1 with β3 and β6 was abolished by the deletion of repeats 23–24, whereas it did not affect binding to β1A or β1D. Interactions of both β1A and β1D with filamin-Bvar-1(19 and 20) could still be demonstrated, although the binding appeared to be weaker than that of a construct containing repeat 21, filamin-Bvar-1(19–21). These data suggest that deletion of the 41–amino acid region in filamin-B leads to either the removal of inhibitory sequences in repeat 19 or to the introduction of a new binding site for β1A and β1D in the remaining part of repeat 20 (amino acids 1995–2185). The presence of repeat 21 increases binding activity, which is consistent with the data showing that this repeat is necessary for efficient binding of filamin-B to β1A and contributes to the binding activity of filamin-Bvar-1 (19–21) to β1 integrins. As may be the case for the binding of filamin-B to β1A, binding of filamin-Bvar-1 to β3 and β6 probably requires dimerization mediated by repeat 24.
Biochemical interaction of filamin variants with integrins
To confirm the interactions between the different filamin splice variants and the β1A and β1D subunits observed in yeast, we expressed the regions containing repeats 19–24 of filamin-Avar-1 or filamin-Bvar-1, or the corresponding regions of wild-type filamin-A(19–24) or filamin-B(19–24), in COS-7 cells. The proteins were tagged at their NH2 terminus with hemagglutinin A (HA) and expression of equivalent amounts of proteins in COS-7 cells was confirmed by immunoblotting with anti-HA antibody ( A). Binding of the filamin constructs to β1A and β1D was tested in a pull-down assay using glutathione-S-transferase (GST) fusion proteins containing the cytoplasmic domains of these integrin subunits, immobilized on glutathione-Sepharose beads ( B). As shown in C, GST–β1A bound to filamin-Avar-1(19–24) and filamin-Bvar-1(19–24), but not to the corresponding fragments of the wild-type filamin isoforms (filamin-A, 19–24, and filamin-B, 19–24). Binding of GST–β1D to the different filamin-A and filamin-B constructs was either undetectable or very weak. The interaction between β1A and filamin-B(19–24) detected in yeast could not be confirmed in the pull-down assay, probably because it is too weak. Interestingly, we found that a truncated filamin-B construct that lacks the first 14 amino acids of repeat 19 (filamin-B, 2009–2602) could be efficiently precipitated with GST–β1A (unpublished data). Thus, it appears that not only the deletion of COOH-terminal residues of repeat 19, as in the variant-1 protein, but also the deletion of NH2-terminal residues of repeat 19 results in stronger binding of filamin-B to β1A. Together, these data suggest that repeat 19 contains an inhibitory element for binding of filamin-B to β subunits.
Figure 3. Binding of filamin isoforms and variants to GST–β1A and –β1D fusion proteins. (A) Detection of the expressed products from HA-tagged filamin-B cDNA constructs (filamin repeats 19–24) in lysates of COS-7 cells, transiently (more ...)
Identification of the binding sites for filamins on β1A and β1D
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 () . 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.
Figure 4. 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 (more ...)
Analysis of deletions of the cytoplasmic domain of β1A shows that for binding to filamin-B, almost the complete cytoplasmic domain of β1A is required. Only the last 10 COOH-terminal amino acids of β1A can be deleted without causing a loss of interaction. The binding site for filamin-Bvar-1 on β1A comprises both the NPXY motifs and the intervening sequence, whereas on β1D additional membrane proximal sequences are needed. Similarly, the minimal binding site for filamin-A(20*–24) on β1A comprises the two NPXY motifs and the intervening sequence, which resembles the binding site for filamin-Bvar-1. Thus, it appears that the binding site on β1A for the low-affinity interaction with filamin-B is distinct from the one mediating high-affinity binding to filamin-Avar-1 and filamin-Bvar-1. The sequence requirements for the binding of β1D to filamin-Bvar-1 resemble those for the low affinity interaction of β1A to filamin-B ().
Alternative splicing of filamins during in vitro myogenesis
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 ( 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
). , 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 ( 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
Figure 5. 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, (more ...)
The expression of filamin-B proteins in C2C12 cells was assessed by immunoblotting with an antibody specific for the H1 region of filamin-B ( C). In agreement with the PCR data, the level of filamin-B protein containing the H1 region decreased during the differentiation of C2C12 cells. In contrast, the expression of β1D and sarcomeric myosin heavy chain (MHC) was induced in differentiating C2C12 cells.
GFP COOH-terminal tags do not interfere with filamin dimerization
Before initiating studies to define the cellular localization of the different splice variants of filamin-B, we examined whether the addition of GFP at the COOH terminus of filamin influences the ability of this protein to form dimers. To this end, a filamin-B(19–24) construct with GFP at the COOH-terminal end, and a control construct, tagged with HA at the NH2-terminal end, were transiently expressed in CHO cells. After 2 d, cell lysates and intact cells were treated with the chemical cross-linking reagent dithiobis-succinimidyl propionate (DSP) at two concentrations. As shown in , the addition of increasing amounts of cross-linker led to a shift from monomeric to dimeric tagged filamins, as visualized by immunoblotting with antibodies against HA or GFP. The similar dimerization capacity of HA- and GFP-tagged filamin-B(19–24) indicates that the GFP tag had no effect on dimerization. The same samples under reducing conditions, which disrupt the disulfide bond, only contained filamin monomers. Specificity of the cross-linking reaction was checked using the NH2-terminal HA-tagged filamin-Bvar-1(19–23) construct, which did not form dimers due to truncation of the COOH-terminal repeat 24, which is required for dimerization.
