The Journal of Membrane Biology

, Volume 194, Issue 1, pp 59–76 | Cite as

Sequence and Phylogenetic Analyses of 4 TMS Junctional Proteins of Animals: Connexins, Innexins, Claudins and Occludins

  • V. B. Hua
  • A. B. Chang
  • J. H. Tchieu
  • N. M. Kumar
  • P. A. Nielsen
  • M. H. SaierJr
Article

Abstract

Connexins and probably innexins are the principal constituents of gap junctions, while claudins and occludins are principal tight junctional constituents. All have similar topologies with four α-helical transmembrane segments (TMSs), and all exhibit well-conserved extracytoplasmic cysteines that either are known to or potentially can form disulfide bridges. We have conducted sequence, topological and phylogenetic analyses of the proteins that comprise the connexin, innexin, claudin and occludin families. A multiple alignment of the sequences of each family was used to derive average hydropathy and similarity plots as well as phylogenetic trees. Analyses of the data generated led to the following evolutionary and functional suggestions: (1) In all four families, the most conserved regions of the proteins from each family are the four TMSs although the extracytoplasmic loops between TMSs 1 and 2, and TMSs 3 and 4 are usually well conserved. (2) The phylogenetic trees revealed sets of orthologues except for the innexins where phylogeny primarily reflects organismal source, probably due to a lack of relevant organismal sequence data. (3) The two halves of the connexins exhibit similarities suggesting that they were derived from a common origin by an internal gene duplication event. (4) Conserved cysteyl residues in the connexins and innexins may point to a similar extracellular structure involved in the docking of hemichannels to create intercellular communication channels. (5) We suggest a similar role in homomeric interactions for conserved extracellular residues in the claudins and occludins. The lack of sequence or motif similarity between the four different families indicates that, if they did evolve from a common ancestral gene, they have diverged considerably to fulfill separate, novel functions. We suggest that internal duplication was a general evolutionary strategy used to generate new families of channels and junctions with unique functions. These findings and suggestions should serve as guides for future studies concerning the structures, functions and evolutionary origins of junctional proteins.

Keywords

Intercellular communication Gap junctions Tight junctions Connexins Innexins Claudins Occludins Evolution 

Notes

Acknowledgements

We thank Mary Beth Miller for assistance in the preparation of this manuscript. This work was supported by NIH grants GM55434 and GM64368 from the National Institute of General Medical Sciences (to MHS), an NEI grant EY13605 (to NMK), an RPB grant of unrestricted funds from Research to Prevent Blindness (to the UIC), and a grant from the Danish Research Council (to PAN).

