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PubMed2003

Direct interaction between the actin-binding protein filamin-A and the inwardly rectifying potassium channel, Kir2.1.

The role of filamins in actin cross-linking and membrane stabilization is well established, but recently their ability to interact with a variety of transmembrane receptors and signaling proteins has led to speculation of additional roles in scaffolding and signal transduction. Here we report a direct interaction between filamin-A and Kir2.1, an isoform of inwardly rectifying potassium channel expressed in vascular smooth muscle and an important regulator of vascular tone. Yeast two-hybrid screening of a porcine coronary artery cDNA library using the carboxyl terminus of Kir2.1 as bait yielded cDNA encoding a fragment of filamin-A (residues 2481-2647). Interaction between filamin-A and Kir2.1 was confirmed by in vitro overlay assay of membrane-bound Kir2.1 with glutathione S-transferase fusion protein of the isolated filamin clone. Additionally, antibodies directed against Kir2.1 coimmunoprecipitated filamin-A from arterial smooth muscle cell lysates, and immunocytochemical analysis of individual arterial smooth muscle cells showed that Kir2.1 and filamin co-localize in "hotspots" at the cell membrane. Interaction with filamin-A was found to have no effect on Kir2.1 channel behavior but, rather, increased the number of functional channels resident within the membrane. We conclude that filamin-A is potentially an important regulator of Kir2.1 surface expression and location within vascular smooth muscle.

Actins↗
PubMed1989

Identification of a filamin isoform enriched at the ends of stress fibers in chicken embryo fibroblasts.

Filamin (actin-binding protein) is a cytoskeletal protein that crosslinks actin filaments in vitro. Filamin is thought to be involved in a variety of cell types in stabilizing actin networks, and in platelets it may play a role in linking actin to the membrane. In this report, we describe a monoclonal antibody (Mab 6E) that was used to immunoprecipitate an isoform of filamin from extracts of chicken fibroblasts revealed an unusual pattern: while other filamin antibodies stained the entire length of stress fibers, the Mab 6E staining was predominantly at the ends of stress fibers. In double-labeling experiments, the distribution of the Mab 6E antigen was found to be strikingly similar to that of alpha-actinin. Mab 6E staining was associated, in part, with focal adhesions, which are sites of actin-membrane attachment. Unlike other focal adhesion proteins, such as vinculin and talin, this filamin isoform is apparently not localized evenly throughout the entire area of adhesion, being absent from or greatly reduced in the distal portion of the area. The Mab 6E antigen was identified as filamin by immunological crossreactivity with a panel of antifilamin monoclonals as well as with a polyclonal anti-filamin. The Mab 6E isoform, however, was found to differ from the major form of filamin both by one-dimensional peptide analysis and slightly slower migration on SDS-containing gels. The Mab 6E antigen was also detected by immunofluorescence in the Z-lines of isolated adult myofibrils. These results suggest that chicken fibroblasts may express different isoforms of filamin that could have specialized roles within the cell.

Actinin↗
PubMed2000

The bi-directional regulation of filamin on the ATPase activity of smooth muscle myosin.

OBJECTIVE: The aim of this study is to investigate the functional relationship between filamin, a known actin binding protein, and myosin and the effects of filamin on the interaction between myosin and actin. METHODS: Ultra-centrifugation method was used to investigate the binding of filamin to both phosphorylated and unphosphorylated myosins. Mg-ATPase activities of both phosphorylated and unphosphorylated myosins in the presence and absence of actin were measured to observe the effects resulted from filamin-actin and filamin-myosin interactions. RESULTS: It was found that filamin is also a myosin binding protein. Filamin inhibited the actin activated Mg-ATPase activity of phosphorylated myosin and stimulated Mg-ATPase of phosphorylated myosin in the absence of actin; in addition, filamin stimulated Mg-ATPase activity of unphosphorylated myosin in both the presence or absence of actin. CONCLUSION: The result suggest that the effects of filamin on the myosin Mg-ATPase activities are bi-directional, i.e., stimulatory via binding to myosin and inhibitory via binding to actin.

