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. 1996 Sep 2;134(6):1531–1541. doi: 10.1083/jcb.134.6.1531

Dominant-negative effect on adhesion by myelin Po protein truncated in its cytoplasmic domain

PMCID: PMC2120993  PMID: 8830780

Abstract

The myelin Po protein is believed to hold myelin together via interactions of both its extracellular and cytoplasmic domains. We have already shown that the extracellular domains of Po can interact in a homophilic manner (Filbin, M.T., F.S. Walsh, B.D. Trapp, J.A. Pizzey, and G.I. Tennekoon. 1990. Nature (Lond.). 344:871-872). In addition, we have shown that for this homophilic adhesion to take place, the cytoplasmic domain of Po must be intact and most likely interacting with the cytoskeleton; Po proteins truncated in their cytoplasmic domains are not adhesive (Wong, M.H., and M.T. Filbin, 1994. J. Cell Biol. 126:1089-1097). To determine if the presence of these truncated forms of Po could have an effect on the functioning of the full-length Po, we coexpressed both molecules in CHO cells. The adhesiveness of CHO cells expressing both full-length Po and truncated Po was then compared to cells expressing only full-length Po. In these coexpressors, both the full-length and the truncated Po proteins were glycosylated. They reached the surface of the cell in approximately equal amounts as shown by an ELISA and surface labeling, followed by immunoprecipitation. Furthermore, the amount of full-length Po at the cell surface was equivalent to other cell lines expressing only full-length Po that we had already shown to be adhesive. Therefore, there should be sufficient levels of full-length Po at the surface of these coexpressors to measure adhesion of Po. However, as assessed by an aggregation assay, the coexpressors were not adhesive. By 60 min they had not formed large aggregates and were indistinguishable from the control transfected cells not expressing Po. In contrast, in the same time, the cells expressing only the full-length Po had formed large aggregates. This indicates that the truncated forms of Po have a dominant-negative effect on the adhesiveness of the full-length Po. Furthermore, from cross-linking studies, full-length Po, when expressed alone but not when coexpressed with truncated Po, appears to cluster in the membrane. We suggest that truncated Po exerts its dominant-negative effect by preventing clustering of full-length Po. We also show that colchicine, which disrupts microtubules, prevents adhesion of cells expressing only the full-length Po. This strengthens our suggestion that an interaction of Po with the cytoskeleton, either directly or indirectly, is required for adhesion to take place.

