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. 1995 Aug 2;130(4):897–907. doi: 10.1083/jcb.130.4.897

Mechanochemistry of protein 4.1's spectrin-actin-binding domain: ternary complex interactions, membrane binding, network integration, structural strengthening

PMCID: PMC2199952  PMID: 7642705

Abstract

Mechanical strength of the red cell membrane is dependent on ternary interactions among the skeletal proteins, spectrin, actin, and protein 4.1. Protein 4.1's spectrin-actin-binding (SAB) domain is specified by an alternatively spliced exon encoding 21 amino acid (aa) and a constitutive exon encoding 59 aa. A series of truncated SAB peptides were engineered to define the sequences involved in spectrin-actin interactions, and also membrane strength. Analysis of in vitro supramolecular assemblies showed that gelation activity of SAB peptides correlates with their ability to recruit a critical amount of spectrin into the complex to cross-link actin filaments. Also, several SAB peptides appeared to exhibit a weak, cooperative actin-binding activity which mapped to the first 26 residues of the constitutive 59 aa. Fluorescence-imaged microdeformation was used to show SAB peptide integration into the elastic skeletal network of spectrin, actin, and protein 4.1. In situ membrane-binding and membrane-strengthening abilities of the SAB peptides correlated with their in vitro gelation activity. The findings imply that sites for strong spectrin binding include both the alternative 21-aa cassette and a conserved region near the middle of the 59 aa. However, it is shown that only weak SAB affinity is necessary for physiologically relevant action. Alternatively spliced exons can thus translate into strong modulation of specific protein interactions, economizing protein function in the cell without, in and of themselves, imparting unique function.