Actins↗
PubMed1976

Purification and properties of filamin, and actin binding protein from chicken gizzard.

Filamin, a protein recently identified in chicken gizzard (Wang, K., Ash, F., and Singer, S. J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 4483-4486), has been purified free of other components and its molecular properties have been examined. Filamin has a sedimentation constant (S020,w) of 8.86 S and a partial specific volume of 0.734 ml/g. Sedimentation equilibrium experiments give a value of 498,000 for the molecular weight of native filamin. From these data a frictional ratio of 2.32 has been calculated. On sodium dodecyl sulfate gel electrophoresis, filamin migrates as a single protein band with an estimated molecular weight of 240,000. Filamin is a soluble protein and under a variety of conditions tested does not by itself form filaments or precipitate from solution. However, filamin binds to rabbit skeletal muscle F-actin, and the complex is readily sedimented by centrifugation to yield a gelatinous pellet containing actin and filamin. These results indicate that filamin is a dimeric protein with a moderate degree of asymmetry that binds to actin. The results also suggest that the distribution of filamin in cells is derived from its interaction with polymerized actin.

Actins↗
PubMed2000

Filamin isogene expression during mouse myogenesis.

The developmental pattern of filamin gene expression has been studied in mouse embryos by using in situ hybridization. The probes used were isoform specific, (35)S-labeled antisense complementary ribonucleic acids (cRNAs) to the 3; untranslated region (3; UTR) of muscle-specific and nonmuscle-specific filamin genes. Northern blot and in situ hybridization results showed that nonmuscle-specific filamin transcripts had a size of 9.5 kb and were expressed in all nonmuscle tissues. Labeling was most intense in tissues containing a substantial proportion of epithelial and smooth muscle cells. Muscle-specific filamin transcripts had a size of 10 kb and were expressed primarily in cardiac and skeletal muscle. The expression of muscle-specific filamin messenger ribonucleicacids (mRNAs) was detected in heart at 8.0 days after coitum, whereas that in the myotomes of somites was not detected until 10.5 days after coitum. The expression of muscle-specific filamin mRNAs in heart and in skeletal muscle continued through the subsequent days of myogenesis. The results showed that muscle-specific filamin gene transcripts are detected before the formation of myotubes in vivo. This is the first study of filamin gene expression at the early stages of skeletal muscle development. Dev Dyn 2000;217:99-108.

Animals↗
PubMed1998

Expression and subcellular distribution of filamin isotypes in endothelial cells and pericytes.

Two principal forms of the actin binding protein, filamin, are expressed in mammalian cells: nonmuscle and muscle isotypes (FLN-1 and FLN-2). A protein that copurifies with an alpha-naphthyl acetate hydrolyzing esterase from human omentum microvessel endothelial cells (EC) is isolated by nondenaturing electrophoresis, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and electroblotting. The purified protein is subjected to in situ trypsin cleavage, reversed-phase high performance liquid chromatography (HPLC) and automated Edman degradation. Six peptide fragments from the protein are identified to have 60-66% identity with nonmuscle filamin (ABP-280). Two of these peptides are 100% identical to a previously sequenced human muscle filamin fragment. Polyclonal antibody is produced using a 16-residue synthetic peptide corresponding to a structural beta-sheet region of muscle filamin. Compared with a variety of vascular cells evaluated, retinal pericytes express an abundance of both muscle and non-muscle filamin isotypes. Pericytes contain at least 10 times more muscle filamin than human umbilical vein EC and at least three times the amount expressed in human omentum microvessel and bovine pulmonary artery EC. Differential detergent fractionation indicates that both filamin isotypes are primarily localized in the cytosol and membrane/organelle fractions of pericytes. Another actin crosslinking protein, alpha-actinin, is primarily found in the cytosol and cytoskeletal fractions. The dynamic regulation of actin microfilament organization in pericytes may be controlled in part by the two filamin isotypes, which in turn may contribute to pericyte contractility.