References

  1. 1.
    Ando-Akatsuka, Y., Saitou, M., Hirase, T., Kishi, M., Sakakibara, A., Itoh, M., Yonemura, S., Furuse, M., Tsukita, S. 1996Interspecies diversity of the occludin sequence: cDNA cloning of human, mouse, dog, and rat-kangaroo homologues.J. Cell Biol.1334347PubMedGoogle Scholar
  2. 2.
    Balda, M.S., Flores-Maldonado, C., Cereijido, M., Matter, K. 2000Multiple domains of occludin are involved in the regulation of paracellular permeability.J. Cell. Biochem.788596CrossRefPubMedGoogle Scholar
  3. 3.
    Bevans, C.G., Kordel, M., Rhee, S.K., Harris, A.L. 1998Gating connexin 43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation.J. Biol. Chem.27455815587Google Scholar
  4. 4.
    Beyer, E.G., Paul, D.L., Goodenough, D.A. 1987Connexin 43: A protein from rat heart homologous to a gap junction protein from liver.J. Cell Biol.10526212629PubMedGoogle Scholar
  5. 5.
    Blaschuk, O.W., Oshima, T., Gour, B.J., Symonds, J.M., Park, J.H., Kevil, C.G., Trocha, S.D., Michaud, S., Okayama, N., Elrod, J.W., Alexander, J.S. 2002Identification of an occludin cell adhesion recognition sequence.Inflammation26193198CrossRefPubMedGoogle Scholar
  6. 6.
    Colegio, O.R., Van Itallie, C.M., McCrea, H.J., Rahner, C., Anderson, J.M. 2002Claudins create charge-selective channels in the paracellular pathway between epithelial cells.Am. J. Physiol.283C142C147Google Scholar
  7. 7.
    Cordenonsi, M., Turco, F., D'atri, F., Hammar, E., Martinucci, G., Meggio, F., Citi, S. 1999 Xenopus laevis occludin. Identification of in vitro phosphorylation sites by protein kinase CK2 and association with cingulin.Eur. J. Biochem.264374384CrossRefPubMedGoogle Scholar
  8. 8.
    Curtin, K.D., Zhang, Z., Wyman, R.J. 1999 Drosophila has several genes for gap junction proteins.Gene232191201PubMedGoogle Scholar
  9. 9.
    D'Andrea, P., Veronesi, V., Bicego, M., Melchionda, S., Zelante, L., Di Iorio, E., Bruzzone, R., Gasparini, P. 2002Hearing loss: frequency and functional studies of the most common connexin26 alleles.Biochem. Biophys. Res. Commun.296685691CrossRefPubMedGoogle Scholar
  10. 10.
    D'Atri, P., Citi, S. 2002Molecular complexity of vertebrate tight junctions.Mol. Membrane Biol.19103112CrossRefGoogle Scholar
  11. 11.
    Delmar, M. 2002Connexin diversity: discriminating the message.Circ. Res.918586CrossRefPubMedGoogle Scholar
  12. 12.
    Eiberger, J., Degen, J., Romualdi, A., Deutsch, U., Willecke, K., Sohl, G. 2001Connexin genes in the mouse and human genome.Cell Adhes. Commun.8163165Google Scholar
  13. 13.
    Epstein, M.L., Gilula, N.B. 1977A study of communication specificity between cells in culture.J. Cell Biol.75769787PubMedGoogle Scholar
  14. 14.
    Evans, W.H., Martin, P.E.M. 2002aGap junctions: structure and function.Mol. Membrane Biol.19121136Google Scholar
  15. 15.
    Evans, W.H., Martin, P.E. 2002bLighting up gap junction channels in a flash.Bioessays24876880Google Scholar
  16. 16.
    Feng, D.-F., Doolittle, R.F. 1990Progressive alignment and phylogenetic tree construction of protein sequences.Methods Enzymol.183375387PubMedGoogle Scholar
  17. 17.
    Ganfornina, M.D., Sanchez, D., Herrera, M., Bastiani, M.J. 1999Developmental expression and molecular characterization of two gap junction channel proteins during embryogenesis in the grasshopper Schistocerca americana. Dev. Genet.24137150CrossRefPubMedGoogle Scholar
  18. 18.
    Ghassemifar, M.R., Sheth, B., Papenbrock, T., Leese, H.J., Houghton, F.D., Fleming, T.P. 2002Occludin TM4: an isoform of the tight junction protein present in primates lacking the fourth transmembrane domain.J. Cell Sci.11531713180PubMedGoogle Scholar
  19. 19.
    Ghosh, P., Ghosh, S., Das, S. 2002Self-regulation of rat liver GAP junction by phosphorylation.Biochim. Biophys. Acta1564500504CrossRefPubMedGoogle Scholar
  20. 20.
    Hand, G.M., Muller, D.J., Nicholson, B.J., Engel, A., Sosinsky, G.E. 2002Isolation and characterization of gap junctions from tissue culture cells.J. Mol. Biol.315587600CrossRefPubMedGoogle Scholar
  21. 21.
    Harris, A.L. 2001Emerging issues of connexin channels: biophysics fills the gap.Q. Rev. Biophys.34325472PubMedGoogle Scholar
  22. 22.
    Heiskala, M., Peterson, P.A., Yang, Y. 2001The roles of claudin superfamily proteins in paracellular transport.Traffic29398CrossRefPubMedGoogle Scholar
  23. 23.
    Jahromi, S.S., Wentlandt, K., Piran, S., Carlen, P.L. 2002Anticonvulsant actions of gap junctional blockers in an in vitro seizure model.J. Neurophysiol.8818931902PubMedGoogle Scholar
  24. 24.
    Kim, D.Y., Kam, Y., Koo, S.K., Joe, C.O. 1999Gating connexin 43 channels reconstituted in lipid vesicles by mitogen activated protein kinase phosphorylation.J. Biol. Chem.2745581CrossRefPubMedGoogle Scholar
