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. 1991 Oct 1;115(1):267–277. doi: 10.1083/jcb.115.1.267

Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin

PMCID: PMC2289929  PMID: 1833409

Abstract

Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2- nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta- spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768- 1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.

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Selected References

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  1. Anderson J. P., Morrow J. S. The interaction of calmodulin with human erythrocyte spectrin. Inhibition of protein 4.1-stimulated actin binding. J Biol Chem. 1987 May 5;262(13):6365–6372. [PubMed] [Google Scholar]
  2. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 7. Microbiol Rev. 1983 Jun;47(2):180–230. doi: 10.1128/mr.47.2.180-230.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bennett V., Stenbuck P. J. Identification and partial purification of ankyrin, the high affinity membrane attachment site for human erythrocyte spectrin. J Biol Chem. 1979 Apr 10;254(7):2533–2541. [PubMed] [Google Scholar]
  5. Bourguignon L. Y., Walker G., Suchard S. J., Balazovich K. A lymphoma plasma membrane-associated protein with ankyrin-like properties. J Cell Biol. 1986 Jun;102(6):2115–2124. doi: 10.1083/jcb.102.6.2115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  7. Cianci C. D., Giorgi M., Morrow J. S. Phosphorylation of ankyrin down-regulates its cooperative interaction with spectrin and protein 3. J Cell Biochem. 1988 Jul;37(3):301–315. doi: 10.1002/jcb.240370305. [DOI] [PubMed] [Google Scholar]
  8. Coleman T. R., Fishkind D. J., Mooseker M. S., Morrow J. S. Functional diversity among spectrin isoforms. Cell Motil Cytoskeleton. 1989;12(4):225–247. doi: 10.1002/cm.970120405. [DOI] [PubMed] [Google Scholar]
  9. Davis L. H., Bennett V. Mapping the binding sites of human erythrocyte ankyrin for the anion exchanger and spectrin. J Biol Chem. 1990 Jun 25;265(18):10589–10596. [PubMed] [Google Scholar]
  10. Harris A. S., Anderson J. P., Yurchenco P. D., Green L. A., Ainger K. J., Morrow J. S. Mechanisms of cytoskeletal regulation: functional and antigenic diversity in human erythrocyte and brain beta spectrin. J Cell Biochem. 1986;30(1):51–69. doi: 10.1002/jcb.240300107. [DOI] [PubMed] [Google Scholar]
  11. Howe C. L., Sacramone L. M., Mooseker M. S., Morrow J. S. Mechanisms of cytoskeletal regulation: modulation of membrane affinity in avian brush border and erythrocyte spectrins. J Cell Biol. 1985 Oct;101(4):1379–1385. doi: 10.1083/jcb.101.4.1379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kalomiris E. L., Bourguignon L. Y. Mouse T lymphoma cells contain a transmembrane glycoprotein (GP85) that binds ankyrin. J Cell Biol. 1988 Feb;106(2):319–327. doi: 10.1083/jcb.106.2.319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Knowles W. J., Morrow J. S., Speicher D. W., Zarkowsky H. S., Mohandas N., Mentzer W. C., Shohet S. B., Marchesi V. T. Molecular and functional changes in spectrin from patients with hereditary pyropoikilocytosis. J Clin Invest. 1983 Jun;71(6):1867–1877. doi: 10.1172/JCI110942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lambert S., Yu H., Prchal J. T., Lawler J., Ruff P., Speicher D., Cheung M. C., Kan Y. W., Palek J. cDNA sequence for human erythrocyte ankyrin. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1730–1734. doi: 10.1073/pnas.87.5.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lu P. W., Soong C. J., Tao M. Phosphorylation of ankyrin decreases its affinity for spectrin tetramer. J Biol Chem. 1985 Dec 5;260(28):14958–14964. [PubMed] [Google Scholar]
  18. Lux S. E., John K. M., Bennett V. Analysis of cDNA for human erythrocyte ankyrin indicates a repeated structure with homology to tissue-differentiation and cell-cycle control proteins. Nature. 1990 Mar 1;344(6261):36–42. doi: 10.1038/344036a0. [DOI] [PubMed] [Google Scholar]
  19. Morrow J. S., Cianci C. D., Ardito T., Mann A. S., Kashgarian M. Ankyrin links fodrin to the alpha subunit of Na,K-ATPase in Madin-Darby canine kidney cells and in intact renal tubule cells. J Cell Biol. 1989 Feb;108(2):455–465. doi: 10.1083/jcb.108.2.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Morrow J. S., Speicher D. W., Knowles W. J., Hsu C. J., Marchesi V. T. Identification of functional domains of human erythrocyte spectrin. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6592–6596. doi: 10.1073/pnas.77.11.6592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nelson W. J., Veshnock P. J. Ankyrin binding to (Na+ + K+)ATPase and implications for the organization of membrane domains in polarized cells. Nature. 1987 Aug 6;328(6130):533–536. doi: 10.1038/328533a0. [DOI] [PubMed] [Google Scholar]
  22. Sahr K. E., Laurila P., Kotula L., Scarpa A. L., Coupal E., Leto T. L., Linnenbach A. J., Winkelmann J. C., Speicher D. W., Marchesi V. T. The complete cDNA and polypeptide sequences of human erythroid alpha-spectrin. J Biol Chem. 1990 Mar 15;265(8):4434–4443. [PubMed] [Google Scholar]
  23. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  24. Srinivasan Y., Elmer L., Davis J., Bennett V., Angelides K. Ankyrin and spectrin associate with voltage-dependent sodium channels in brain. Nature. 1988 May 12;333(6169):177–180. doi: 10.1038/333177a0. [DOI] [PubMed] [Google Scholar]
  25. Steiner J. P., Bennett V. Ankyrin-independent membrane protein-binding sites for brain and erythrocyte spectrin. J Biol Chem. 1988 Oct 5;263(28):14417–14425. [PubMed] [Google Scholar]
  26. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tyler J. M., Reinhardt B. N., Branton D. Associations of erythrocyte membrane proteins. Binding of purified bands 2.1 and 4.1 to spectrin. J Biol Chem. 1980 Jul 25;255(14):7034–7039. [PubMed] [Google Scholar]
  28. White R. A., Birkenmeier C. S., Lux S. E., Barker J. E. Ankyrin and the hemolytic anemia mutation, nb, map to mouse chromosome 8: presence of the nb allele is associated with a truncated erythrocyte ankyrin. Proc Natl Acad Sci U S A. 1990 Apr;87(8):3117–3121. doi: 10.1073/pnas.87.8.3117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Winkelmann J. C., Chang J. G., Tse W. T., Scarpa A. L., Marchesi V. T., Forget B. G. Full-length sequence of the cDNA for human erythroid beta-spectrin. J Biol Chem. 1990 Jul 15;265(20):11827–11832. [PubMed] [Google Scholar]
  30. Winkelmann J. C., Leto T. L., Watkins P. C., Eddy R., Shows T. B., Linnenbach A. J., Sahr K. E., Kathuria N., Marchesi V. T., Forget B. G. Molecular cloning of the cDNA for human erythrocyte beta-spectrin. Blood. 1988 Jul;72(1):328–334. [PubMed] [Google Scholar]

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