Smooth Muscle Alpha-actinin Research Articles

Smooth muscle α-actinin binds tightly to fesselin and attenuates its activity toward actin polymerization

Fesselin is an actin binding protein from smooth muscle that nucleates actin polymerization in a Ca(++)-calmodulin dependent manner, bundles actin and inhibits the actin-activated ATPase activity of myosin S1. We now report that fesselin binds to smooth muscle alpha-actinin. Binding was measured by blot overlay, affinity chromatography and sedimentation methods. Binding was moderate with an association constant of 1-4 x 10(7) M(-1) assuming a 1:1 association of fesselin with alpha-actinin. Fesselin binds to the central spectrin domain repeat region of alpha-actinin but not to the CH1-CH2 domain. Fesselin accelerates the polymerization of actin. This activity of fesselin was attenuated by alpha-actinin. These observations support the role of fesselin in organizing the cytoskeleton.

Structure of the α-Actinin–Vinculin Head Domain Complex Determined by Cryo-electron Microscopy

The vinculin binding site on alpha-actinin was determined by cryo-electron microscopy of 2D arrays formed on phospholipid monolayers doped with a nickel chelating lipid. Chicken smooth muscle alpha-actinin was cocrystallized with the beta1-integrin cytoplasmic domain and a vinculin fragment containing residues 1-258 (vinculin(D1)). Vinculin(D1) was located at a single site on alpha-actinin with 60-70% occupancy. In these arrays, alpha-actinin lacks molecular 2-fold symmetry and the two ends of the molecule, which contain the calmodulin-like and actin binding domains, are held in distinctly different environments. The vinculin(D1) difference density has a shape very suggestive of the atomic structure. The atomic model of the complex juxtaposes the alpha-actinin binding site on vinculin(D1) with the N-terminal lobe of the calmodulin-like domain on alpha-actinin. The results show that the interaction between two species with weak affinity can be visualized in a membrane-like environment.

Dynamic Cross-linking by α-Actinin Determines the Mechanical Properties of Actin Filament Networks

We used smooth muscle alpha-actinin to evaluate the contribution of cross-linker dynamics to the mechanical properties of actin filament networks. Recombinant actin-binding domain (residues 2-269) binds actin filaments with a Kd of 1 microM at 25 degrees C, 20 times stronger than actin-binding domain produced by thermolysin digestion of native alpha-actinin (residues 25-257). Between 8 and 25 degrees C the rate constants for recombinant actin-binding domain to bind to (0.8-2.7 microM-1 s-1) and dissociate from (0.2-2.4 s-1) actin filaments depend on temperature. At 8 degrees C actin filaments cross-linked with alpha-actinin are stiff and nearly solid, whereas at 25 degrees C the mechanical properties approach those of actin filaments alone. In these experiments, high actin concentrations kept most of the alpha-actinin bound to actin and temperature varied a single parameter, cross-linker dynamics, because the mechanical properties of pure actin filaments (a viscoelastic gel) or biotinylated actin filaments cross-linked irreversibly by avidin (a stiff viscoelastic solid) depend little on temperature. These results show that the rate of exchange of dynamic cross-links between actin filaments is an important determinant of the mechanical properties of the networks.

Interactions between smooth muscle alpha-actinin and lipid bilayers.

alpha-Actinin has been proposed to be the actin-plasma membrane linker. This assumption is based on the discovery of direct interaction of alpha-actinin with two specific lipids, diacylglycerol and palmitic acid [Burn, P. (1988) Trends Biochem. Sci. 13, 79-83]. In our study, the binding of alpha-actinin with vesicles containing negatively charged phospholipids was measured by the method of 90 degrees light-scattering. Our results show that alpha-actinin is able to bind membranes containing negatively charged phospholipids, but not to bind membranes composed of neutral lipids only. Diacylglycerol and palmitic acid, on the other hand, have little effect on the binding of alpha-actinin to lipid vesicles. Analysis of binding isotherms in terms of a membrane binding model gave apparent dissociation constants which varied between 0.2 and 3 microM over a range of 5-20 mol % negatively charged phospholipid. Comparing the kinetics of alpha-chymotrypsin digestion of alpha-actinin in solution to those of vesicle-bound alpha-actinin, it can be seen that the cleavage site at the junction between the C-terminal and the central rod domain of alpha-actinin and another cleavage site on the C-terminal domain can be most effectively protected by its membrane binding. Analysis of the amide I and II regions of Fourier-transform infrared spectra of alpha-actinin revealed that the association of alpha-actinin with negatively charged phospholipid vesicles resulted in some perturbation of the protein secondary structure. Monolayers containing negatively charged phospholipid were layered and incubated on the surface of a polymerization solution of actin and alpha-actinin, and observed with an electron microscope. The results show that the bundle structure of actin filaments can be formed if diacylglycerol and palmitic acid are present in lipid layers.