Amino Acid Sequence↗
PubMed2006

Expression pattern of Filamin-240 in Drosophila blood cells.

The expression pattern of Filamin-240 was studied in subsets of Drosophila blood cells by means of immunofluorescent staining and Western blot analysis with use of an antibody specific to a "filamin-folding domain", a consensus motif profile generated from the 20 existing filamin repeats. Expression of Filamin-240 is restricted to lamellocytes - a special blood cell type of the cellular immune response - and is involved in the regulation of lamellocyte development. In the cher1 homozygous larvae, which lack Filamin-240 protein, a vigorous lamellocyte differentiation occurs which is further enhanced upon in vivo immune challenge by a parasitic wasp, Leptopilina boulardi. By introducing a full-length transgene encoding the Drosophila Filamin-240 protein into the cher1 Filamin-deficient homozygous mutant, the mutant blood cell phenotype was rescued. These data demonstrate that the expression of Filamin-240 is strictly lamellocyte specific in Drosophila blood cells and that the protein is a suppressor of lamellocyte development.

Animals↗
PubMed2003

Filamin C accumulation is a strong but nonspecific immunohistochemical marker of core formation in muscle.

Filamin C is the muscle isoform of a group of large actin-crosslinking proteins. On the one hand, filamin C is associated with the Z-disk of the myofibrillar apparatus and binds to myotilin; on the other hand, it interacts with the sarcoglycan complex at the sarcolemma. Filamin C may be involved in reorganizing the cytoskeleton in response to signalling events and in muscle it may, in addition, fulfill structural functions at the Z-disk. An examination of biopsies from patients with multi-minicore myopathy, central core myopathy and neurogenic target fibers with core-like target formations (TF) revealed strong reactivity of all the cores and target formations with two different anti-filamin C antibodies. In all three conditions, the immunoreactivity in the cores for filamin C was considerably stronger than that for desmin. Only for alphaB-crystallin were comparable levels of immunoreactivity detected. There was no difference in intensity for filamin C between the three pathological conditions. Thus, filamin C along with alphaB-crystallin is a strong and robust, but nonspecific marker of core formation. The reason why filamin C accumulates in cores is unclear at present, but we postulate that it may be critically involved in the chain of events eventually leading to myofibrillar degeneration.

Biomarkers↗
PubMed1999

Drosophila filamin encoded by the cheerio locus is a component of ovarian ring canals.

BACKGROUND: The ring canals in the ovary of the fruit fly Drosophila provide a versatile system in which to study the assembly and regulation of membrane-associated actin structures. Derived from arrested cleavage furrows, ring canals allow direct communication between cells. The robust inner rim of filamentous actin that attaches to the ring-canal plasma membrane contains cytoskeletal proteins encoded by the hu-li-tao shao (hts) and kelch genes, and is regulated by the Src64 and Tec29 tyrosine kinases. Female sterile cheerio mutants fail to recruit actin to ring canals, disrupting the flow of cytoplasm to oocytes. RESULTS: We have cloned cheerio and found that it encodes a member of the Filamin/ABP-280 family of actin-binding proteins, known to bind transmembrane proteins and crosslink actin filaments into parallel or orthogonal arrays. Antibodies to Drosophila Filamin revealed that Filamin is an abundant ring-canal protein and the first known component of both the outer and inner rims of the ring canal. The cheerio gene also encodes a new Filamin isoform that lacks the actin-binding domain. CONCLUSIONS: Localization of Filamin to nascent ring canals is necessary for the recruitment of actin filaments. We propose that Filamin links filamentous actin to the plasma membrane of the ring canal. Although loss of Filamin in human cells supports a role for Filamin in organizing orthogonal actin arrays at the cell cortex, the cheerio mutant provides the first evidence that Filamin is required in membrane-associated parallel actin bundles, such as those found in ring canals, contractile rings and stress fibers.

