Structural and functional aspects of filamins

Filamins are a family of high molecular mass cytoskeletal proteins that organize filamentous actin in networks and stress fibers. Over the past few years it has become clear that filamins anchor various transmembrane proteins to the actin cytoskeleton and provide a scaffold for a wide range of cytoplasmic signaling proteins. The recent cloning of three human filamins and studies on filamin orthologues from chicken and Drosophila revealed unexpected complexity of the filamin family, the biological implications of which have just started to be addressed. Expression of dysfunctional filamin-A leads to the genetic disorder of ventricular heterotopia and gives reason to expect that abnormalities in the other isogenes may also be connected with human disease. In this review aspects of filamin structure, its splice variants, binding partners and biological function will be discussed.     

The ABP-120 C-end region from Entamoeba histolytica interacts with sulfatide, a new lipid target

EhABP-120 is the first filamin identified in the parasitic protozoan Entamoeba histolytica. Filamins are a family of cross-linking actin-binding proteins that promote a dynamic orthogonal web. They have been reported to interact directly with more than 30 cellular proteins and some phosphoinositides. The biochemical consequences of these interactions may have either positive or negative effects on the cross-linking function and also form a link between the cytoskeleton and plasma membrane. In this study, the EhABP-120 carboxy-terminal domain (END) was biochemically characterized. This domain was able to associate to 3-sulfate galactosyl ceramide, a new lipid target for a member of the filamin family. Also, the END domain was able to dimerize “in vitro.” Molecular modeling analysis showed that the dimeric region is stabilized by a disulfide bond. Electrostatic and docking studies suggest that an electropositive concave pocket at the dimeric END domain interacts simultaneously with several sulfogalactose moieties of the sulfatide.     

Filamins: promiscuous organizers of the cytoskeleton

Filamins are elongated homodimeric proteins that crosslink F-actin. Each monomer chain of filamin comprises an actin-binding domain, and a rod segment consisting of six (Dictyostelium filamin) up to 24 (human filamin) highly homologous repeats of approximately 96 amino acid residues, which adopt an immunoglobulin-like fold. Two hinges in the rod segment, together with the reversible unfolding of single repeats, might be the structural basis for the intrinsic flexibility of the actin networks generated by filamins. There are numerous filamin-binding proteins that associate, in most cases, along the repeats of the rod repeats. This rather promiscuous behaviour renders filamin a versatile scaffold between the actin network and finely tuned molecular cascades from the membrane to the cytoskeleton.     

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.     

Homology modeling and consensus protein disorder prediction of human filamin

Filamins are dimeric actin-binding proteins participating in the organization of the actin-based cytoskeleton. Their modular domain organization is made up of an N-terminal actin-binding domain composed of two CH domains followed by flexible rod regions that consist of 24 Ig-like domains. Homology modeling was used to model human filamin using Modeller 9v5. The resulting model assessed by Verify 3D and PROCHECK showed that the final model is reliable. The conformational disorder prediction of human filamin residues were also mapped on the validated structure of human filamin. Prediction of protein disorder in filamin structures will help structural biologists to find suitable targets to be analyzed and for understanding protein function.     

The molecular basis of filamin binding to integrins and competition with talin

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Filamins are large, actin-crosslinking proteins that connect multiple transmembrane and signaling proteins to the cytoskeleton. Here, we describe the high-resolution structure of an interface between filamin A and an integrin adhesion receptor. When bound, the integrin beta cytoplasmic tail forms an extended beta strand that interacts with beta strands C and D of the filamin immunoglobulin-like domain (IgFLN) 21. This interface is common to many integrins, and we suggest it is a prototype for other IgFLN domain interactions. Notably, the structurally defined filamin binding site overlaps with that of the integrin-regulator talin, and these proteins compete for binding to integrin tails, allowing integrin-filamin interactions to impact talin-dependent integrin activation. Phosphothreonine-mimicking mutations inhibit filamin, but not talin, binding, indicating that kinases may modulate this competition and provide additional means to control integrin functions.     