Flexibility and fine structure of smooth-muscle alpha-actinin.

The microfilament protein alpha-actinin exists as a dimer. The N-terminal regions of both polypeptides, arranged in antiparallel orientation, comprise the actin-binding regions, while the C-terminal, larger parts consist of four spectrin-like repeats that interact to form a rod-like structure. To elucidate the fine structure of smooth-muscle alpha-actinin, we used energy-filtered transmission electron microscopy in conjunction with negative staining. Survey pictures of the protein purified from chicken gizzard revealed discrete, elongated particles whose length and width varied with the ionic strength of the buffer. It was determined to to 29.3 nm x 4.8 nm in 0.05 M KCl and 32.6 nm x 4.4 nm in 0.15 M KCl. Both ends of the molecule displayed hook-like structures consisting of globular domains, which were highly variable in their orientation with respect to the long axis of the molecule. Their location at the ends of the molecule, and the finding that these hooks were missing from particles obtained by thermolysin digestion indicated that they probably correspond to the N-terminal actin-binding regions. The rod-like center of the molecule revealed discrete globular masses which probably comprise the spectrin-like repeats. Their arrangement was compatible with the interpretation that three spectrin repeats of each polypeptide chain can form pairs with the respective sequences of the other chain. The rod-like 53-kDa fragment obtained after thermolysin digestion largely retained this structural organization but appeared wider (22.5 nm x 5.9 nm). Our results help to clarify previous discrepancies on the quatenary organization of alpha-actinin and suggest that effective actin-binding and cross-linking of alpha-actinin is based on the high flexibility of the terminal hooks.

Phosphatidylinositol 4,5-bisphosphate phosphatase regulates the rearrangement of actin filaments.

Phosphatidylinositol 4,5-bisphosphate (PIP2) reorganizes actin filaments by modulating the functions of a variety of actin-regulatory proteins. Until now, it was thought that bound PIP2 is hydrolyzed only by tyrosine-phosphorylated phospholipase Cgamma (PLCgamma) after the activation of tyrosine kinases. Here, we show a new mechanism for the hydrolysis of bound PIP2 and the regulation of actin filaments by PIP2 phosphatase (synaptojanin). We isolated a 150-kDa protein (p150) from brains that binds the SH3 domains of Ash/Grb2. The sequence of this protein was found to be homologous to that of synaptojanin. The expression of p150 in COS 7 cells produces a decrease in the number of actin stress fibers in the center of the cells and causes the cells to become multinuclear. On the other hand, the expression of a PIP2 phosphatase-negative mutant does not disrupt actin stress fibers or produce the multinuclear phenotype. We have also shown that p150 forms the complexes with Ash/Grb2 and epidermal growth factor (EGF) receptors only when the cells are treated with EGF and that it reorganizes actin filaments in an EGF-dependent manner. Moreover, the PIP2 phosphatase activity of native p150 purified from bovine brains is not inhibited by profilin, cofilin, or alpha-actinin, although PLCdelta1 activity is markedly inhibited by these proteins. Furthermore, p150 suppresses actin gelation, which is induced by smooth muscle alpha-actinin. All these data suggest that p150 (synaptojanin) hydrolyzes PIP2 bound to actin regulatory proteins, resulting in the rearrangement of actin filaments downstream of tyrosine kinase and Ash/Grb2.