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Selected References

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  1. Achler C., Filmer D., Merte C., Drenckhahn D. Role of microtubules in polarized delivery of apical membrane proteins to the brush border of the intestinal epithelium. J Cell Biol. 1989 Jul;109(1):179–189. doi: 10.1083/jcb.109.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akiyama S. K., Yamada S. S., Yamada K. M., LaFlamme S. E. Transmembrane signal transduction by integrin cytoplasmic domains expressed in single-subunit chimeras. J Biol Chem. 1994 Jun 10;269(23):15961–15964. [PubMed] [Google Scholar]
  3. Balzac F., Retta S. F., Albini A., Melchiorri A., Koteliansky V. E., Geuna M., Silengo L., Tarone G. Expression of beta 1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility. J Cell Biol. 1994 Oct;127(2):557–565. doi: 10.1083/jcb.127.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cammer W., Brosnan C. F., Bloom B. R., Norton W. T. Degradation of the P0, P1, and Pr proteins in peripheral nervous system myelin by plasmin: implications regarding the role of macrophages in demyelinating diseases. J Neurochem. 1981 Apr;36(4):1506–1514. doi: 10.1111/j.1471-4159.1981.tb00593.x. [DOI] [PubMed] [Google Scholar]
  5. Carpén O., Pallai P., Staunton D. E., Springer T. A. Association of intercellular adhesion molecule-1 (ICAM-1) with actin-containing cytoskeleton and alpha-actinin. J Cell Biol. 1992 Sep;118(5):1223–1234. doi: 10.1083/jcb.118.5.1223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chance P. F., Fischbeck K. H. Molecular genetics of Charcot-Marie-Tooth disease and related neuropathies. Hum Mol Genet. 1994;3(Spec No):1503–1507. doi: 10.1093/hmg/3.suppl_1.1503. [DOI] [PubMed] [Google Scholar]
  7. Chance P. F., Pleasure D. Charcot-Marie-Tooth syndrome. Arch Neurol. 1993 Nov;50(11):1180–1184. doi: 10.1001/archneur.1993.00540110060006. [DOI] [PubMed] [Google Scholar]
  8. Cheung E., Juliano R. L. CHO cell aggregation induced by fibronectin-coated beads. Differences between wild-type and adhesion-variant cells. Exp Cell Res. 1984 May;152(1):127–133. doi: 10.1016/0014-4827(84)90236-2. [DOI] [PubMed] [Google Scholar]
  9. D'Urso D., Brophy P. J., Staugaitis S. M., Gillespie C. S., Frey A. B., Stempak J. G., Colman D. R. Protein zero of peripheral nerve myelin: biosynthesis, membrane insertion, and evidence for homotypic interaction. Neuron. 1990 Mar;4(3):449–460. doi: 10.1016/0896-6273(90)90057-m. [DOI] [PubMed] [Google Scholar]
  10. Ding Y., Brunden K. R. The cytoplasmic domain of myelin glycoprotein P0 interacts with negatively charged phospholipid bilayers. J Biol Chem. 1994 Apr 8;269(14):10764–10770. [PubMed] [Google Scholar]
  11. Doherty P., Fruns M., Seaton P., Dickson G., Barton C. H., Sears T. A., Walsh F. S. A threshold effect of the major isoforms of NCAM on neurite outgrowth. Nature. 1990 Feb 1;343(6257):464–466. doi: 10.1038/343464a0. [DOI] [PubMed] [Google Scholar]
  12. Domnina L. V., Rovensky J. A., Vasiliev J. M., Gelfand I. M. Effect of microtubule-destroying drugs on the spreading and shape of cultured epithelial cells. J Cell Sci. 1985 Mar;74:267–282. doi: 10.1242/jcs.74.1.267. [DOI] [PubMed] [Google Scholar]
  13. Doyle J. P., Stempak J. G., Cowin P., Colman D. R., D'Urso D. Protein zero, a nervous system adhesion molecule, triggers epithelial reversion in host carcinoma cells. J Cell Biol. 1995 Oct;131(2):465–482. doi: 10.1083/jcb.131.2.465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Everly J. L., Brady R. O., Quarles R. H. Evidence that the major protein in rat sciatic nerve myelin is a glycoprotein. J Neurochem. 1973 Aug;21(2):329–334. doi: 10.1111/j.1471-4159.1973.tb04253.x. [DOI] [PubMed] [Google Scholar]
  15. Filbin M. T., Tennekoon G. I. High level of expression of the myelin protein P0 in Chinese hamster ovary cells. J Neurochem. 1990 Aug;55(2):500–505. doi: 10.1111/j.1471-4159.1990.tb04163.x. [DOI] [PubMed] [Google Scholar]
  16. Filbin M. T., Tennekoon G. I. Homophilic adhesion of the myelin P0 protein requires glycosylation of both molecules in the homophilic pair. J Cell Biol. 1993 Jul;122(2):451–459. doi: 10.1083/jcb.122.2.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Filbin M. T., Tennekoon G. I. Myelin P0-protein, more than just a structural protein? Bioessays. 1992 Aug;14(8):541–547. doi: 10.1002/bies.950140808. [DOI] [PubMed] [Google Scholar]
  18. Filbin M. T., Tennekoon G. I. The role of complex carbohydrates in adhesion of the myelin protein, P0. Neuron. 1991 Nov;7(5):845–855. doi: 10.1016/0896-6273(91)90286-9. [DOI] [PubMed] [Google Scholar]