Filamin-regulated F-actin assembly is essential for morphogenesis and controls phototaxis in Dictyostelium

Dictyostelium strains lacking the F-actin cross-linking protein filamin (ddFLN) have a severe phototaxis defect at the multicellular slug stage. Filamins are rod-shaped homodimers that cross-link the actin cytoskeleton into highly viscous, orthogonal networks. Each monomer chain of filamin is comprised of an F-actin-binding domain and a rod domain. In rescue experiments only intact filamin re-established correct phototaxis in filamin minus mutants, whereas C-terminally truncated filamin proteins that had lost the dimerization domain and molecules lacking internal repeats but retaining the dimerization domain did not rescue the phototaxis defect. Deletion of individual rod repeats also changed their subcellular localization, and mutant filamins in general were less enriched at the cell cortex as compared with the full-length protein and were increasingly present in the cytoplasm. For correct phototaxis ddFLN is only required at the tip of the slug because expression under control of the cell type-specific extracellular-matrix protein A (ecmA) promoter and mixing experiments with wild type cells supported phototactic orientation. Likewise, in chimeric slugs wild type cells were primarily found at the tip of the slug, which acts as an organizer in Dictyostelium morphogenesis.     

Assembly of a filamin four-domain fragment and the influence of splicing variant-1 on the structure

Filamins are scaffold proteins that bind to various proteins, including the actin cytoskeleton, integrin adhesion receptors, and adaptor proteins such as migfilin. Alternative splicing of filamin, largely constructed from 24 Ig-like domains, is thought to have a role in regulating its interactions with other proteins. The filamin A splice variant-1 (FLNa var-1) lacks 41 amino acids, including the last β-strand of domain 19, FLNa(19), and the first β-strand of FLNa(20) that was previously shown to mask a key binding site on FLNa(21). Here, we present a structural characterization of domains 18-21, FLNa(18-21), in the FLNa var-1 as well as its nonspliced counterpart. A model of nonspliced FLNa(18-21), obtained from small angle x-ray scattering data, shows that these four domains form an L-shaped structure, with one arm composed of a pair of domains. NMR spectroscopy reveals that in the splice variant, FLNa(19) is unstructured whereas the other domains retain the same fold as in their canonical counterparts. The maximum dimensions predicted by small angle x-ray scattering data are increased upon migfilin binding in the FLNa(18-21) but not in the splice variant, suggesting that migfilin binding is able to displace the masking β-strand and cause a rearrangement of the structure. Possible function roles for the spliced variants are discussed.     

Novel structural and functional findings of the ehFLN protein from Entamoeba histolytica

The ehFLN protein (previously known as EhABP-120) is the first filamin to be identified in the parasitic protozoan Entamoeba histolytica. Filamins are a family of cross-linking actin-binding proteins that organize filamentous actin in networks and stress fibers. It has been reported that filamins of different organisms directly interact with more than 30 cellular proteins and some PPIs. The biochemical consequences of such interactions may have either positive or negative effects on the cross-linking function. Besides, filamins form a link between cytoskeleton and plasma membrane. In this work, the ehFLN protein was biochemically characterized; amoebae filamin was found to associate with both PA and PI(3)P in vitro, new lipid targets for a member of the filamins. By molecular modeling analysis and protein-lipid overlay assays, K-609, 709, and 710 were determined to be essential for the PA-ehFLN1 complex stability. Also, the integrity of the 4th repeat of ehFLN is essential to keep interaction with the PI(3)P. Transfected trophozoites that overexpressed the d100, d50NH(2), and d50COOH regions of ehFLN1 displayed both increased motility and chemotactic response to TYI-S-33 media. Together, these results suggest that short regions of ehFLN are involved in signaling events that, in cooperation with phosphatidic acid, EhPLD2 and EhPI3K, could promote cell motility.     

Structural interaction and functional regulation of polycystin-2 by filamin

Filamins are important actin cross-linking proteins implicated in scaffolding, membrane stabilization and signal transduction, through interaction with ion channels, receptors and signaling proteins. Here we report the physical and functional interaction between filamins and polycystin-2, a TRP-type cation channel mutated in 10-15% patients with autosomal dominant polycystic kidney disease. Yeast two-hybrid and GST pull-down experiments demonstrated that the C-termini of filamin isoforms A, B and C directly bind to both the intracellular N- and C-termini of polycystin-2. Reciprocal co-immunoprecipitation experiments showed that endogenous polycystin-2 and filamins are in the same complexes in renal epithelial cells and human melanoma A7 cells. We then examined the effect of filamin on polycystin-2 channel function by electrophysiology studies with a lipid bilayer reconstitution system and found that filamin-A substantially inhibits polycystin-2 channel activity. Our study indicates that filamins are important regulators of polycystin-2 channel function, and further links actin cytoskeletal dynamics to the regulation of this channel protein.     