Identification of a Phosphatidylinositol 4,5-Bisphosphate-binding Site in Chicken Skeletal Muscle α-Actinin

We previously reported that phosphatidylinositol 4,5-bisphosphate (PIP2) dramatically increases the gelating activity of smooth muscle alpha-actinin (Fukami, K., Furuhashi, K., Inagaki, M., Endo, T., Hatano, S., and Takenawa, T. (1992) Nature 359, 150-152) and that the hydrolysis of PIP2 on alpha-actinin by tyrosine kinase activation may be important in cytoskeletal reorganization (Fukami, K., Endo, T., Imamura, M., and Takenawa, T. (1994) J. Biol. Chem. 269, 1518-1522). Here we report that a proteolytic fragment with lysylendopeptidase comprising amino acids 168-184 (TAPYRNVNIQNFHLSWK) from striated muscle alpha-actinin contains a PIP2-binding site. A synthetic peptide composed of the 17 amino acids remarkably inhibited the activities of phospholipase C (PLC)-gamma 1 and -delta 1. Furthermore, we detected an interaction between PIP2 and a bacterially expressed alpha-actinin fragment (amino acids 137-259) by PLC inhibition assay. Point mutants in which arginine 172 or lysine 184 of alpha-actinin were replaced by isoleucine reduced the inhibitory effect on PLC activity by nearly half. Direct interactions between PIP2 and the peptide (amino acids 168-184) or the bacterially expressed protein (amino acids 137-259) were confirmed by enzyme-linked immunosorvent assay. We also found this region homologous to the sequence of the PIP2-binding site in spectrin and the pleckstrin homology domains of PLC-delta 1 and Grb7. Synthetic peptides from the homologous regions in spectrin and PLC-delta 1 inhibited PLC activities. These results indicate that residues 168-184 comprise a binding site for PIP2 in alpha-actinin and that similar sequences found in spectrin and PLC-delta 1 may be involved in the interaction with PIP2.

Minimum folding unit of dystrophin rod domain.

Fragments of the rod domain of dystrophin, which consists of spectrin-like repeating sequences, have been prepared by expression in Escherichia coli. The phasing established earlier for the dystrophin rod, as well as for Drosophila spectrin and smooth muscle alpha-actinin, suggested a length of less than 113 residues for the dystrophin repeat that we have chosen. Fragments with a common N-terminus and lengths between 113 and 119 residues were prepared. The formation of the stable native tertiary fold could be recognized by resistance to proteolysis, the circular dichroism spectrum in the regions of both peptide and aromatic absorption bands and the resolution of the long-wavelength component in the tryptophan absorption spectrum. It was found that the critical length for folding was 117 residues: shortening the chain by 1 further residue resulted in loss of the capacity to form a defined tertiary structure. Residue 117 is a glutamine; replacement of this by a methionine residue did not impair the ability of the chain to enter the folded conformation, implying that it is the length of the C-terminal alpha-helix, rather than any specific side-chain interaction, that is critical in determining the stability of the native structure. The fragment of 119 residues forms a significantly more stable structure than that of 117. It appears that the minimum unit capable of forming the native fold extends some residues into the adjoining sequence repeat.

Formation of two-dimensional complexes of F-actin and crosslinking proteins on lipid monolayers: demonstration of unipolar alpha-actinin-F-actin crosslinking

A method is described for forming two-dimensional (2-D) paracrystalline complexes of F-actin and bundling/gelation proteins on positively charged lipid monolayers. These arrays facilitate detailed structural studies of protein interactions with F-actin by eliminating superposition effects present in 3-D bundles. Bundles of F-actin have been produced using the glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase, the cytoskeletal protein erythrocyte adducin as well as smooth muscle alpha-actinin from chicken gizzard. All of the 2-D bundles formed contain F-actin with a 13/6 helical structure. F-actin-aldolase bundles have an interfilament spacing of 12.6 nm and a superlattice arrangement of actin filaments that can be explained by expression of a local twofold axis in the neighborhood of the aldolase. Well ordered F-actin-alpha-actinin 2-D bundles have an interfilament spacing of 36 nm and contain crosslinks 33 nm in length angled approximately 25-35 degrees to the filament axis. Images and optical diffraction patterns of these bundles suggest that they consist of parallel, unipolar arrays of actin filaments. This observation is consistent with an actin crosslinking function at adhesion plaques where actin filaments are bound to the cell membrane with uniform polarity.

Determination of the alpha-actinin-binding site on actin filaments by cryoelectron microscopy and image analysis.