Figure 6. The GFP tag on the COOH-terminal part of filamin-B does not interfere with dimerization in vivo. CHO cells were transfected with either the NH2-terminal HA- or the COOH-terminal GFP-tagged FLN-B(19–24) or with the NH2-terminal HA-tagged FLN-B (more ...)
Subcellular distribution of full-length filamin-B variants and their interaction with β1 subunits
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 ( 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 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, 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 ( C). GST pull-down assays confirmed that deletion of the variant-1 region from full-length filamin-B increases binding to β1 integrins ( 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 ( 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.
Figure 7. 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 (more ...)
The subcellular distribution of the different GFP-tagged filamin-B variants was examined in GD25-β1A mouse fibroblasts stably expressing these proteins. The localization of GFP-tagged filamin-B () was identical to that of endogenous filamin-B, as revealed by immunostaining using an antibody against the H1 region of filamin-B (). Thus, the GFP tag did not interfere with the normal localization of filamin-B. Filamin-B was localized at actin stress fibers but was not appreciably concentrated in focal contacts. The distribution of filamin-B(ΔH1) and filamin-Bvar-1 variants was similar to that of filamin-B (). Interestingly, filamin-Bvar-1(ΔH1) was associated with a proportion of the peripheral focal contacts positive for vinculin (, G–O, and E). There was also GFP fluorescence in the nucleus in many of these cells, the significance of which is unknown. As anticipated, the filamin-Bvar-1(ΔH1) variant did not react with anti–filamin-B H1 antibody. However, its presence in focal contacts was revealed by GFP fluorescence, whereas endogenous filamin-B, which does react with this antibody, was found associated with actin stress fibers (, M–O). Neither endogenous filamin-B ( B) nor any of the filamin-B variants (unpublished data) were enriched in lamellipodia. The expression of filamin-Bvar-1(ΔH1) in focal contacts did not have an apparent effect on the composition and localization of these structures. They were confined at the end of actin stress fibers (, J–L, and F) and in addition to vinculin, they contained talin, phospho-tyrosine, and paxillin (). We did not detect α-actinin in focal contacts ( C). Lastly, in GD25-β1D cells expressing filamin-Bvar-1(ΔH1) there was also a prominent staining of some focal contacts (unpublished data).
Figure 8. Expression and localization of filamin-B variants in GD25–β1A mouse fibroblasts. GD25–β1A cells (A and B) and GD25–β1A cells stably expressing GFP-tagged filamin-B (C and D), filamin-Bvar-1 (E), or filamin-B (more ...)
Figure 9. Localization of filamin-Bvar-1(ΔH1) in focal contacts. Confocal microscopy of GD25-β1A cells showing the green fluorescence of filamin-Bvar-1(ΔH1) compared with red talin (A), phosphotyrosine (B), α-actinin (C), paxillin (more ...)
We conclude that the localization of filamin-Bvar-1(ΔH1) in focal contacts requires, in addition to the characterized variant-1 high-affinity binding site for β1 integrins, a function that is induced by the loss of the H1 region.
Filamin-B variants affect myoblast differentiation in vitro
The functional significance of the developmentally regulated splicing of the H1 region in filamin-B, as well as the deletion of the 41–amino acid region, was explored by analyzing the effects of ectopic expression of the four different filamin-B splice variants on myogenesis of C2C12 cells. Myogenic differentiation was induced by switching the culture to a medium containing 2% horse serum (differentiation medium). Interestingly, C2C12 cells expressing filamin-Bvar-1(ΔH1) fused into myotubes within 2 to 3 d after the medium switch, which is 1–2 d earlier than the fusing of cells from the other transduced cell lines ( A). Furthermore, the myotubes formed by the cells expressing the filamin-B variants lacking the H1 region (filamin-B[ΔH1] and filamin-Bvar-1[ΔH1]) were thinner than those formed by the other transduced cell lines or GFP control cells. This difference in morphology was more obvious when the myotubes were stained for MHC ( and ) .
Figure 10. Effects of the ectopic expression of filamin-B variants on myogenesis. C2C12 myoblasts stably expressing GFP and GFP fusions of the indicated filamin-B variants were grown to confluence and then switched to differentiation medium. (A) The phase contrast (more ...)
Figure 11. Localization of GFP or GFP fusions of filamin-B or filamin- Bvar-1(ΔH1) in undifferentiated and differentiated C2C12 cells. (A–C) Confocal microscopy of undifferentiated C2C12 myoblasts showing the green fluorescence of GFP and two GFP (more ...)
Figure 12. Effects of filamin-B variants on myogenesis. Confocal microscopy of C2C12 myoblasts stably expressing GFP fusions of filamin-B variants, 6 d after induction of differentiation. Sarcomeric MHC was immunolabeled in cells to facilitate myotube identification (more ...)
In the differentiating myotubes, filamin-B, filamin-Bvar-1, and filamin-B(ΔH1) were localized diffusely throughout the cytoplasm, with regions of enrichment at the longitudinal actin stress fibers (). In contrast, the localization of filamin-Bvar-1(ΔH1) was typically polarized and dotted at the periphery of tubes (). GFP alone was mainly localized in the nucleus (). Occasionally, depending on the culture conditions, in well-differentiated tubes with clear sarcomeric organization, filamin-B was localized at the Z-lines, as shown by costaining for sarcomeric α-actinin, and at intermediate M-bands ().
Immunoblot analysis of MHC demonstrated that the morphological differentiation is accompanied by biochemical changes. The induction of MHC in the filamin-Bvar-1(ΔH1)–expressing C2C12 cells was faster than in the other cells, where the induction was similar to that in GFP control cells ( B). Taken together, these results show that expression of the filamin-double variant, filamin-Bvar-1(ΔH1), accelerates muscle differentiation in vitro.