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Biochem J. Jul 1, 1994; 301(Pt 1): 225–233.
PMCID: PMC1137166

Identification of the vinculin-binding site in the cytoskeletal protein alpha-actinin.

Abstract

Using low-speed sedimentation equilibrium we have established that vinculin binds to alpha-actinin with a Kd of 1.3 x 10(-5) M. Electron microscopy of negatively stained preparations of vinculin revealed spherical particles (diameter 11.2 nm; S.D. 1.7 nm, n = 21), whereas alpha-actinin appeared as a rod-shaped particle (length 33 nm; S.D. 3.3 nm, n = 23). Mixtures of the two proteins contained both 'lollipop'- and 'dumbell'-shaped particles which we interpret as either one or two spherical vinculin molecules associated with the ends of the alpha-actinin rod. We have further defined the vinculin-binding site in alpha-actinin using 125I-vinculin and a gel-blot assay in which proteolytic fragments of alpha-actinin and fragments of alpha-actinin expressed in Escherichia coli were resolved by SDS/PAGE and blotted to nitrocellulose. 125I-vinculin bound to polypeptides derived from the spectrin-like repeat region of alpha-actinin, but did not bind to the actin-binding domain. Binding was inhibited by a 100-fold molar excess of unlabelled vinculin. Using a series of glutathione S-transferase fusion proteins we have mapped the vinculin-binding site to a region toward the C-terminal end of the molecule (alpha-actinin residues 713-749). 125I-vinculin also bound to fusion proteins containing this sequence which had been immobilized on glutathione-agarose beads. The vinculin-binding site is localized in a highly conserved region of the molecule close to the first of two EF-hand calcium-binding motifs.