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Selected References

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  1. Alloisio N., Dalla Venezia N., Rana A., Andrabi K., Texier P., Gilsanz F., Cartron J. P., Delaunay J., Chishti A. H. Evidence that red blood cell protein p55 may participate in the skeleton-membrane linkage that involves protein 4.1 and glycophorin C. Blood. 1993 Aug 15;82(4):1323–1327. [PubMed] [Google Scholar]
  2. Becker P. S., Schwartz M. A., Morrow J. S., Lux S. E. Radiolabel-transfer cross-linking demonstrates that protein 4.1 binds to the N-terminal region of beta spectrin and to actin in binary interactions. Eur J Biochem. 1990 Nov 13;193(3):827–836. doi: 10.1111/j.1432-1033.1990.tb19406.x. [DOI] [PubMed] [Google Scholar]
  3. Bennett V. The membrane skeleton of human erythrocytes and its implications for more complex cells. Annu Rev Biochem. 1985;54:273–304. doi: 10.1146/annurev.bi.54.070185.001421. [DOI] [PubMed] [Google Scholar]
  4. Cohen C. M., Korsgren C. Band 4.1 causes spectrin-actin gels to become thixiotropic. Biochem Biophys Res Commun. 1980 Dec 31;97(4):1429–1435. doi: 10.1016/s0006-291x(80)80025-8. [DOI] [PubMed] [Google Scholar]
  5. Conboy J. G., Chan J., Mohandas N., Kan Y. W. Multiple protein 4.1 isoforms produced by alternative splicing in human erythroid cells. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9062–9065. doi: 10.1073/pnas.85.23.9062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cooper J. A., Pollard T. D. Methods to measure actin polymerization. Methods Enzymol. 1982;85(Pt B):182–210. doi: 10.1016/0076-6879(82)85021-0. [DOI] [PubMed] [Google Scholar]
  7. Correas I., Leto T. L., Speicher D. W., Marchesi V. T. Identification of the functional site of erythrocyte protein 4.1 involved in spectrin-actin associations. J Biol Chem. 1986 Mar 5;261(7):3310–3315. [PubMed] [Google Scholar]
  8. Discher D. E., Mohandas N., Evans E. A. Molecular maps of red cell deformation: hidden elasticity and in situ connectivity. Science. 1994 Nov 11;266(5187):1032–1035. doi: 10.1126/science.7973655. [DOI] [PubMed] [Google Scholar]
  9. Discher D., Parra M., Conboy J. G., Mohandas N. Mechanochemistry of the alternatively spliced spectrin-actin binding domain in membrane skeletal protein 4.1. J Biol Chem. 1993 Apr 5;268(10):7186–7195. [PubMed] [Google Scholar]
  10. Evans E. A. Structure and deformation properties of red blood cells: concepts and quantitative methods. Methods Enzymol. 1989;173:3–35. doi: 10.1016/s0076-6879(89)73003-2. [DOI] [PubMed] [Google Scholar]
  11. Fowler V., Taylor D. L. Spectrin plus band 4.1 cross-link actin. Regulation by micromolar calcium. J Cell Biol. 1980 May;85(2):361–376. doi: 10.1083/jcb.85.2.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gascard P., Cohen C. M. Absence of high-affinity band 4.1 binding sites from membranes of glycophorin C- and D-deficient (Leach phenotype) erythrocytes. Blood. 1994 Feb 15;83(4):1102–1108. [PubMed] [Google Scholar]
  13. Golan D. E., Brown C. S., Cianci C. M., Furlong S. T., Caulfield J. P. Schistosomula of Schistosoma mansoni use lysophosphatidylcholine to lyse adherent human red blood cells and immobilize red cell membrane components. J Cell Biol. 1986 Sep;103(3):819–828. doi: 10.1083/jcb.103.3.819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goodman S. R., Krebs K. E., Whitfield C. F., Riederer B. M., Zagon I. S. Spectrin and related molecules. CRC Crit Rev Biochem. 1988;23(2):171–234. doi: 10.3109/10409238809088319. [DOI] [PubMed] [Google Scholar]
  15. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  16. Horne W. C., Huang S. C., Becker P. S., Tang T. K., Benz E. J., Jr Tissue-specific alternative splicing of protein 4.1 inserts an exon necessary for formation of the ternary complex with erythrocyte spectrin and F-actin. Blood. 1993 Oct 15;82(8):2558–2563. [PubMed] [Google Scholar]
  17. Horne W. C., Prinz W. C., Tang E. K. Identification of two cAMP-dependent phosphorylation sites on erythrocyte protein 4.1. Biochim Biophys Acta. 1990 Oct 15;1055(1):87–92. doi: 10.1016/0167-4889(90)90095-u. [DOI] [PubMed] [Google Scholar]
  18. Huang J. P., Tang C. J., Kou G. H., Marchesi V. T., Benz E. J., Jr, Tang T. K. Genomic structure of the locus encoding protein 4.1. Structural basis for complex combinational patterns of tissue-specific alternative RNA splicing. J Biol Chem. 1993 Feb 15;268(5):3758–3766. [PubMed] [Google Scholar]
  19. Kishino A., Yanagida T. Force measurements by micromanipulation of a single actin filament by glass needles. Nature. 1988 Jul 7;334(6177):74–76. doi: 10.1038/334074a0. [DOI] [PubMed] [Google Scholar]
  20. Lieber M. R., Steck T. L. A description of the holes in human erythrocyte membrane ghosts. J Biol Chem. 1982 Oct 10;257(19):11651–11659. [PubMed] [Google Scholar]
  21. Ling E., Danilov Y. N., Cohen C. M. Modulation of red cell band 4.1 function by cAMP-dependent kinase and protein kinase C phosphorylation. J Biol Chem. 1988 Feb 15;263(5):2209–2216. [PubMed] [Google Scholar]
  22. Lombardo C. R., Willardson B. M., Low P. S. Localization of the protein 4.1-binding site on the cytoplasmic domain of erythrocyte membrane band 3. J Biol Chem. 1992 May 15;267(14):9540–9546. [PubMed] [Google Scholar]