The three-dimensional structure of actin filaments decorated with the actin-binding domain of chick smooth muscle alpha-actinin (alpha A1-2) has been determined to 21-A resolution. The shape and location of alpha A1-2 was determined by subtracting maps of F-actin from the reconstruction of decorated filaments. alpha A1-2 resembles a bell that measures approximately 38 A at its base and extends 42 A from its base to its tip. In decorated filaments, the base of alpha A1-2 is centered about the outer face of subdomain 2 of actin and contacts subdomain 1 of two neighboring monomers along the long-pitch (two-start) helical strands. Using the atomic model of F-actin (Lorenz, M., D. Popp, and K. C. Holmes. 1993. J. Mol. Biol. 234:826-836.), we have been able to test directly the likelihood that specific actin residues, which have been previously identified by others, interact with alpha A1-2. Our results indicate that residues 86-117 and 350-375 comprise distinct binding sites for alpha-actinin on adjacent actin monomers.

Cross-linker dynamics determine the mechanical properties of actin gels

To evaluate the contributions of cross-linker dynamics and polymer deformation to the frequency-dependent stiffness of actin filament gels, we compared the rheological properties of actin gels with three types of cross-linkers: a weak one, Acanthamoeba alpha-actinin (dissociation rate constant 5.2 s-1, association rate constant 1.1 x 10(6) M-1 s-1); a strong one, chicken smooth muscle alpha-actinin (dissociation rate constant 0.66 s-1, association rate constant 1.20 x 10(6) M-1 s-1); and an extremely strong one, biotin/avidin (dissociation rate constant approximately zero). The biotin/avidin cross-linked gel, whose behavior is determined by polymer bending alone, behaves like a solid and shows no frequency dependence. The amoeba alpha-actinin cross-linked gel behaves like a viscoelastic fluid, and the frequency dependence of the stiffness can be explained by a mathematical model for dynamically cross-linked gels. The stiffness of the chicken alpha-actinin cross-linked gel is independent of frequency, and has viscoelastic properties intermediate between the two. The two alpha-actinins have similar association rate constants for binding to actin filaments, consistent with a diffusion-limited reaction. Rigid cross-links make the gel stiff, but make it elastic without the ability to deform permanently. Dynamically cross-linked actin filaments should allow the cell to react passively to various outside forces without any sort of signaling. Slower, signal-mediated pathways, such as severing filaments or changing the affinity of cross-linkers, could alter the nature of these passive reactions.

Expression of alpha-actinin in the stereocilia of hair cells of the inner ear: immunohistochemical localization.

The distribution of alpha-actinin was studied on surface preparations and cryosections from the organ of Corti of the adult rat by using polyclonal and monoclonal antibodies to smooth muscle alpha-actinin. The polyclonal antibody used gave a positive immunoreactivity in the cuticular plate of the hair cells, pillar cells and supporting cells. A specific labelling of the stereocilia of the outer hair cells (OHCs) and inner hair cells (IHCs) on surface preparations was highly visible. The monoclonal antibody used produces similar patterns of labelling in the organ of Corti. The possible roles of this protein in hair cells are discussed.

Requirement of phosphatidylinositol 4,5-bisphosphate for alpha-actinin function.

Inositol phospholipid turnover is enhanced during mitogenic stimulation of cells by growth factors and the breakdown of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) may be important in triggering cell proliferation. PtdInsP2 also binds actin-binding proteins to regulate their activity, but it is not yet understood how this control is achieved. The protein alpha-actinin from striated muscle contains large amounts of endogenous PtdInsP2, whereas that from smooth muscle has only a little but will bind exogenously added PtdInsP2. In vitro alpha-actinin binds to F-actin and will crosslink actin filaments, increasing the viscosity of F-actin solutions. We report here that alpha-actinin from striated muscle is an endogenous PtdInsP2-bound protein and that the specific interaction between alpha-actinin and PtdInsP2 regulates the F-actin-gelating activity of alpha-actinin. Although the F-actin-gelating activity of alpha-actinin from smooth muscle is much reduced compared with that from striated muscle, exogenous PtdInsP2 can enhance the activity of smooth muscle alpha-actinin to the level seen in striated muscles. These results show that PtdInsP2 is present in striated muscle alpha-actinin and that it is necessary for alpha-actinin to realize its maximum gelating activity.

Analysis of the actin-binding domain of alpha-actinin by mutagenesis and demonstration that dystrophin contains a functionally homologous domain.