  19. Filbin M. T., Walsh F. S., Trapp B. D., Pizzey J. A., Tennekoon G. I. Role of myelin P0 protein as a homophilic adhesion molecule. Nature. 1990 Apr 26;344(6269):871–872. doi: 10.1038/344871a0. [DOI] [PubMed] [Google Scholar]
  20. Frost E., Williams J. Mapping temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescue. Virology. 1978 Nov;91(1):39–50. doi: 10.1016/0042-6822(78)90353-7. [DOI] [PubMed] [Google Scholar]
  21. Fujimori T., Takeichi M. Disruption of epithelial cell-cell adhesion by exogenous expression of a mutated nonfunctional N-cadherin. Mol Biol Cell. 1993 Jan;4(1):37–47. doi: 10.1091/mbc.4.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Funayama N., Fagotto F., McCrea P., Gumbiner B. M. Embryonic axis induction by the armadillo repeat domain of beta-catenin: evidence for intracellular signaling. J Cell Biol. 1995 Mar;128(5):959–968. doi: 10.1083/jcb.128.5.959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Giese K. P., Martini R., Lemke G., Soriano P., Schachner M. Mouse P0 gene disruption leads to hypomyelination, abnormal expression of recognition molecules, and degeneration of myelin and axons. Cell. 1992 Nov 13;71(4):565–576. doi: 10.1016/0092-8674(92)90591-y. [DOI] [PubMed] [Google Scholar]
  24. Gilbert T., Le Bivic A., Quaroni A., Rodriguez-Boulan E. Microtubular organization and its involvement in the biogenetic pathways of plasma membrane proteins in Caco-2 intestinal epithelial cells. J Cell Biol. 1991 Apr;113(2):275–288. doi: 10.1083/jcb.113.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  26. Gumbiner B. M. Proteins associated with the cytoplasmic surface of adhesion molecules. Neuron. 1993 Oct;11(4):551–564. doi: 10.1016/0896-6273(93)90068-3. [DOI] [PubMed] [Google Scholar]
  27. Hayasaka K., Himoro M., Sato W., Takada G., Uyemura K., Shimizu N., Bird T. D., Conneally P. M., Chance P. F. Charcot-Marie-Tooth neuropathy type 1B is associated with mutations of the myelin P0 gene. Nat Genet. 1993 Sep;5(1):31–34. doi: 10.1038/ng0993-31. [DOI] [PubMed] [Google Scholar]
  28. Heasman J., Crawford A., Goldstone K., Garner-Hamrick P., Gumbiner B., McCrea P., Kintner C., Noro C. Y., Wylie C. Overexpression of cadherins and underexpression of beta-catenin inhibit dorsal mesoderm induction in early Xenopus embryos. Cell. 1994 Dec 2;79(5):791–803. doi: 10.1016/0092-8674(94)90069-8. [DOI] [PubMed] [Google Scholar]
  29. Hirano S., Kimoto N., Shimoyama Y., Hirohashi S., Takeichi M. Identification of a neural alpha-catenin as a key regulator of cadherin function and multicellular organization. Cell. 1992 Jul 24;70(2):293–301. doi: 10.1016/0092-8674(92)90103-j. [DOI] [PubMed] [Google Scholar]
  30. Huang M. M., Lipfert L., Cunningham M., Brugge J. S., Ginsberg M. H., Shattil S. J. Adhesive ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK. J Cell Biol. 1993 Jul;122(2):473–483. doi: 10.1083/jcb.122.2.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hugon J. S., Bennett G., Pothier P., Ngoma Z. Loss of microtubules and alteration of glycoprotein migration in organ cultures of mouse intestine exposed to nocodazole or colchicine. Cell Tissue Res. 1987 Jun;248(3):653–662. doi: 10.1007/BF00216496. [DOI] [PubMed] [Google Scholar]
  32. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
  33. Kinch M. S., Strominger J. L., Doyle C. Cell adhesion mediated by CD4 and MHC class II proteins requires active cellular processes. J Immunol. 1993 Nov 1;151(9):4552–4561. [PubMed] [Google Scholar]
  34. Kintner C. Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain. Cell. 1992 Apr 17;69(2):225–236. doi: 10.1016/0092-8674(92)90404-z. [DOI] [PubMed] [Google Scholar]
  35. Kitamura K., Suzuki M., Uyemura K. Purification and partial characterization of two glycoproteins in bovine peripheral nerve myelin membrane. Biochim Biophys Acta. 1976 Dec 14;455(3):806–816. doi: 10.1016/0005-2736(76)90050-x. [DOI] [PubMed] [Google Scholar]
  36. Kulkens T., Bolhuis P. A., Wolterman R. A., Kemp S., te Nijenhuis S., Valentijn L. J., Hensels G. W., Jennekens F. G., de Visser M., Hoogendijk J. E. Deletion of the serine 34 codon from the major peripheral myelin protein P0 gene in Charcot-Marie-Tooth disease type 1B. Nat Genet. 1993 Sep;5(1):35–39. doi: 10.1038/ng0993-35. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Lee S. J., Nathans D. Proliferin secreted by cultured cells binds to mannose 6-phosphate receptors. J Biol Chem. 1988 Mar 5;263(7):3521–3527. [PubMed] [Google Scholar]