Animals↗
PubMed2003

The limits of promiscuity: isoform-specific dimerization of filamins.

Filamins are a family of actin cross-linking proteins that are primarily localized in the cortical cytoplasm of all mammalian cells. Until now, three major isoforms (filamins a, b, and c) have been identified, that were shown to be differentially expressed and/or localized in different tissues. An amino-terminal double CH-domain actin binding domain, and a dimerization region in the carboxy-terminal portion of the protein are the molecular basis for its actin cross-linking activity. Chemical cross-linking of bacterially expressed recombinant proteins was used to demonstrate that in all three filamin isoforms the most carboxy-terminally situated immunoglobulinlike domain is required and sufficient for dimerization. The efficiency of the dimerization was increased upon inclusion of the preceding hinge 2 region, indicating a function for this region in the regulation of dimerization. By mixing recombinant proteins derived from different filamin isoforms, we found that heterodimer formation is possible between filamins b and c but not between filamin a and the other two filamins. This selectivity of dimerization might provide a further molecular explanation for the differential intracellular sorting of filamin isoforms and their distinct properties.

Amino Acid Sequence↗
PubMed2001

Increased filamin binding to beta-integrin cytoplasmic domains inhibits cell migration.

Multicellular animal development depends on integrins. These adhesion receptors link to the actin cytoskeleton, transmitting biochemical signals and force during cell migration and interactions with the extracellular matrix. Many integrin-cytoskeleton connections are formed by filamins and talin. The beta7 integrin tail binds strongly to filamin and supports less migration, fibronectin matrix assembly and focal adhesion formation than either the beta1D tail, which binds strongly to talin, or the beta1A tail, which binds modestly to both filamin and talin. To probe the role of filamin binding, we mapped the filamin-binding site of integrin tails and identified amino acid substitutions that led to selective loss of filamin binding to the beta7 tail and gain of filamin binding to the beta1A tail. These changes affected cell migration and membrane protrusions but not fibronectin matrix assembly or focal adhesion formation. Thus, tight filamin binding restricts integrin-dependent cell migration by inhibiting transient membrane protrusion and cell polarization.

Amino Acid Substitution↗
PubMed2004

Filamin A binding stabilizes nascent glycoprotein Ibalpha trafficking and thereby enhances its surface expression.

The glycoprotein (Gp) Ib-IX-V complex is essential for platelet-mediated hemostasis and thrombosis. The cytoplasmic domain of its largest polypeptide subunit GpIbalpha possesses a binding region for filamin A, which links GpIb-IX-V to the platelet cytoskeleton. There is evidence that filamin A binding to GpIbalpha directs the surface expression of GpIb-IX. To investigate the mechanism of this effect, we examined GpIbalpha biosynthesis in Chinese hamster ovary (CHO) cells stably co-expressing wild-type or mutant GpIbalpha with GpIbbeta, GpIX with and without filamin A. We observed that surface GpIbalpha expression is enhanced in CHO cells co-expressing human filamin A. In comparison with cells expressing only GpIbalpha, GpIbbeta, and GpIX (CHO-GpIbalpha/betaIX), lysates from CHO-GpIbalpha/betaIX + filamin A-expressing cells showed greater amounts of immature, incompletely O-glycosylated and fully mature GpIbalpha, but lesser amounts of the approximately 15-kDa C-terminal peptide released when the extracellular domain of GpIbalpha is cleaved by proteases. When filamin A binding is eliminated by truncation of GpIbalpha at C-terminal residue 557 or by a deletion between amino acids 560-570, the decreased synthesis of mature GpIbalpha is accompanied by decreased immature GpIbalpha and by an increased immunodetectable C-terminal peptide. The synthesis of mature GpIbalpha in CHO-GpIbalpha/betaIX cells is eliminated by brefeldin A (which inhibits transport out of the endoplasmic reticulum (ER)) and restored by lactacystin (which inhibits proteasomal degradation). These results suggest that GpIbalpha binds to filamin A within the ER and that filamin A binding directs post-ER trafficking of GpIbalpha to the cell surface.