To define the actin-binding site within the NH2-terminal domain (residues 1-245) of chick smooth muscle alpha-actinin, we expressed a series of alpha-actinin deletion mutants in monkey Cos cells. Mutant alpha-actinins in which residues 2-19, 217-242, and 196-242 were deleted still retained the ability to target to actin filaments and filament ends, suggesting that the actin-binding site is located within residues 20-195. When a truncated alpha-actinin (residues 1-290) was expressed in Cos cells, the protein localized exclusively to filament ends. This activity was retained by a deletion mutant lacking residues 196-242, confirming that these are not essential for actin binding. The actin-binding site in alpha-actinin was further defined by expressing both wild-type and mutant actin-binding domains as fusion proteins in E. coli. Analysis of the ability of such proteins to bind to F-actin in vitro showed that the binding site was located between residues 108 and 189. Using both in vivo and in vitro assays, we have also shown that the sequence KTFT, which is conserved in several members of the alpha-actinin family of actin-binding proteins (residues 36-39 in the chick smooth muscle protein) is not essential for actin binding. Finally, we have established that the NH2-terminal domain of dystrophin is functionally as well as structurally homologous to that in alpha-actinin. Thus, a chimeric protein containing the NH2-terminal region of dystrophin (residues 1-233) fused to alpha-actinin residues 244-888 localized to actin-containing structures when expressed in Cos cells. Furthermore, an E. coli-expressed fusion protein containing dystrophin residues 1-233 was able to bind to F-actin in vitro.

Mutually exclusive splicing of calcium-binding domain exons in chick alpha-actinin.

We have determined the complete sequence of chick brain alpha-actinin (892 amino acids; 107,644 Da). The sequence differs from that of smooth muscle alpha-actinin only in the region of the first EF-hand calcium-binding motif, where 27 residues in brain alpha-actinin are replaced by just 22 residues in the smooth muscle isoform. This probably accounts for the different calcium sensitivities of the two isoforms with respect to actin binding. Analysis of the gene structure showed that this region of sequence divergence is encoded by two separate exons whose incorporation is mutually exclusive. We have determined the proportion of the two transcripts in various tissues and cell lines using poly(A)+ RNA and a quantitative assay based on the polymerase chain reaction. MRC-5 fibroblasts and HeLa cells express mRNAs encoding both isoforms, whereas Namalwa lymphoblastoid cells, which lack actin stress fibers, express only the non-muscle mRNA. Both isoforms of alpha-actinin became incorporated into stress fibers and cell-matrix junctions when full-length chick alpha-actinin cDNAs were expressed in monkey COS cells. The levels of chick alpha-actinin mRNAs were found to be serum-inducible, suggesting that alpha-actinin may be an early response gene.

Properties of the spectrin‐like structural element of smooth‐muscle α‐actinin

The fragment of smooth muscle alpha-actinin, comprising the four spectrin-like structural repeating units, has a high alpha-helix content, similar to that of spectrin, and a hydrodynamic frictional coefficient, indicative of an elongated, probably bent or kinked rod-like structure, as found for spectrin dimer and tetramer. The fragment exists in solution as an extremely stable dimer, which is dissociated only under denaturing conditions and is much more resistant to dissociation by urea than is the spectrin heterodimer. High-resolution proton magnetic resonance spectra reveal that a part of the polypeptide chain gives rise to sharp resonances; this is also true of spectrin and it implies that the individual structural repeating units contain segmentally mobile elements, which may be required to generate the elastic properties of the spectrin family of proteins. Again like spectrin, the alpha-actinin fragment contains multiple binding sites for long-chain fatty acids, as revealed by quenching of tryptophan fluorescence by 2-bromostearate (though not by 9(10)-bromostearate). The results point to extensive structural and functional similarities between the repeating units of all the proteins of the spectrin family.

Primary structure of chicken skeletal muscle and fibroblast alpha-actinins deduced from cDNA sequences.

