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. 1995 Jul 2;130(2):313–330. doi: 10.1083/jcb.130.2.313

Ank3 (epithelial ankyrin), a widely distributed new member of the ankyrin gene family and the major ankyrin in kidney, is expressed in alternatively spliced forms, including forms that lack the repeat domain

PMCID: PMC2199924  PMID: 7615634

Abstract

We cloned a novel ankyrin, Ank3, from mouse kidney cDNA. The full- length transcript is predicted to encode a 214-kD protein containing an 89 kD, NH2 terminal "repeat" domain; a 65 kD, central "spectrin- binding" domain; and a 56 kD, COOH-terminal "regulatory" domain. The Ank3 gene maps to mouse Chromosome 10, approximately 36 cM from the centromere, a locus distinct from Ank1 and Ank2. Ank3 is the major kidney ankyrin. Multiple transcripts of approximately 7.5, 6.9, 6.3, 5.7, 5.1, and 4.6 kb are highly expressed in kidney where Ank1 and Ank2 mRNAs are barely detectable. The smaller mRNAs (< or = 6.3 kb) lack the entire repeat domain. These transcripts have a unique 5'untranslated region and NH2-terminal sequence and encode a predicted protein of 121 kD. Two small sequences of 21 and 18 amino acids are alternatively spliced at the junction of the repeat and spectrin-binding domains in the larger (> or = 6.9 kb) RNAs. Alternative splicing of a 588 bp sequence (corresponding to a 21.5-kD acidic amino acid sequence) within the regulatory domain also occurs. Ank3 is much more widely expressed than previously described ankyrins. By Northern hybridization or immunocytochemistry, it is present in most epithelial cells, in neuronal axons, in muscle cells, and in megakaryocytes/platelets, macrophages, and the interstitial cells of Leydig (testis). On immunoblots, an antibody raised to a unique regions of the regulatory domain detects multiple Ank3 isoforms in the kidney (215, 200, 170, 120, 105 kD) and in other tissues. The 215/200 kD and 120/105-kD kidney proteins are close to the sizes predicted for the 7.5/6.9- and 6.3/5.7- kb RNAs (with/without the 588-bp acidic insert). Interestingly, it appears that Ank3 exhibits a polarized distribution only in tissues that express the approximately 7.0-kb isoforms, the only isoforms in the kidney that contain the repeat domain. In tissues where smaller transcripts (< or = 6.3 kb) are expressed. Ank3 is diffusely distributed in some or all cells and may be associated with cytoplasmic structures. We conclude that Ank3 is a broadly distributed epithelial ankyrin and is the major ankyrin in the kidney and other tissues, where it plays an important role in the polarized distribution of many integral membrane proteins.