Acetylcysteine↗
PubMed1980

Filamin concentration in cleavage furrow and midbody region: frequency of occurrence compared with that of alpha-actinin and myosin.

Affinity-purified rabbit antibody to purified chicken gizzard filamin was used in indirect immunofluorescence to localize filamin in dividing chick embryo cells. The antibody was shown to bind only chick embryo cell filamin when whole cell extracts were analyzed by the sensitive sodium dodecyl sulfate-polyacrylamide gel electrophoresis overlay technique described by Adair et al. (1978, J. Cell Biol. 790:281-285). The results show that filamin is located in stress fibers and membrane ruffles during interphase. As cells prophase, the condensing chromosomes are surrounded by diffuse antifilamin staining. No stress fibers are apparent. During metaphase and anaphase, the staining is bright but diffuse. There is often peripheral membrane staining. Filamin is not concentrated in the spindle region but neither is it excluded from the spindle. During cytokinesis, filamin is found highly concentrated in the cleavage furrow in 16 out of 100 cells examined. This frequency of concentration in the furrow is comparable to that observed for alpha-actinin (14%). Myosin concentration in the furrow is more frequent; it is observed in 37% of the cells examined. Neither myosin, alpha-actinin, nor filamin is observed concentrated in the furrow 100% of the time. We conclude that the results are consistent with, but not sufficient to prove, the hypothesis that alpha-actinin and filamin are essential components of the mechanism of cytokinesis.

Actinin↗
PubMed1987

The role of tropomyosin in the interactions of F-actin with caldesmon and actin-binding protein (or filamin).

The interactions of actin filaments with actin-binding protein (filamin) and caldesmon under the influence of tropomyosin were studied in detail using falling-ball viscometry, binding assay and electron microscopy. Caldesmon decreased the binding constant of filamin with F-actin. In contrast, the maximum binding ability of filamin to F-actin was decreased by tropomyosin. The filamin-induced gelation of actin filaments was inhibited by caldesmon. Tropomyosin also inhibited this gelation. The effect of caldesmon became stronger under the influence of tropomyosin. Furthermore, both caldesmon and tropomyosin additionally decreased the filamin binding to F-actin. From these results, caldesmon and tropomyosin appeared to influence filamin binding to F-actin with different modes of actin. In addition, there was no sign of direct interactions between filamin, caldesmon and tropomyosin as judged from gel filtration. Under the influence of caldesmon and tropomyosin, calmodulin conferred Ca2+ sensitivity on the filamin-induced gelation of actin filaments.

Actins↗
PubMed1985

The filamins: properties and functions.

The filamins are a group of homologous proteins defined by their high native molecular weight (500,000), their amino acid compositions, their cross-reactivity to antibodies to heterologous filamins, their localization to actin networks and bundles in situ, and their ability to cross-link actin filaments in vitro into three-dimensional networks and bundles. Native filamins contain two subunits (relative mass = 250 000). Each subunit carries at least one actin-binding site and formation of bivalent dimers is therefore believed to explain filamin's ability to cross-link actin filaments. Formation of networks in vitro (corresponding to formation of macroscopic gels) has been analyzed using the theory of Flory. As predicted, a sharp transition to gel (at the critical gelation concentration of filamin) is observed when actin is mixed with increasing concentrations of filamin and the critical gelation concentration is found to vary inversely with the length of actin filaments. However, the measured values of the critical gelation concentration are all higher (2- to 14-fold) than predicted by the theory and the prediction that the critical concentration varies directly with the actin concentration was verified with only one of two techniques used. Filamin's length (160-190 nm) and flexibility (1000-fold greater than actin filaments) may make it especially well fitted to cross-link actin filaments into three-dimensional networks when present in low molar ratios (1:200 to 1:50) relative to actin. At higher molar ratios (greater than 1:20) it also cross-links actin filaments into bundles. Assuming that filamin actually helps organize supramolecular structures inside cells (not yet tested directly), then its concentration relative to actin may help determine whether networks or bundles are formed. Other factors that may influence its localization and function inside cells include competition with other actin-binding proteins (such as myosin and tropomyosin) for binding sites on actin and phosphorylation, which may alter its ability to bind to actin.