  25. 25.
    Kitamura, K., Takahashi, K., Tamagawa, Y., Noguchi, Y., Kuroishikawa, Y., Ishikawa, K., Hagiwara, H. 2000Deafness genes.J. Med. Dent. Sci.47111PubMedGoogle Scholar
  26. 26.
    Kiuchi-Saishin, Y., Gotoh, S., Furuse, M., Takasuga, A., Tano, Y., Tsukita, S. 2002Differential expression patterns of claudins, tight junction membrane proteins, in mouse nephron segments.J. Am. Soc. Nephrol.13875886PubMedGoogle Scholar
  27. 27.
    Kollmar, R., Nakamura, S.K., Kappler, J.A., Hudspeth, A.J. 2001Expression and phylogeny of claudins in vertebrate primordia.Proc. Natl. Acad. Sci. USA981019610201CrossRefPubMedGoogle Scholar
  28. 28.
    Kumar, N.M., Gilula, N.B. 1996The gap junction communication channel.Cell84381388PubMedGoogle Scholar
  29. 29.
    Kyte, J., Doolittle, R.F. 1982A simple method for displaying the hydropathic character of a protein.J. Mol. Biol.157105132PubMedGoogle Scholar
  30. 30.
    Landesman, Y., White, T.W., Starich, T.A., Shaw, I.E., Goodenough, D.A., Paul, D.L. 1999Innexin-3 forms connexin-like intercellular channels.J. Cell Sci.11223912396PubMedGoogle Scholar
  31. 31.
    Langbein, L., Grund, C., Kuhn, C., Praetzel, S., Kartenbeck, J., Brandner, J.M., Moll, I., Franke, W.W. 2002Tight junctions and compositionally related junctional structures in mammalian stratified epithelia and cell cultures derived therefrom.Eur. J. Cell Biol.81419435PubMedGoogle Scholar
  32. 32.
    Le, T., Tseng, T.T., Saier Jr., M.H. 1999Flexible programs for the prediction of average amphipathicity of multiply aligned homologous proteins: Application to integral membrane transport proteins.Mol. Membr. Biol.16173179CrossRefPubMedGoogle Scholar
  33. 33.
    Loewenstein, W.R. 1987The cell-to-cell channel of gap junctions.Cell48725726PubMedGoogle Scholar
  34. 34.
    Long, H., Crean, C.D., Lee, W.H., Cummings, O.W., Gabig, T.G. 2001Expression of Clostridium perfringens enterotoxin receptors claudin-3 and claudin-4 in prostate cancer epithelium.Cancer Res.6178787881PubMedGoogle Scholar
  35. 35.
    Lopez, P., Balicki, D., Buehler, L.K., Falk, M.M., Chen, S.C. 2001Distribution and dynamics of gap junction channels revealed in living cells.Cell Adhes. Commun.8237242Google Scholar
  36. 36.
    Mackay, D., Ionides, A., Kibar, Z., Rouleau, G., Berry, V., Moore, A., Shiels, A., Bhattacharya, S. 1999Connexin46 mutations in autosomal dominant congenital cataract.Am. J. Hum. Genet.6413571364CrossRefPubMedGoogle Scholar
  37. 37.
    McClane, B.A. 2000 Clostridium perfringens enterotoxin and intestinal tight junctions.Trends Microbiol.8145146CrossRefPubMedGoogle Scholar
  38. 38.
    Milks, L.C., Kumar, N.M., Houghten, R., Unwin, N., Gilula, N.B. 1988Topology of the 32-kd liver gap junction protein determined by site-directed antibody localizations.EMBO J.729672975PubMedGoogle Scholar
  39. 39.
    Morcos, Y., Hosie, M.J., Bauer, H.C., Chan-Ling, T. 2001Immunolocalization of occludin and claudin-1 to tight junctions in intact CNS vessels of mammalian retina.J. Neurocytol.30107123CrossRefPubMedGoogle Scholar
  40. 40.
    Nies, D.H., Koch, S., Wachi, S., Peitzsch, N., Saier Jr., M.H. 1998CHR, a novel family of prokaryotic proton motive force-driven transporters probably containing chromate/sulfate antiporters.J. Bacteriol.18057995802PubMedGoogle Scholar
  41. 41.
    Omori, Y., Mesnil, M., Yamasaki, H. 1996Connexin 32 mutations from X-linked Charcot-Marie tooth disease patients: functional defects and dominant negative effects.Mol. Biol. Cell7907916PubMedGoogle Scholar
  42. 42.
    Panchin, Y., Kelmanson, I., Matz, M., Lukyanov, K., Usman, N., Lukyanov, S. 2000A ubiquitous family of putative gap junction molecules.Curr. Biol.10R473R474CrossRefPubMedGoogle Scholar
  43. 43.
    Pao, S.S., Paulsen, I.T., Saier Jr., M.H. 1998The major facilitator superfamily.Microbiol. Mol. Biol. Rev.62132PubMedGoogle Scholar
  44. 44.
    Phelan, P., Stanch, T.A. 2001Innexins get into the gap.Bioessays23388396PubMedGoogle Scholar
  45. 45.
    Potenza, N., del Gaudio, R., Rivieccio, L., Russo, G.M., Geraci, G. 2002Cloning and molecular characterization of the first innexin of the phylum annelida—expression of the gene during development.J. Mol. Evol.54312321PubMedGoogle Scholar
  46. 46.
    Richard, G., Smith, L.E., Bailey, R.A., Itin, P., Hohl, D., Epstein Jr., E.H., DiGiovanna, J.J., Compton, J.G., Bale, S.J. 1998Mutations in the human connexin gene GJB3 cause erythrokeratodermia variabilis.Nature Genet.20366369CrossRefPubMedGoogle Scholar
  47. 47.
    Saier Jr., M.H. 2000Vectorial metabolism and the evolution of transport systems.J. Bacteriol.18250295035CrossRefPubMedGoogle Scholar
  48. 48.
    Saier Jr., M.H. 2001