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Selected References

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  1. Barnes W. M. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2216–2220. doi: 10.1073/pnas.91.6.2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bennett V., Davis J. Erythrocyte ankyrin: immunoreactive analogues are associated with mitotic structures in cultured cells and with microtubules in brain. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7550–7554. doi: 10.1073/pnas.78.12.7550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bennett V., Davis J., Fowler W. E. Brain spectrin, a membrane-associated protein related in structure and function to erythrocyte spectrin. Nature. 1982 Sep 9;299(5879):126–131. doi: 10.1038/299126a0. [DOI] [PubMed] [Google Scholar]
  4. Bennett V. Immunoreactive forms of human erythrocyte ankyrin are present in diverse cells and tissues. Nature. 1979 Oct 18;281(5732):597–599. doi: 10.1038/281597a0. [DOI] [PubMed] [Google Scholar]
  5. Bennett V., Stenbuck P. J. Association between ankyrin and the cytoplasmic domain of band 3 isolated from the human erythrocyte membrane. J Biol Chem. 1980 Jul 10;255(13):6424–6432. [PubMed] [Google Scholar]
  6. 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]
  7. Birkenmeier C. S., White R. A., Peters L. L., Hall E. J., Lux S. E., Barker J. E. Complex patterns of sequence variation and multiple 5' and 3' ends are found among transcripts of the erythroid ankyrin gene. J Biol Chem. 1993 May 5;268(13):9533–9540. [PubMed] [Google Scholar]
  8. Bork P. Hundreds of ankyrin-like repeats in functionally diverse proteins: mobile modules that cross phyla horizontally? Proteins. 1993 Dec;17(4):363–374. doi: 10.1002/prot.340170405. [DOI] [PubMed] [Google Scholar]
  9. Bourguignon L. Y., Jin H., Iida N., Brandt N. R., Zhang S. H. The involvement of ankyrin in the regulation of inositol 1,4,5-trisphosphate receptor-mediated internal Ca2+ release from Ca2+ storage vesicles in mouse T-lymphoma cells. J Biol Chem. 1993 Apr 5;268(10):7290–7297. [PubMed] [Google Scholar]
  10. Chan W., Kordeli E., Bennett V. 440-kD ankyrinB: structure of the major developmentally regulated domain and selective localization in unmyelinated axons. J Cell Biol. 1993 Dec;123(6 Pt 1):1463–1473. doi: 10.1083/jcb.123.6.1463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  12. Davis J. Q., Bennett V. Brain ankyrin. A membrane-associated protein with binding sites for spectrin, tubulin, and the cytoplasmic domain of the erythrocyte anion channel. J Biol Chem. 1984 Nov 10;259(21):13550–13559. [PubMed] [Google Scholar]
  13. Davis J. Q., Bennett V. Brain ankyrin. Purification of a 72,000 Mr spectrin-binding domain. J Biol Chem. 1984 Feb 10;259(3):1874–1881. [PubMed] [Google Scholar]
  14. Davis J. Q., McLaughlin T., Bennett V. Ankyrin-binding proteins related to nervous system cell adhesion molecules: candidates to provide transmembrane and intercellular connections in adult brain. J Cell Biol. 1993 Apr;121(1):121–133. doi: 10.1083/jcb.121.1.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Davis J., Davis L., Bennett V. Diversity in membrane binding sites of ankyrins. Brain ankyrin, erythrocyte ankyrin, and processed erythrocyte ankyrin associate with distinct sites in kidney microsomes. J Biol Chem. 1989 Apr 15;264(11):6417–6426. [PubMed] [Google Scholar]
  16. 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]
  17. Davis L. H., Davis J. Q., Bennett V. Ankyrin regulation: an alternatively spliced segment of the regulatory domain functions as an intramolecular modulator. J Biol Chem. 1992 Sep 15;267(26):18966–18972. [PubMed] [Google Scholar]
  18. Del Sal G., Manfioletti G., Schneider C. The CTAB-DNA precipitation method: a common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing. Biotechniques. 1989 May;7(5):514–520. [PubMed] [Google Scholar]
  19. Devarajan P., Scaramuzzino D. A., Morrow J. S. Ankyrin binds to two distinct cytoplasmic domains of Na,K-ATPase alpha subunit. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):2965–2969. doi: 10.1073/pnas.91.8.2965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Drenckhahn D., Bennett V. Polarized distribution of Mr 210,000 and 190,000 analogs of erythrocyte ankyrin along the plasma membrane of transporting epithelia, neurons and photoreceptors. Eur J Cell Biol. 1987 Jun;43(3):479–486. [PubMed] [Google Scholar]
  22. Drenckhahn D., Schlüter K., Allen D. P., Bennett V. Colocalization of band 3 with ankyrin and spectrin at the basal membrane of intercalated cells in the rat kidney. Science. 1985 Dec 13;230(4731):1287–1289. doi: 10.1126/science.2933809. [DOI] [PubMed] [Google Scholar]
  23. Dubreuil R. R., Yu J. Ankyrin and beta-spectrin accumulate independently of alpha-spectrin in Drosophila. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10285–10289. doi: 10.1073/pnas.91.22.10285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Flucher B. E., Daniels M. P. Distribution of Na+ channels and ankyrin in neuromuscular junctions is complementary to that of acetylcholine receptors and the 43 kd protein. Neuron. 1989 Aug;3(2):163–175. doi: 10.1016/0896-6273(89)90029-9. [DOI] [PubMed] [Google Scholar]
  25. Flucher B. E., Morton M. E., Froehner S. C., Daniels M. P. Localization of the alpha 1 and alpha 2 subunits of the dihydropyridine receptor and ankyrin in skeletal muscle triads. Neuron. 1990 Sep;5(3):339–351. doi: 10.1016/0896-6273(90)90170-k. [DOI] [PubMed] [Google Scholar]
  26. Gallagher P. G., Tse W. T., Scarpa A. L., Lux S. E., Forget B. G. Large numbers of alternatively spliced isoforms of the regulatory region of human erythrocyte ankyrin. Trans Assoc Am Physicians. 1992;105:268–277. [PubMed] [Google Scholar]
  27. Georgatos S. D., Marchesi V. T. The binding of vimentin to human erythrocyte membranes: a model system for the study of intermediate filament-membrane interactions. J Cell Biol. 1985 Jun;100(6):1955–1961. doi: 10.1083/jcb.100.6.1955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Gregorio C. C., Repasky E. A., Fowler V. M., Black J. D. Dynamic properties of ankyrin in T lymphocytes: colocalization with spectrin and protein kinase C beta. J Cell Biol. 1994 Apr;125(2):345–358. doi: 10.1083/jcb.125.2.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Gundersen D., Orlowski J., Rodriguez-Boulan E. Apical polarity of Na,K-ATPase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton. J Cell Biol. 1991 Mar;112(5):863–872. doi: 10.1083/jcb.112.5.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hammerton R. W., Krzeminski K. A., Mays R. W., Ryan T. A., Wollner D. A., Nelson W. J. Mechanism for regulating cell surface distribution of Na+,K(+)-ATPase in polarized epithelial cells. Science. 1991 Nov 8;254(5033):847–850. doi: 10.1126/science.1658934. [DOI] [PubMed] [Google Scholar]
  31. Kaelin W. G., Jr, Krek W., Sellers W. R., DeCaprio J. A., Ajchenbaum F., Fuchs C. S., Chittenden T., Li Y., Farnham P. J., Blanar M. A. Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell. 1992 Jul 24;70(2):351–364. doi: 10.1016/0092-8674(92)90108-o. [DOI] [PubMed] [Google Scholar]
  32. Katz A. I., Doucet A., Morel F. Na-K-ATPase activity along the rabbit, rat, and mouse nephron. Am J Physiol. 1979 Aug;237(2):F114–F120. doi: 10.1152/ajprenal.1979.237.2.F114. [DOI] [PubMed] [Google Scholar]
  33. Koob R., Zimmermann M., Schoner W., Drenckhahn D. Colocalization and coprecipitation of ankyrin and Na+,K+-ATPase in kidney epithelial cells. Eur J Cell Biol. 1988 Feb;45(2):230–237. [PubMed] [Google Scholar]
  34. Kordeli E., Bennett V. Distinct ankyrin isoforms at neuron cell bodies and nodes of Ranvier resolved using erythrocyte ankyrin-deficient mice. J Cell Biol. 1991 Sep;114(6):1243–1259. doi: 10.1083/jcb.114.6.1243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kordeli E., Davis J., Trapp B., Bennett V. An isoform of ankyrin is localized at nodes of Ranvier in myelinated axons of central and peripheral nerves. J Cell Biol. 1990 Apr;110(4):1341–1352. doi: 10.1083/jcb.110.4.1341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kordeli E., Lambert S., Bennett V. AnkyrinG. A new ankyrin gene with neural-specific isoforms localized at the axonal initial segment and node of Ranvier. J Biol Chem. 1995 Feb 3;270(5):2352–2359. doi: 10.1074/jbc.270.5.2352. [DOI] [PubMed] [Google Scholar]
  37. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Lambert S., Bennett V. Postmitotic expression of ankyrinR and beta R-spectrin in discrete neuronal populations of the rat brain. J Neurosci. 1993 Sep;13(9):3725–3735. doi: 10.1523/JNEUROSCI.13-09-03725.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Li Z. P., Burke E. P., Frank J. S., Bennett V., Philipson K. D. The cardiac Na+-Ca2+ exchanger binds to the cytoskeletal protein ankyrin. J Biol Chem. 1993 Jun 5;268(16):11489–11491. [PubMed] [Google Scholar]
  42. Lokeshwar V. B., Fregien N., Bourguignon L. Y. Ankyrin-binding domain of CD44(GP85) is required for the expression of hyaluronic acid-mediated adhesion function. J Cell Biol. 1994 Aug;126(4):1099–1109. doi: 10.1083/jcb.126.4.1099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Lux S. E., Tse W. T., Menninger J. C., John K. M., Harris P., Shalev O., Chilcote R. R., Marchesi S. L., Watkins P. C., Bennett V. Hereditary spherocytosis associated with deletion of human erythrocyte ankyrin gene on chromosome 8. Nature. 1990 Jun 21;345(6277):736–739. doi: 10.1038/345736a0. [DOI] [PubMed] [Google Scholar]
  44. Marrs J. A., Napolitano E. W., Murphy-Erdosh C., Mays R. W., Reichardt L. F., Nelson W. J. Distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different Na+,K(+)-ATPase distributions in polarized epithelia. J Cell Biol. 1993 Oct;123(1):149–164. doi: 10.1083/jcb.123.1.149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Morgans C. W., Kopito R. R. Association of the brain anion exchanger, AE3, with the repeat domain of ankyrin. J Cell Sci. 1993 Aug;105(Pt 4):1137–1142. doi: 10.1242/jcs.105.4.1137. [DOI] [PubMed] [Google Scholar]
  46. Nelson W. J., Hammerton R. W. A membrane-cytoskeletal complex containing Na+,K+-ATPase, ankyrin, and fodrin in Madin-Darby canine kidney (MDCK) cells: implications for the biogenesis of epithelial cell polarity. J Cell Biol. 1989 Mar;108(3):893–902. doi: 10.1083/jcb.108.3.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Nelson W. J., Shore E. M., Wang A. Z., Hammerton R. W. Identification of a membrane-cytoskeletal complex containing the cell adhesion molecule uvomorulin (E-cadherin), ankyrin, and fodrin in Madin-Darby canine kidney epithelial cells. J Cell Biol. 1990 Feb;110(2):349–357. doi: 10.1083/jcb.110.2.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Otto E., Kunimoto M., McLaughlin T., Bennett V. Isolation and characterization of cDNAs encoding human brain ankyrins reveal a family of alternatively spliced genes. J Cell Biol. 1991 Jul;114(2):241–253. doi: 10.1083/jcb.114.2.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Peters L. L., Birkenmeier C. S., Bronson R. T., White R. A., Lux S. E., Otto E., Bennett V., Higgins A., Barker J. E. Purkinje cell degeneration associated with erythroid ankyrin deficiency in nb/nb mice. J Cell Biol. 1991 Sep;114(6):1233–1241. doi: 10.1083/jcb.114.6.1233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Peters L. L., Lux S. E. Ankyrins: structure and function in normal cells and hereditary spherocytes. Semin Hematol. 1993 Apr;30(2):85–118. [PubMed] [Google Scholar]
  52. Platt O. S., Lux S. E., Falcone J. F. A highly conserved region of human erythrocyte ankyrin contains the capacity to bind spectrin. J Biol Chem. 1993 Nov 15;268(32):24421–24426. [PubMed] [Google Scholar]
  53. Rowe L. B., Nadeau J. H., Turner R., Frankel W. N., Letts V. A., Eppig J. T., Ko M. S., Thurston S. J., Birkenmeier E. H. Maps from two interspecific backcross DNA panels available as a community genetic mapping resource. Mamm Genome. 1994 May;5(5):253–274. doi: 10.1007/BF00389540. [DOI] [PubMed] [Google Scholar]
  54. Shibayama T., Nakaya K., Nakamura Y. Differential binding activity of erythrocyte ankyrin to the alpha-subunits of Na+, K(+)-ATPases from rat cerebral and axonal membrane. Cell Struct Funct. 1993 Feb;18(1):79–85. doi: 10.1247/csf.18.79. [DOI] [PubMed] [Google Scholar]
  55. Smith P. R., Bradford A. L., Joe E. H., Angelides K. J., Benos D. J., Saccomani G. Gastric parietal cell H(+)-K(+)-ATPase microsomes are associated with isoforms of ankyrin and spectrin. Am J Physiol. 1993 Jan;264(1 Pt 1):C63–C70. doi: 10.1152/ajpcell.1993.264.1.C63. [DOI] [PubMed] [Google Scholar]
  56. Smith P. R., Saccomani G., Joe E. H., Angelides K. J., Benos D. J. Amiloride-sensitive sodium channel is linked to the cytoskeleton in renal epithelial cells. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):6971–6975. doi: 10.1073/pnas.88.16.6971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. 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]
  58. Tse W. T., Menninger J. C., Yang-Feng T. L., Francke U., Sahr K. E., Lux S. E., Ward D. C., Forget B. G. Isolation and chromosomal localization of a novel nonerythroid ankyrin gene. Genomics. 1991 Aug;10(4):858–866. doi: 10.1016/0888-7543(91)90173-c. [DOI] [PubMed] [Google Scholar]
  59. Wang R. F., Cao W. W., Johnson M. G. A simplified, single tube, single buffer system for RNA-PCR. Biotechniques. 1992 May;12(5):702–704. [PubMed] [Google Scholar]
  60. 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]
  61. White R. A., Birkenmeier C. S., Peters L. L., Barker J. E., Lux S. E. Murine erythrocyte ankyrin cDNA: highly conserved regions of the regulatory domain. Mamm Genome. 1992;3(5):281–285. doi: 10.1007/BF00292156. [DOI] [PubMed] [Google Scholar]

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