Actins↗
PubMed2003

[The changes of filamin distribution in renal tubular epithelial cells during kidney ischemia/reperfusion injury in neonatal rats].

OBJECTIVE: By investigating the changes of filamin distribution in renal tubular epithelial cells at different ischemia/reperfusion time stages we studied the role of filamin in kidney ischemia/reperfusion injury. METHODS: The neonatal SD rat ischemia/reperfusion model was set up. Immunofluorescent staining was used to show the distribution of filamin in renal tubular epithelial cells and the quantities of filamin were counted by ImagePlus-Pro system. RESULTS: Filamin was located around the basal plasma membrane in normal renal tubular epithelial cells. After ischemia for 0.5 h, the redistribution was not significant. After reperfusion for 0.5 h, filamin came into the cell plasma, appeared at the apex of cells and in the lumen. This kind of change was most apparent after reperfusion for 2 h and accompanied with destruction of renal tubules. The regeneration started 24 h post-ischemia. Filamin was redistributed to the basal plasma membrane. After reperfusion for 120 h, the regeneration ended and the structure of renal tubules became normal. CONCLUSION: The distribution of filamin was changed during ischemia/reperfusion injury, this change was prior to the change of actin cytoskeleton and integrin.

Actins↗
PubMed1993

Molecular characterization of a multi-promoter gene encoding a chicken filamin protein.

We report the cloning and sequencing of cDNA encoding a chicken filamin protein. The 2,567 amino acid protein contains an NH2-terminal 267 amino acid actin-binding domain followed by a series of 24 repeating units that are each approximately 95 amino acids in length. The overall primary structure of filamin closely resembles that of human actin-binding protein (ABP). However, filamin lacks a 24-amino-acid insertion prior to repeat 16 that is contained within ABP. This region of human ABP is a site of calpain cleavage and is thought to confer flexibility on the molecule. Hence, it is possible that the properties of actin gels formed with either human ABP or filamin reflect the presence or absence of this insertion. Filamin is encoded within a multi-promoter transcription unit. A downstream filamin promoter (Fil1) resembles those of certain housekeeping genes and has a putative binding site for the transcription factor E2F. Thus, transcription from this promoter may be linked to the cell cycle. A second filamin promoter (Fil2) is located at least 8 kilobases upstream from the Fil1 promoter. This structural arrangement suggests that regulation of filamin gene expression is likely to be complex.

Amino Acid Sequence↗
PubMed1978

Purification of mammalian filamin. Similarity to high molecular weight actin-binding protein in macrophages, platelets, fibroblasts, and other tissues.

We have purified the high molecular weight actin-binding protein, filamin from guinea pig vas deferens. We find this mammalian filamin is very similar to chicken gizzard filamin in subunit molecular weight, amnio acid composition, actin-binding properties, immunological cross-reactivity, and the ability to be phosphorylated by cyclic AMP-dependent protein kinase. Anti-filamin antibodies cross-react with a high molecular weight macrophage actin-binding protein, and with a high molecular weight protein in platelets and fibroblasts. Furthermore like filamin, these proteins are also phosphorylated and cyclic AMP stimulates their phosphorylation. Anti-filamin antibodies do not cross-react with the erythrocyte membrane protein spectrin or with high molecular weight proteins in brain extracts. We conclude that filamin from avian and mammalian smooth muscle are very similar proteins and furthermore that many, but not all, non-muscle cells contain a protein closely related to filamin.

Actins↗