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  • Arimura C, Suzuki T, Yanagisawa M, Imamura M, Hamada Y, Masaki T. Primary structure of chicken skeletal muscle and fibroblast alpha-actinins deduced from cDNA sequences. Eur J Biochem. 1988 Nov 15;177(3):649–655. [PubMed]
  • Baron MD, Davison MD, Jones P, Critchley DR. The sequence of chick alpha-actinin reveals homologies to spectrin and calmodulin. J Biol Chem. 1987 Dec 25;262(36):17623–17629. [PubMed]
  • Baron MD, Davison MD, Jones P, Patel B, Critchley DR. Isolation and characterization of a cDNA encoding a chick alpha-actinin. J Biol Chem. 1987 Feb 25;262(6):2558–2561. [PubMed]
  • Beck K. Structural model of vinculin: correlation of amino acid sequence with electron-microscopical shape. FEBS Lett. 1989 May 22;249(1):1–4. [PubMed]
  • Beggs AH, Byers TJ, Knoll JH, Boyce FM, Bruns GA, Kunkel LM. Cloning and characterization of two human skeletal muscle alpha-actinin genes located on chromosomes 1 and 11. J Biol Chem. 1992 May 5;267(13):9281–9288. [PubMed]
  • Belkin AM, Koteliansky VE. Interaction of iodinated vinculin, metavinculin and alpha-actinin with cytoskeletal proteins. FEBS Lett. 1987 Aug 17;220(2):291–294. [PubMed]
  • Bennett JP, Zaner KS, Stossel TP. Isolation and some properties of macrophage alpha-actinin: evidence that it is not an actin gelling protein. Biochemistry. 1984 Oct 9;23(21):5081–5086. [PubMed]
  • Blanchard A, Ohanian V, Critchley D. The structure and function of alpha-actinin. J Muscle Res Cell Motil. 1989 Aug;10(4):280–289. [PubMed]
  • Bretscher A, Vandekerckhove J, Weber K. alpha-Actinins from chicken skeletal muscle and smooth muscle show considerable chemical and immunological differences. Eur J Biochem. 1979 Oct;100(1):237–243. [PubMed]
  • Burridge K, Feramisco JR. Non-muscle alpha actinins are calcium-sensitive actin-binding proteins. Nature. 1981 Dec 10;294(5841):565–567. [PubMed]
  • Burridge K, Fath K, Kelly T, Nuckolls G, Turner C. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol. 1988;4:487–525. [PubMed]
  • Crawford AW, Michelsen JW, Beckerle MC. An interaction between zyxin and alpha-actinin. J Cell Biol. 1992 Mar;116(6):1381–1393. [PMC free article] [PubMed]
  • Davison MD, Baron MD, Critchley DR, Wootton JC. Structural analysis of homologous repeated domains in alpha-actinin and spectrin. Int J Biol Macromol. 1989 Apr;11(2):81–90. [PubMed]
  • Duhaiman AS, Bamburg JR. Isolation of brain alpha-actinin. Its characterization and a comparison of its properties with those of muscle alpha-actinins. Biochemistry. 1984 Apr 10;23(8):1600–1608. [PubMed]
  • Feramisco JR, Burridge K. A rapid purification of alpha-actinin, filamin, and a 130,000-dalton protein from smooth muscle. J Biol Chem. 1980 Feb 10;255(3):1194–1199. [PubMed]
  • Fyrberg E, Kelly M, Ball E, Fyrberg C, Reedy MC. Molecular genetics of Drosophila alpha-actinin: mutant alleles disrupt Z disc integrity and muscle insertions. J Cell Biol. 1990 Jun;110(6):1999–2011. [PMC free article] [PubMed]
  • Gimona M, Fürst DO, Small JV. Metavinculin and vinculin from mammalian smooth muscle: bulk isolation and characterization. J Muscle Res Cell Motil. 1987 Aug;8(4):329–341. [PubMed]
  • Hemmings L, Kuhlman PA, Critchley DR. Analysis of the actin-binding domain of alpha-actinin by mutagenesis and demonstration that dystrophin contains a functionally homologous domain. J Cell Biol. 1992 Mar;116(6):1369–1380. [PMC free article] [PubMed]
  • Imamura M, Endo T, Kuroda M, Tanaka T, Masaki T. Substructure and higher structure of chicken smooth muscle alpha-actinin molecule. J Biol Chem. 1988 Jun 5;263(16):7800–7805. [PubMed]
  • Isenberg G, Leonard K, Jockusch BM. Structural aspects of vinculin-actin interactions. J Mol Biol. 1982 Jun 25;158(2):231–249. [PubMed]
  • Kuhlman PA, Hemmings L, Critchley DR. The identification and characterisation of an actin-binding site in alpha-actinin by mutagenesis. FEBS Lett. 1992 Jun 15;304(2-3):201–206. [PubMed]
  • Landon F, Gache Y, Touitou H, Olomucki A. Properties of two isoforms of human blood platelet alpha-actinin. Eur J Biochem. 1985 Dec 2;153(2):231–237. [PubMed]
  • Luna EJ, Hitt AL. Cytoskeleton--plasma membrane interactions. Science. 1992 Nov 6;258(5084):955–964. [PubMed]
  • Milam LM. Electron microscopy of rotary shadowed vinculin and vinculin complexes. J Mol Biol. 1985 Aug 5;184(3):543–545. [PubMed]
  • Mimura N, Asano A. Isolation and characterization of a conserved actin-binding domain from rat hepatic actinogelin, rat skeletal muscle, and chicken gizzard alpha-actinins. J Biol Chem. 1986 Aug 15;261(23):10680–10687. [PubMed]
  • Noegel A, Witke W, Schleicher M. Calcium-sensitive non-muscle alpha-actinin contains EF-hand structures and highly conserved regions. FEBS Lett. 1987 Sep 14;221(2):391–396. [PubMed]
  • O'Halloran T, Molony L, Burridge K. Purification and assay of vinculin, metavinculin, and talin. Methods Enzymol. 1986;134:69–77. [PubMed]
  • Otey CA, Pavalko FM, Burridge K. An interaction between alpha-actinin and the beta 1 integrin subunit in vitro. J Cell Biol. 1990 Aug;111(2):721–729. [PMC free article] [PubMed]
  • Otto JJ. Detection of vinculin-binding proteins with an 125I-vinculin gel overlay technique. J Cell Biol. 1983 Oct;97(4):1283–1287. [PMC free article] [PubMed]
  • Parr T, Waites GT, Patel B, Millake DB, Critchley DR. A chick skeletal-muscle alpha-actinin gene gives rise to two alternatively spliced isoforms which differ in the EF-hand Ca(2+)-binding domain. Eur J Biochem. 1992 Dec 15;210(3):801–809. [PubMed]
  • Pavalko FM, Burridge K. Disruption of the actin cytoskeleton after microinjection of proteolytic fragments of alpha-actinin. J Cell Biol. 1991 Aug;114(3):481–491. [PMC free article] [PubMed]
  • Podlubnaya ZA, Tskhovrebova LA, Zaalishtsbvili MM, Stefanenko GA. Electron microscopic study of alpha-actinin. J Mol Biol. 1975 Feb 25;92(2):357–359. [PubMed]
  • Price GJ, Jones P, Davison MD, Patel B, Bendori R, Geiger B, Critchley DR. Primary sequence and domain structure of chicken vinculin. Biochem J. 1989 Apr 15;259(2):453–461. [PMC free article] [PubMed]
  • Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. [PubMed]
  • Squire PG, Himmel ME. Hydrodynamics and protein hydration. Arch Biochem Biophys. 1979 Aug;196(1):165–177. [PubMed]
  • Suzuki A, Goll DE, Singh I, Allen RE, Robson RM, Stromer MH. Some properties of purified skeletal muscle alpha-actinin. J Biol Chem. 1976 Nov 10;251(21):6860–6870. [PubMed]
  • Wachsstock DH, Wilkins JA, Lin S. Specific interaction of vinculin with alpha-actinin. Biochem Biophys Res Commun. 1987 Jul 31;146(2):554–560. [PubMed]
  • Waites GT, Graham IR, Jackson P, Millake DB, Patel B, Blanchard AD, Weller PA, Eperon IC, Critchley DR. Mutually exclusive splicing of calcium-binding domain exons in chick alpha-actinin. J Biol Chem. 1992 Mar 25;267(9):6263–6271. [PubMed]
  • Wallraff E, Schleicher M, Modersitzki M, Rieger D, Isenberg G, Gerisch G. Selection of Dictyostelium mutants defective in cytoskeletal proteins: use of an antibody that binds to the ends of alpha-actinin rods. EMBO J. 1986 Jan;5(1):61–67. [PMC free article] [PubMed]
  • Way M, Pope B, Gooch J, Hawkins M, Weeds AG. Identification of a region in segment 1 of gelsolin critical for actin binding. EMBO J. 1990 Dec;9(12):4103–4109. [PMC free article] [PubMed]
  • Winograd E, Hume D, Branton D. Phasing the conformational unit of spectrin. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10788–10791. [PMC free article] [PubMed]
  • Wilkins JA, Chen KY, Lin S. Detection of high molecular weight vinculin binding proteins in muscle and nonmuscle tissues with an electroblot-overlay technique. Biochem Biophys Res Commun. 1983 Nov 15;116(3):1026–1032. [PubMed]
  • Witke W, Hofmann A, Köppel B, Schleicher M, Noegel AA. The Ca(2+)-binding domains in non-muscle type alpha-actinin: biochemical and genetic analysis. J Cell Biol. 1993 May;121(3):599–606. [PMC free article] [PubMed]

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