The complete 897-amino-acid sequence of chicken skeletal muscle alpha-actinin and the 856-amino-acid sequence (97% of the entire sequence) of chicken fibroblast alpha-actinin have been determined by cloning and sequencing the cDNAs. Genomic Southern analysis with the cDNA sequences shows that skeletal and fibroblast alpha-actinins are encoded by separate single-copy genes. RNA blot analyzes show that the skeletal alpha-actinin gene is expressed in the pectoralis muscle and that the fibroblast gene is expressed in the gizzard smooth muscle as well as in the fibroblast. The deduced skeletal alpha-actinin molecule has a calculated Mr of 104 x 10(3), and each alpha-actinin can be divided into three domains: (1) the NH2-terminal highly conserved actin-binding domain, which shows similarity to the product of the Duchenne's muscular dystrophy locus; (2) the middle rod-shaped dimer-forming domain, which contains the spectrin-type repeat units; and (3) the COOH-terminal two EF-hand consensus regions. Comparison of the skeletal alpha-actinin sequence with the fibroblast and smooth muscle alpha-actinin sequences demonstrated that the EF-hand structure was conserved in all of these alpha-actinin sequences, despite the reported variability of the Ca2+ sensitivities of the actin-gelation by various alpha-actinin isoforms.

Substructure and higher structure of chicken smooth muscle alpha-actinin molecule.

Substructure of chicken gizzard smooth muscle alpha-actinin molecule was deduced by domainal mapping of the proteolytic fragments with alpha-chymotrypsin. There were three chymotryptic cleavage sites (Sites I, II, and III, from the amino terminus). Cleavage at Site I generated two fragments, i.e. an NH2-terminal 36-kDa fragment and a COOH-terminal 70-kDa fragment. The 70-kDa fragment generated either a 55-kDa fragment by cleavage at Site II or a 65-kDa fragment by cleavage at Site III. Purified NH2-terminal 36-kDa fragment bound to F-actin, whereas the 55-kDa fragment formed a dimeric molecule. Circular dichroism and electron microscopic experiments demonstrated that the alpha-helical content of the 55-kDa fragment was 14% higher than that of native gizzard alpha-actinin, coinciding with the apparently rod-shaped configuration of this fragment. A 110-kDa product was generated from two 55-kDa fragments in a cross-linking study with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Two cross-linkable sites in the 55-kDa, A- and B-site, were shown to be involved in this reaction. Further, it was demonstrated by using N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide labeling and immunoblotting analyses that the A-site on one 55-kDa fragment was cross-linked to the B-site on the other. These results suggest that smooth muscle alpha-actinin formed an antiparallel dimeric molecule in which the 55-kDa fragments connected the two actin-binding domains composed of the 36-kDa fragments.

The sequence of chick alpha-actinin reveals homologies to spectrin and calmodulin.

We have sequenced a cDNA, isolated from a chick embryo fibroblast lambda gt11 library, that encodes all 887 amino acids of alpha-actinin. Sequence from 10 different peptides from chick smooth muscle alpha-actinin was found to match that derived from the cDNA. The deduced protein sequence can be divided into three distinct domains: (a) the N-terminal 240 amino acid contains a highly conserved region (compared with Dictyostelium alpha-actinin) which probably represents the actin-binding domain, (b) amino acids 270-740 contain four repeats of a spectrin-like sequence, and (c) the C-terminal sequence contains two EF-hand Ca2+-binding sites. Each of these sites is defective in at least one oxygen-containing Ca2+-chelating amino acid side chain, suggesting that they are nonfunctional. Southern blots suggest that the alpha-actinin cDNA described here hybridizes to only one gene in chicken. Northern blots reveal only one size class of mRNA in fibroblasts and smooth muscle, but no hybridizing species could be detected in skeletal muscle poly(A+) RNA. The results are consistent with the view that smooth and skeletal muscle alpha-actinins are encoded by separate genes, which are considerably divergent.

Isolation and characterization of a cDNA encoding a chick alpha-actinin.

We have isolated and sequenced a 2.1-kilobase cDNA encoding 86% of the sequence of alpha-actinin. The cDNA clone was isolated from a chick embryo fibroblast cDNA library constructed in the expression vector lambda gt11. Identification of this sequence as alpha-actinin was confirmed by immunological methods and by comparing the deduced protein sequence with the sequence of several CNBr fragments obtained from adult chicken smooth muscle (gizzard) alpha-actinin. The deduced protein sequence shows two distinct domains, one of which consists of four repeats of approximately 120 amino acids. This region corresponds to a previously identified 50-kDa tryptic peptide involved in formation of the alpha-actinin dimer. The last 19 residues of C-terminal sequence display an homology with the so-called E-F hand of Ca2+-binding proteins. Hybridization analysis reveals only one size of mRNA (approximately 3.5 kilobases) in fibroblasts, but multiple bands in genomic cDNA.