  39. Lemke G., Axel R. Isolation and sequence of a cDNA encoding the major structural protein of peripheral myelin. Cell. 1985 Mar;40(3):501–508. doi: 10.1016/0092-8674(85)90198-9. [DOI] [PubMed] [Google Scholar]
  40. Lemke G., Lamar E., Patterson J. Isolation and analysis of the gene encoding peripheral myelin protein zero. Neuron. 1988 Mar;1(1):73–83. doi: 10.1016/0896-6273(88)90211-5. [DOI] [PubMed] [Google Scholar]
  41. Levine E., Lee C. H., Kintner C., Gumbiner B. M. Selective disruption of E-cadherin function in early Xenopus embryos by a dominant negative mutant. Development. 1994 Apr;120(4):901–909. doi: 10.1242/dev.120.4.901. [DOI] [PubMed] [Google Scholar]
  42. Lukashev M. E., Sheppard D., Pytela R. Disruption of integrin function and induction of tyrosine phosphorylation by the autonomously expressed beta 1 integrin cytoplasmic domain. J Biol Chem. 1994 Jul 15;269(28):18311–18314. [PubMed] [Google Scholar]
  43. Martini R., Zielasek J., Toyka K. V., Giese K. P., Schachner M. Protein zero (P0)-deficient mice show myelin degeneration in peripheral nerves characteristic of inherited human neuropathies. Nat Genet. 1995 Nov;11(3):281–286. doi: 10.1038/ng1195-281. [DOI] [PubMed] [Google Scholar]
  44. Murray B. A., Jensen J. J. Evidence for heterophilic adhesion of embryonic retinal cells and neuroblastoma cells to substratum-adsorbed NCAM. J Cell Biol. 1992 Jun;117(6):1311–1320. doi: 10.1083/jcb.117.6.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Nagafuchi A., Takeichi M. Cell binding function of E-cadherin is regulated by the cytoplasmic domain. EMBO J. 1988 Dec 1;7(12):3679–3684. doi: 10.1002/j.1460-2075.1988.tb03249.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Nagafuchi A., Takeichi M., Tsukita S. The 102 kd cadherin-associated protein: similarity to vinculin and posttranscriptional regulation of expression. Cell. 1991 May 31;65(5):849–857. doi: 10.1016/0092-8674(91)90392-c. [DOI] [PubMed] [Google Scholar]
  47. Patel P. I., Lupski J. R. Charcot-Marie-Tooth disease: a new paradigm for the mechanism of inherited disease. Trends Genet. 1994 Apr;10(4):128–133. doi: 10.1016/0168-9525(94)90214-3. [DOI] [PubMed] [Google Scholar]
  48. Pavalko F. M., Otey C. A. Role of adhesion molecule cytoplasmic domains in mediating interactions with the cytoskeleton. Proc Soc Exp Biol Med. 1994 Apr;205(4):282–293. doi: 10.3181/00379727-205-43709. [DOI] [PubMed] [Google Scholar]
  49. Sakamoto Y., Kitamura K., Yoshimura K., Nishijima T., Uyemura K. Complete amino acid sequence of PO protein in bovine peripheral nerve myelin. J Biol Chem. 1987 Mar 25;262(9):4208–4214. [PubMed] [Google Scholar]
  50. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schneider-Schaulies J., von Brunn A., Schachner M. Recombinant peripheral myelin protein P0 confers both adhesion and neurite outgrowth-promoting properties. J Neurosci Res. 1990 Nov;27(3):286–297. doi: 10.1002/jnr.490270307. [DOI] [PubMed] [Google Scholar]
  52. Smilenov L., Briesewitz R., Marcantonio E. E. Integrin beta 1 cytoplasmic domain dominant negative effects revealed by lysophosphatidic acid treatment. Mol Biol Cell. 1994 Nov;5(11):1215–1223. doi: 10.1091/mbc.5.11.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Trapp B. D., Kidd G. J., Hauer P., Mulrenin E., Haney C. A., Andrews S. B. Polarization of myelinating Schwann cell surface membranes: role of microtubules and the trans-Golgi network. J Neurosci. 1995 Mar;15(3 Pt 1):1797–1807. doi: 10.1523/JNEUROSCI.15-03-01797.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Tímár J., Bazaz R., Kimler V., Haddad M., Tang D. G., Robertson D., Tovari J., Taylor J. D., Honn K. V. Immunomorphological characterization and effects of 12-(S)-HETE on a dynamic intracellular pool of the alpha IIb beta 3-integrin in melanoma cells. J Cell Sci. 1995 Jun;108(Pt 6):2175–2186. doi: 10.1242/jcs.108.6.2175. [DOI] [PubMed] [Google Scholar]
  55. Urlaub G., Chasin L. A. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4216–4220. doi: 10.1073/pnas.77.7.4216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wong M. H., Filbin M. T. The cytoplasmic domain of the myelin P0 protein influences the adhesive interactions of its extracellular domain. J Cell Biol. 1994 Aug;126(4):1089–1097. doi: 10.1083/jcb.126.4.1089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zhang K., Filbin M. T. Formation of a disulfide bond in the immunoglobulin domain of the myelin P0 protein is essential for its adhesion. J Neurochem. 1994 Jul;63(1):367–370. doi: 10.1046/j.1471-4159.1994.63010367.x. [DOI] [PubMed] [Google Scholar]

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