Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1995 Feb 1;128(3):363–371. doi: 10.1083/jcb.128.3.363

Syntrophin binds to an alternatively spliced exon of dystrophin

PMCID: PMC2120343  PMID: 7844150

Abstract

Dystrophin, the protein product of the Duchenne muscular dystrophy locus, is a protein of the membrane cytoskeleton that associates with a complex of integral and membrane-associated proteins. Of these, the 58- kD intracellular membrane-associated protein, syntrophin, was recently shown to consist of a family of three related but distinct genes. We expressed the cDNA of human beta 1-syntrophin and the COOH terminus of human dystrophin in reticulocyte lysates using an in vitro transcription/translation system. Using antibodies to dystrophin we immunoprecipitated these two interacting proteins in a variety of salt and detergent conditions. We demonstrate that the 53 amino acids encoded on exon 74 of dystrophin, an alternatively spliced exon, are necessary and sufficient for interaction with translated beta 1- syntrophin in our assay. On the basis of its alternative splicing, dystrophin may thus be present in two functionally distinct populations. In this recombinant expression system, the dystrophin relatives, human dystrophin related protein (DRP or utrophin) and the 87K postsynaptic protein from Torpedo electric organ, also bind to translated beta 1-syntrophin. We have found a COOH-terminal 37-kD fragment of beta 1-syntrophin sufficient to interact with translated dystrophin and its homologues, suggesting that the dystrophin binding site on beta 1-syntrophin occurs on a region that is conserved among the three syntrophin homologues.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams M. E., Butler M. H., Dwyer T. M., Peters M. F., Murnane A. A., Froehner S. C. Two forms of mouse syntrophin, a 58 kd dystrophin-associated protein, differ in primary structure and tissue distribution. Neuron. 1993 Sep;11(3):531–540. doi: 10.1016/0896-6273(93)90157-m. [DOI] [PubMed] [Google Scholar]
  2. Ahn A. H., Yoshida M., Anderson M. S., Feener C. A., Selig S., Hagiwara Y., Ozawa E., Kunkel L. M. Cloning of human basic A1, a distinct 59-kDa dystrophin-associated protein encoded on chromosome 8q23-24. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4446–4450. doi: 10.1073/pnas.91.10.4446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arahata K., Ishiura S., Ishiguro T., Tsukahara T., Suhara Y., Eguchi C., Ishihara T., Nonaka I., Ozawa E., Sugita H. Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide. Nature. 1988 Jun 30;333(6176):861–863. doi: 10.1038/333861a0. [DOI] [PubMed] [Google Scholar]
  4. Bies R. D., Phelps S. F., Cortez M. D., Roberts R., Caskey C. T., Chamberlain J. S. Human and murine dystrophin mRNA transcripts are differentially expressed during skeletal muscle, heart, and brain development. Nucleic Acids Res. 1992 Apr 11;20(7):1725–1731. doi: 10.1093/nar/20.7.1725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Butler M. H., Douville K., Murnane A. A., Kramarcy N. R., Cohen J. B., Sealock R., Froehner S. C. Association of the Mr 58,000 postsynaptic protein of electric tissue with Torpedo dystrophin and the Mr 87,000 postsynaptic protein. J Biol Chem. 1992 Mar 25;267(9):6213–6218. [PubMed] [Google Scholar]
  6. Byers T. J., Kunkel L. M., Watkins S. C. The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle. J Cell Biol. 1991 Oct;115(2):411–421. doi: 10.1083/jcb.115.2.411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Byers T. J., Lidov H. G., Kunkel L. M. An alternative dystrophin transcript specific to peripheral nerve. Nat Genet. 1993 May;4(1):77–81. doi: 10.1038/ng0593-77. [DOI] [PubMed] [Google Scholar]
  8. Campbell K. P., Kahl S. D. Association of dystrophin and an integral membrane glycoprotein. Nature. 1989 Mar 16;338(6212):259–262. doi: 10.1038/338259a0. [DOI] [PubMed] [Google Scholar]
  9. Carr C., Fischbach G. D., Cohen J. B. A novel 87,000-Mr protein associated with acetylcholine receptors in Torpedo electric organ and vertebrate skeletal muscle. J Cell Biol. 1989 Oct;109(4 Pt 1):1753–1764. doi: 10.1083/jcb.109.4.1753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cartaud A., Ludosky M. A., Tomé F. M., Collin H., Stetzkowski-Marden F., Khurana T. S., Kunkel L. M., Fardeau M., Changeux J. P., Cartaud J. Localization of dystrophin and dystrophin-related protein at the electromotor synapse and neuromuscular junction in Torpedo marmorata. Neuroscience. 1992 Jun;48(4):995–1003. doi: 10.1016/0306-4522(92)90288-d. [DOI] [PubMed] [Google Scholar]