    Evolution of transport proteins.

    Setlow, J.K. eds. Genetic Engineering. Principles and Methods, Vol. 23.Kluwer Academic/Plenum PublishersNew York19
    Google Scholar
  49. 49.
    Sakaguchi, T., Kohler, H., Gu, X., McCormick, B.A., Reinecker, H.C. 2002 Shigella flexneri regulates tight junction-associated proteins in human intestinal epithelial cells.Cell Microbiol.4367381CrossRefPubMedGoogle Scholar
  50. 50.
    Shibata, Y., Kumai, M., Nishii, K., Nakamura, K. 2001Diversity and molecular anatomy of gap junctions.Med. Electron Microsc.34153159CrossRefPubMedGoogle Scholar
  51. 51.
    Sotkis, A., Wang, X.G., Yasumura, T., Peracchia, L.L., Persechini, A., Rash, J.E., Peracchia, C. 2001Calmodulin colocalizes with connexins and plays a direct role in gap junction channel gating.Cell Adhes. Commun.8277281Google Scholar
  52. 52.
    Starich, T., Sheehan, M., Jadrich, J., Shaw, J. 2001Innexins in C. elegans. Cell Adhes. Commun.8311314Google Scholar
  53. 53.
    Stebbings, L.A., Todman, M.G., Phelan, P., Bacon, J.P., Davies, J.A. 2000Two Drosophila innexins are expressed in overlapping domains and cooperate to form gap-junction channels.Mol. Biol. Cell1124592470PubMedGoogle Scholar
  54. 54.
    Stebbings, L.A., Todman, M.G., Phillips, R., Greer, C.E., Tam, J., Phelan, P., Jacobs, K., Bacon, J.P., Davies, J.A. 2002Gap junctions in Drosophila: developmental expression of the entire innexin gene family.Mech. Dev.113197205CrossRefPubMedGoogle Scholar
  55. 55.
    Tepass, U., Tanentzapf, G., Ward, R., Fehon, R. 2001Epithelial cell polarity and cell junctions in Drosophila. Annu. Rev. Genet.35747784CrossRefPubMedGoogle Scholar
  56. 56.
    Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G. 1997The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.Nucleic Acids Res.2548764882CrossRefPubMedGoogle Scholar
  57. 57.
    Tseng, T.-T., Gratwick, K.S., Kollman, J., Park, D., Nies, D.H., Goffeau, A., Saier Jr., M.H. 1999The RND permease superfamily: An ancient, ubiquitous and diverse family that includes human disease and development proteins.J. Mol. Microbiol. Biotechnol.1107125PubMedGoogle Scholar
  58. 58.
    Tsukita, S., Furuse, M. 2000The structure and function of claudins, cell adhesion molecules at tight junctions.Ann. N.Y. Acad. Sci.915129135PubMedGoogle Scholar
  59. 59.
    Tsukita, S., Furuse, M. 2002Claudin-based barrier in simple and stratified cellular sheets.Curr. Opin. Cell. Biol.14531CrossRefPubMedGoogle Scholar
  60. 60.
    Unger, V.M., Kumar, N.M., Gilula, N.B., Yeager, M. 1999Three-dimensional structure of a recombinant gap junction membrane channel.Sci. Mag.28311761180CrossRefGoogle Scholar
  61. 61.
    White, T.W., Paul, D.L. 1999Genetic diseases and gene knockouts reveal diverse connexin functions.Annu. Rev. Physiol.61283310CrossRefPubMedGoogle Scholar
  62. 62.
    Wiliecke, K., Eiberger, J., Degen, J., Eckardt, D., Romualdi, A., Guldenagel, M., Deutsch, U., Sohl, G. 2002Structural and functional diversity of connexin genes in the mouse and human genome.Biol. Chem.383725737PubMedGoogle Scholar
  63. 63.
    Yeager, M., Unger, V.M., Falk, M.M. 1998Synthesis, assembly and structure of gap junction intercellular channels.Curr. Opin. Struct. Biol.8517524CrossRefPubMedGoogle Scholar
  64. 64.
    Zhai, Y., Saier Jr., M.H. 2001The AveHAS program for the determination of average hydrophobicity, amphipathicity, and similarity.J. Mol. Microbiol. Biotechnol.3285286PubMedGoogle Scholar

Copyright information

© Springer-Verlag New York Inc. 2003

Authors and Affiliations

  • V. B. Hua
    • 1
  • A. B. Chang
    • 1
  • J. H. Tchieu
    • 1
  • N. M. Kumar
    • 2
  • P. A. Nielsen
    • 2
  • M. H. SaierJr
    • 1
  1. 1.Division of BiologyUniversity of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0116USA
  2. 2.Department of Cell BiologyThe Scripps Research Institute, La Jolla, CA 92037USA

Personalised recommendations