Structural basis for vertebrate filamin dimerization

Filamins are essential in cell motility and many developmental processes. They are large actin cross linking proteins that contain actin binding domains in their N termini and a long rod region constructed from 24 tandem Ig domains. Dimerization is crucial for the actin crosslinking function of filamins and requires the most C-terminal Ig domain. We describe here the crystal structure of this 24th Ig domain (Ig24) of human filamin C and show how it mediates dimerization. The dimer interface is novel and quite different to that seen in the Dictyostelium discoideum filamin analog. The sequence signature of the dimerization interface suggests that the C-terminal domains of all vertebrate filamins share the same dimerization mechanism. Furthermore, we show that point mutations in the dimerization interface disrupt the dimer and that the dissociation constant for recombinant Ig24 is in the micromolar range.     

Switching of filamin polypeptides during myogenesis in vitro

During chicken skeletal myogenesis in vitro, the actin-binding protein filamin is present at first in association with actin filament bundles both in myoblasts and in myotubes early after fusion. Later in mature myotubes it is found in association with myofibril Z disks. These two associations of filamin are separated by a period of several days, during which the protein is absent from the cytoplasm of differentiating myotubes (Gomer, R., and E. Lazarides, 1981, Cell, 23:524-532). To characterize the two classes of filamin polypeptides we have compared, by two-dimensional peptide mapping, 125I-labeled filamin immunoprecipitated from myoblasts and fibroblasts to filamin immunoprecipitated from mature myotubes and adult skeletal myofibrils. Myoblast filamin is highly homologous to fibroblast and purified chicken gizzard filamins. Mature myotube and adult myofibril filamins are highly homologous but exhibit extensive peptide differences with respect to the other three classes of filamin. Comparison of peptide maps from immunoprecipitated 35S-methionine-labeled filamins also shows that fibroblast and myoblast filamins are highly homologous but show substantial peptide differences with respect to mature myotube filamin. Filamins from both mature myotubes and skeletal myofibrils exhibit a slightly higher electrophoretic mobility than gizzard, fibroblast, and myoblast filamins. Short pulse-labeling studies show that mature myotube filamin is synthesized as a lower molecular weight variant and is not derived from a higher molecular weight precursor. These results suggest that myoblast and mature myotube filamins are distinct gene products and that during skeletal myogenesis in vitro one class of filamin polypeptides is replaced by a new class of filamin polypeptides, and that the latter is maintained into adulthood.     

The C-terminal rod 2 fragment of filamin A forms a compact structure that can be extended

Filamins are large proteins that cross-link actin filaments and connect to other cellular components. The C-terminal rod 2 region of FLNa (filamin A) mediates dimerization and interacts with several transmembrane receptors and intracellular signalling adaptors. SAXS (small-angle X-ray scattering) experiments were used to make a model of a six immunoglobulin-like domain fragment of the FLNa rod 2 (domains 16-21). This fragment had a surprising three-branched structural arrangement, where each branch was made of a tightly packed two-domain pair. Peptides derived from transmembrane receptors and intracellular signalling proteins induced a more open structure of the six domain fragment. Mutagenesis studies suggested that these changes are caused by peptides binding to the CD faces on domains 19 and 21 which displace the preceding domain A-strands (18 and 20 respectively), thus opening the individual domain pairs. A single particle cryo-EM map of a nine domain rod 2 fragment (domains 16-24), showed a relatively compact dimeric particle and confirmed the three-branched arrangement as well as the peptide-induced conformation changes. These findings reveal features of filamin structure that are important for its interactions and mechanical properties.     

1H, 13C and 15N resonance assignments of the human filamin A tandem immunoglobulin-like domains 16–17 and 18–19

Filamins are large actin-binding and cross-linking proteins which act as linkers between the cytoskeleton and various signaling proteins. Filamin A (FLNa) is the most abundant of the three filamin isoforms found in humans. FLNa contains an N-terminal actin-binding domain and 24 immunoglobulin-like (Ig) domains. The Ig domains are responsible for the FLNa dimerization and most of the interactions that FLNa has with numerous other proteins. There are several crystal and solution structures from isolated single Ig domains of filamins in the PDB database, but only few from longer constructs. Here, we present nearly complete chemical shift assignments of FLNa tandem Ig domains 16–17 and 18–19. Chemical shift mapping between FLNa tandem Ig domain 16–17 and isolated domain 17 suggests a novel domain–domain interaction mode.     