  11. Cartaud A., Stetzkowski-Marden F., Cartaud J. Identification of dystrophin-binding protein(s) in membranes from Torpedo electrocyte and rat muscle. J Biol Chem. 1993 Jun 25;268(18):13019–13022. [PubMed] [Google Scholar]
  12. Ervasti J. M., Campbell K. P. Membrane organization of the dystrophin-glycoprotein complex. Cell. 1991 Sep 20;66(6):1121–1131. doi: 10.1016/0092-8674(91)90035-w. [DOI] [PubMed] [Google Scholar]
  13. Feener C. A., Koenig M., Kunkel L. M. Alternative splicing of human dystrophin mRNA generates isoforms at the carboxy terminus. Nature. 1989 Apr 6;338(6215):509–511. doi: 10.1038/338509a0. [DOI] [PubMed] [Google Scholar]
  14. Froehner S. C., Murnane A. A., Tobler M., Peng H. B., Sealock R. A postsynaptic Mr 58,000 (58K) protein concentrated at acetylcholine receptor-rich sites in Torpedo electroplaques and skeletal muscle. J Cell Biol. 1987 Jun;104(6):1633–1646. doi: 10.1083/jcb.104.6.1633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gallie D. R., Sleat D. E., Watts J. W., Turner P. C., Wilson T. M. Mutational analysis of the tobacco mosaic virus 5'-leader for altered ability to enhance translation. Nucleic Acids Res. 1988 Feb 11;16(3):883–893. doi: 10.1093/nar/16.3.883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gallie D. R., Sleat D. E., Watts J. W., Turner P. C., Wilson T. M. The 5'-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo. Nucleic Acids Res. 1987 Apr 24;15(8):3257–3273. doi: 10.1093/nar/15.8.3257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Khurana T. S., Watkins S. C., Chafey P., Chelly J., Tomé F. M., Fardeau M., Kaplan J. C., Kunkel L. M. Immunolocalization and developmental expression of dystrophin related protein in skeletal muscle. Neuromuscul Disord. 1991;1(3):185–194. doi: 10.1016/0960-8966(91)90023-l. [DOI] [PubMed] [Google Scholar]
  18. Koenig M., Hoffman E. P., Bertelson C. J., Monaco A. P., Feener C., Kunkel L. M. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell. 1987 Jul 31;50(3):509–517. doi: 10.1016/0092-8674(87)90504-6. [DOI] [PubMed] [Google Scholar]
  19. Koenig M., Kunkel L. M. Detailed analysis of the repeat domain of dystrophin reveals four potential hinge segments that may confer flexibility. J Biol Chem. 1990 Mar 15;265(8):4560–4566. [PubMed] [Google Scholar]
  20. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  21. Kramarcy N. R., Vidal A., Froehner S. C., Sealock R. Association of utrophin and multiple dystrophin short forms with the mammalian M(r) 58,000 dystrophin-associated protein (syntrophin). J Biol Chem. 1994 Jan 28;269(4):2870–2876. [PubMed] [Google Scholar]
  22. 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]
  23. Lederfein D., Levy Z., Augier N., Mornet D., Morris G., Fuchs O., Yaffe D., Nudel U. A 71-kilodalton protein is a major product of the Duchenne muscular dystrophy gene in brain and other nonmuscle tissues. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5346–5350. doi: 10.1073/pnas.89.12.5346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lidov H. G., Byers T. J., Kunkel L. M. The distribution of dystrophin in the murine central nervous system: an immunocytochemical study. Neuroscience. 1993 May;54(1):167–187. doi: 10.1016/0306-4522(93)90392-s. [DOI] [PubMed] [Google Scholar]
  25. Lidov H. G., Byers T. J., Watkins S. C., Kunkel L. M. Localization of dystrophin to postsynaptic regions of central nervous system cortical neurons. Nature. 1990 Dec 20;348(6303):725–728. doi: 10.1038/348725a0. [DOI] [PubMed] [Google Scholar]
  26. Love D. R., Hill D. F., Dickson G., Spurr N. K., Byth B. C., Marsden R. F., Walsh F. S., Edwards Y. H., Davies K. E. An autosomal transcript in skeletal muscle with homology to dystrophin. Nature. 1989 May 4;339(6219):55–58. doi: 10.1038/339055a0. [DOI] [PubMed] [Google Scholar]
  27. Love D. R., Morris G. E., Ellis J. M., Fairbrother U., Marsden R. F., Bloomfield J. F., Edwards Y. H., Slater C. P., Parry D. J., Davies K. E. Tissue distribution of the dystrophin-related gene product and expression in the mdx and dy mouse. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3243–3247. doi: 10.1073/pnas.88.8.3243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Madhavan R., Jarrett H. W. Calmodulin-activated phosphorylation of dystrophin. Biochemistry. 1994 May 17;33(19):5797–5804. doi: 10.1021/bi00185a018. [DOI] [PubMed] [Google Scholar]