  23. Lorenzo F., Dalla Venezia N., Morlé L., Baklouti F., Alloisio N., Ducluzeau M. T., Roda L., Lefrançois P., Delaunay J. Protein 4.1 deficiency associated with an altered binding to the spectrin-actin complex of the red cell membrane skeleton. J Clin Invest. 1994 Oct;94(4):1651–1656. doi: 10.1172/JCI117508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mach H., Middaugh C. R., Lewis R. V. Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins. Anal Biochem. 1992 Jan;200(1):74–80. doi: 10.1016/0003-2697(92)90279-g. [DOI] [PubMed] [Google Scholar]
  25. McGuire M., Smith B. L., Agre P. Distinct variants of erythrocyte protein 4.1 inherited in linkage with elliptocytosis and Rh type in three white families. Blood. 1988 Jul;72(1):287–293. [PubMed] [Google Scholar]
  26. Mehegan J. P., Tobacman L. S. Cooperative interactions between troponin molecules bound to the cardiac thin filament. J Biol Chem. 1991 Jan 15;266(2):966–972. [PubMed] [Google Scholar]
  27. Menkel A. R., Kroemker M., Bubeck P., Ronsiek M., Nikolai G., Jockusch B. M. Characterization of an F-actin-binding domain in the cytoskeletal protein vinculin. J Cell Biol. 1994 Sep;126(5):1231–1240. doi: 10.1083/jcb.126.5.1231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mische S. M., Mooseker M. S., Morrow J. S. Erythrocyte adducin: a calmodulin-regulated actin-bundling protein that stimulates spectrin-actin binding. J Cell Biol. 1987 Dec;105(6 Pt 1):2837–2845. doi: 10.1083/jcb.105.6.2837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mohandas N., Evans E. Mechanical properties of the red cell membrane in relation to molecular structure and genetic defects. Annu Rev Biophys Biomol Struct. 1994;23:787–818. doi: 10.1146/annurev.bb.23.060194.004035. [DOI] [PubMed] [Google Scholar]
  30. Ohanian V., Wolfe L. C., John K. M., Pinder J. C., Lux S. E., Gratzer W. B. Analysis of the ternary interaction of the red cell membrane skeletal proteins spectrin, actin, and 4.1. Biochemistry. 1984 Sep 11;23(19):4416–4420. doi: 10.1021/bi00314a027. [DOI] [PubMed] [Google Scholar]
  31. Pardee J. D., Spudich J. A. Purification of muscle actin. Methods Enzymol. 1982;85(Pt B):164–181. doi: 10.1016/0076-6879(82)85020-9. [DOI] [PubMed] [Google Scholar]
  32. Podgórski A., Elbaum D. Properties of red cell membrane proteins: mechanism of spectrin and band 4.1 interaction. Biochemistry. 1985 Dec 31;24(27):7871–7876. doi: 10.1021/bi00348a004. [DOI] [PubMed] [Google Scholar]
  33. Ralston G. B. Physico-chemical characterization of the spectrin tetramer from bovine erythrocyte membranes. Biochim Biophys Acta. 1976 Nov 11;455(1):163–172. doi: 10.1016/0005-2736(76)90161-9. [DOI] [PubMed] [Google Scholar]
  34. Reid M. E., Chasis J. A., Mohandas N. Identification of a functional role for human erythrocyte sialoglycoproteins beta and gamma. Blood. 1987 Apr;69(4):1068–1072. [PubMed] [Google Scholar]
  35. Shields M., La Celle P., Waugh R. E., Scholz M., Peters R., Passow H. Effects of intracellular Ca2+ and proteolytic digestion of the membrane skeleton on the mechanical properties of the red blood cell membrane. Biochim Biophys Acta. 1987 Nov 27;905(1):181–194. doi: 10.1016/0005-2736(87)90022-8. [DOI] [PubMed] [Google Scholar]
  36. Subrahmanyam G., Bertics P. J., Anderson R. A. Phosphorylation of protein 4.1 on tyrosine-418 modulates its function in vitro. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5222–5226. doi: 10.1073/pnas.88.12.5222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Takakuwa Y., Tchernia G., Rossi M., Benabadji M., Mohandas N. Restoration of normal membrane stability to unstable protein 4.1-deficient erythrocyte membranes by incorporation of purified protein 4.1. J Clin Invest. 1986 Jul;78(1):80–85. doi: 10.1172/JCI112577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tchernia G., Mohandas N., Shohet S. B. Deficiency of skeletal membrane protein band 4.1 in homozygous hereditary elliptocytosis. Implications for erythrocyte membrane stability. J Clin Invest. 1981 Aug;68(2):454–460. doi: 10.1172/JCI110275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tyler J. M., Hargreaves W. R., Branton D. Purification of two spectrin-binding proteins: biochemical and electron microscopic evidence for site-specific reassociation between spectrin and bands 2.1 and 4.1. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5192–5196. doi: 10.1073/pnas.76.10.5192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ungewickell E., Bennett P. M., Calvert R., Ohanian V., Gratzer W. B. In vitro formation of a complex between cytoskeletal proteins of the human erythrocyte. Nature. 1979 Aug 30;280(5725):811–814. doi: 10.1038/280811a0. [DOI] [PubMed] [Google Scholar]
  41. Ungewickell E., Gratzer W. Self-association of human spectrin. A thermodynamic and kinetic study. Eur J Biochem. 1978 Aug 1;88(2):379–385. doi: 10.1111/j.1432-1033.1978.tb12459.x. [DOI] [PubMed] [Google Scholar]
  42. Waugh R. E. Effects of abnormal cytoskeletal structure on erythrocyte membrane mechanical properties. Cell Motil. 1983;3(5-6):609–622. doi: 10.1002/cm.970030526. [DOI] [PubMed] [Google Scholar]

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