Structural determinants of LL5beta subcellular localisation and association with filamin C

PI3K signalling pathways link cell surface receptors to the control of several intracellular functions including cell growth, survival and movement. Filamins are important regulators of cortical actin structure and function. LL5beta is a filamin binding protein that is an effector of the PI3K signalling pathway. We define an N-terminal region of LL5beta that is responsible for binding to the C-terminus of filamins. Under conditions of very low PI3K activity, we show that this region, together with an additional domain of the protein, is responsible for localising the complex to punctate structures that are also decorated by L-FILIP (a protein previously characterised to bind filamin and accelerate its destruction). Under conditions of significant PI3K activity, PtdIns(3,4,5)P(3) binding to the C-terminal PH domain in LL5beta prevents localisation to these structures. These observations start to define the basis for PI3K regulation of filamin through LL5beta.     

Interaction of the pacemaker channel HCN1 with filamin A

Pacemaker channels are encoded by the HCN gene family and are responsible for a variety of cellular functions including control of spontaneous activity in cardiac myocytes and control of excitability in different types of neurons. Some of these functions require specific membrane localization. Although several voltage-gated channels are known to interact with intracellular proteins exerting auxiliary functions, no cytoplasmic proteins have been found so far to modulate HCN channels. Through the use of a yeast two-hybrid technique, here we showed that filamin A interacts with HCN1, an HCN isoform widely expressed in the brain, but not with HCN2 or HCN4. Filamin A is a cytoplasmic scaffold protein with actin-binding domains whose main function is to link transmembrane proteins to the actin cytoskeleton. Using several HCN1 C-terminal constructs, we identified a filamin A-interacting region of 22 amino acids located downstream from the cyclic nucleotide-binding domain; this region is not conserved in HCN2, HCN3, or HCN4. We also verified by immunoprecipitation from bovine brain that the filamin A-HCN1 interaction is functional in vivo. In filamin A-expressing cells (filamin+), HCN1 (but not HCN4) channels were expressed in hot spots, whereas they were evenly distributed on the membrane of cells lacking filamin A (filamin-) indicating that interaction with filamin A affects membrane localization. Also, in filamin- cells the gating kinetics of HCN1 were strongly accelerated relative to filamin+ cells. The interaction with filamin A may contribute to localizing HCN1 channels to specific neuronal areas and to modulating channel activity.     

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.     

Filamin 2 (FLN2): A muscle-specific sarcoglycan interacting protein

Mutations in genes encoding for the sarcoglycans, a subset of proteins within the dystrophin-glycoprotein complex, produce a limb-girdle muscular dystrophy phenotype; however, the precise role of this group of proteins in the skeletal muscle is not known. To understand the role of the sarcoglycan complex, we looked for sarcoglycan interacting proteins with the hope of finding novel members of the dystrophin-glycoprotein complex. Using the yeast two-hybrid method, we have identified a skeletal muscle-specific form of filamin, which we term filamin 2 (FLN2), as a gamma- and delta-sarcoglycan interacting protein. In addition, we demonstrate that FLN2 protein localization in limb-girdle muscular dystrophy and Duchenne muscular dystrophy patients and mice is altered when compared with unaffected individuals. Previous studies of filamin family members have determined that these proteins are involved in actin reorganization and signal transduction cascades associated with cell migration, adhesion, differentiation, force transduction, and survival. Specifically, filamin proteins have been found essential in maintaining membrane integrity during force application. The finding that FLN2 interacts with the sarcoglycans introduces new implications for the pathogenesis of muscular dystrophy.     

The bimodal role of filamin in controlling the architecture and mechanics of F-actin networks

Reconstituted actin filament networks have been used extensively to understand the mechanics of the actin cortex and decipher the role of actin cross-linking proteins in the maintenance and deformation of cell shape. However, studies of the mechanical role of the F-actin cross-linking protein filamin have led to seemingly contradictory conclusions, in part due to the use of ill-defined mechanical assays. Using quantitative rheological methods that avoid the pitfalls of previous studies, we systematically tested the complex mechanical response of reconstituted actin filament networks containing a wide range of filamin concentrations and compared the mechanical function of filamin with that of the cross-linking/bundling proteins alpha-actinin and fascin. At steady state and within a well defined linear regime of small non-destructive deformations, F-actin solutions behave as highly dynamic networks (actin polymers are still sufficiently mobile to relax the stress) below the cross-linking-to-bundling threshold filamin concentration, and they behave as covalently cross-linked gels above that threshold. Under large deformations, F-actin networks soften at low filamin concentrations and strain-harden at high filamin concentrations. Filamin cross-links F-actin into networks that are more resilient, stiffer, more solid-like, and less dynamic than alpha-actinin and fascin. These results resolve the controversy by showing that F-actin/filamin networks can adopt diametrically opposed rheological behaviors depending on the concentration in cross-linking proteins.