  29. Matsumura K., Ervasti J. M., Ohlendieck K., Kahl S. D., Campbell K. P. Association of dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle. Nature. 1992 Dec 10;360(6404):588–591. doi: 10.1038/360588a0. [DOI] [PubMed] [Google Scholar]
  30. Milner R. E., Busaan J. L., Holmes C. F., Wang J. H., Michalak M. Phosphorylation of dystrophin. The carboxyl-terminal region of dystrophin is a substrate for in vitro phosphorylation by p34cdc2 protein kinase. J Biol Chem. 1993 Oct 15;268(29):21901–21905. [PubMed] [Google Scholar]
  31. Ohlendieck K., Ervasti J. M., Matsumura K., Kahl S. D., Leveille C. J., Campbell K. P. Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle. Neuron. 1991 Sep;7(3):499–508. doi: 10.1016/0896-6273(91)90301-f. [DOI] [PubMed] [Google Scholar]
  32. Panayotatos N., Wells R. D. Recognition and initiation site for four late promoters of phage T7 is a 22-base pair DNA sequence. Nature. 1979 Jul 5;280(5717):35–39. doi: 10.1038/280035a0. [DOI] [PubMed] [Google Scholar]
  33. Peters M. F., Kramarcy N. R., Sealock R., Froehner S. C. beta 2-Syntrophin: localization at the neuromuscular junction in skeletal muscle. Neuroreport. 1994 Aug 15;5(13):1577–1580. [PubMed] [Google Scholar]
  34. Rafael J. A., Sunada Y., Cole N. M., Campbell K. P., Faulkner J. A., Chamberlain J. S. Prevention of dystrophic pathology in mdx mice by a truncated dystrophin isoform. Hum Mol Genet. 1994 Oct;3(10):1725–1733. doi: 10.1093/hmg/3.10.1725. [DOI] [PubMed] [Google Scholar]
  35. Suzuki A., Yoshida M., Hayashi K., Mizuno Y., Hagiwara Y., Ozawa E. Molecular organization at the glycoprotein-complex-binding site of dystrophin. Three dystrophin-associated proteins bind directly to the carboxy-terminal portion of dystrophin. Eur J Biochem. 1994 Mar 1;220(2):283–292. doi: 10.1111/j.1432-1033.1994.tb18624.x. [DOI] [PubMed] [Google Scholar]
  36. Suzuki A., Yoshida M., Ozawa E. Mammalian alpha 1- and beta 1-syntrophin bind to the alternative splice-prone region of the dystrophin COOH terminus. J Cell Biol. 1995 Feb;128(3):373–381. doi: 10.1083/jcb.128.3.373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Suzuki A., Yoshida M., Yamamoto H., Ozawa E. Glycoprotein-binding site of dystrophin is confined to the cysteine-rich domain and the first half of the carboxy-terminal domain. FEBS Lett. 1992 Aug 17;308(2):154–160. doi: 10.1016/0014-5793(92)81265-n. [DOI] [PubMed] [Google Scholar]
  38. Tinsley J. M., Blake D. J., Roche A., Fairbrother U., Riss J., Byth B. C., Knight A. E., Kendrick-Jones J., Suthers G. K., Love D. R. Primary structure of dystrophin-related protein. Nature. 1992 Dec 10;360(6404):591–593. doi: 10.1038/360591a0. [DOI] [PubMed] [Google Scholar]
  39. Wagner K. R., Cohen J. B., Huganir R. L. The 87K postsynaptic membrane protein from Torpedo is a protein-tyrosine kinase substrate homologous to dystrophin. Neuron. 1993 Mar;10(3):511–522. doi: 10.1016/0896-6273(93)90338-r. [DOI] [PubMed] [Google Scholar]
  40. Wagner K. R., Huganir R. L. Tyrosine and serine phosphorylation of dystrophin and the 58-kDa protein in the postsynaptic membrane of Torpedo electric organ. J Neurochem. 1994 May;62(5):1947–1952. doi: 10.1046/j.1471-4159.1994.62051947.x. [DOI] [PubMed] [Google Scholar]
  41. Waksman G., Shoelson S. E., Pant N., Cowburn D., Kuriyan J. Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms. Cell. 1993 Mar 12;72(5):779–790. doi: 10.1016/0092-8674(93)90405-f. [DOI] [PubMed] [Google Scholar]
  42. Yamamoto H., Hagiwara Y., Mizuno Y., Yoshida M., Ozawa E. Heterogeneity of dystrophin-associated proteins. J Biochem. 1993 Jul;114(1):132–139. doi: 10.1093/oxfordjournals.jbchem.a124128. [DOI] [PubMed] [Google Scholar]
  43. Yang B., Ibraghimov-Beskrovnaya O., Moomaw C. R., Slaughter C. A., Campbell K. P. Heterogeneity of the 59-kDa dystrophin-associated protein revealed by cDNA cloning and expression. J Biol Chem. 1994 Feb 25;269(8):6040–6044. [PubMed] [Google Scholar]
  44. Yoshida M., Ozawa E. Glycoprotein complex anchoring dystrophin to sarcolemma. J Biochem. 1990 Nov;108(5):748–752. doi: 10.1093/oxfordjournals.jbchem.a123276. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES