Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research
  • Published:

Expression of the Human Muscarinic Receptor Gene m2 in Dictyostelium discoideum

Bio/Technology volume 13, pages 1225–1229 (1995)Cite this article

Abstract

We have expressed a functional human muscarinic M2 receptor, under the control of the homologous discoidin Iγ promoter, in the cellular slime mold Dictyostelium discoideum. The use of a contact site A leader peptide ensured insertion of the newly synthesized receptor protein into the plasma membrane. Due to the characteristics of the discoidin Iγ promoter, the M2 receptor is expressed during late growth and early development. The heterologously expressed M2 receptors show binding characteristics similar to authentic receptors. Membranes as well as whole cells can be used hi ligand binding assays.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Hodgson, J. 1992. Receptor screening and the search for new Pharmaceuticals. Bio/Technology 10: 973–980.

    CAS  PubMed  Google Scholar 

  2. Grimm, U., Moser, U., Mutschler, E. and Lambrecht, G. 1994. Muscarinic receptors: Focus on presynaptic mechanisms and recently developed novel agonists and antagonists. Pharmazie 49: 711–726.

    CAS  PubMed  Google Scholar 

  3. Hulme, E.C., Birdsall, J.M. and Buckley, N.J. 1990. Muscarinic receptor subtypes. Ann. Rev. in Pharm. and Toxicol. 30: 633–673.

    Article  CAS  Google Scholar 

  4. Gross, J.D. 1994. Developmental decisions in Dictyostelium discoideum. Microbiol. Rev. 58: 330–351.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Takeuchi, I., Tasaka, M., Okamoto, K. and Maeda, Y. 1994. Regulation of cell differentiation and pattern formation in Dictyostelium development. Int J. Dev.Biol. 38: 311–319.

    CAS  PubMed  Google Scholar 

  6. Weeks, G. and Weijer, C.J. 1994. The Dictyostelium cell cycle and its relationship to differentiation. FEMS Microbiol. Lett 124: 123–130.

    Article  CAS  Google Scholar 

  7. Devreotes, P.N. 1994. G Protein-linked signaling pathways control the developmental program of Dictyostelium. Neuron 12: 235–241.

    Article  CAS  Google Scholar 

  8. Hereld, D. and Devreotes, P.N. 1992. The cAMP Receptor Family of Dictyostelium. p. 35–47. In: The cAMP Receptor Family of Dictyostelium. Friedlander, M. and Mueckler, M. (Eds.). International Review of Cytology, Vol 137B. 137, Academic Press Inc. 525 B Street, Suite 1900, San Diego, CA 92101-4495

    Google Scholar 

  9. Klein, P.S., Sun, T.J., Saxe, C.L., Kimmel, A.R., Johnson, R.L. and Devreotes, P.N. 1988. A chemoattractant receptor controls development in Dictyostelium discoideum. Science 241: 1467–1472.

    Article  CAS  Google Scholar 

  10. Vauti, F., Morandini, P., Blusch, J., Sachse, A. and Nellen, W. 1990. Regulation of the Discoidin-I Gamma-Gene in Dictyoslelium-Discoideum-Identification of Individual Promoter Elements Mediating Induction of Transcription and Repression by Cyclic AMP. Mol. Cell Biol. 10: 4080–4088.

    Article  CAS  Google Scholar 

  11. Noegel, A.A., Gerisch, G., Stadler, J. and Westphal, M. 1986. Complete sequence and transcript of a cell adhesion protein from aggregating Dictyostelium cells. EMBO J. 5: 1473–1476.

    Article  CAS  Google Scholar 

  12. Blusch, J., Morandini, P. and Nellen, W. 1992. Transcriptional regulation by folate: inducible gene expression in Dictyostelium transformants during growth and early development. Nucl. Acids Res. 20: 6235–6238.

    Article  CAS  Google Scholar 

  13. Maniak, M. and Nellen, W. 1990. Evidence for a feedback regulated back-up promoter which controls permanent expression of a Dictyostelium gene. Nucl. Acids Res. 18: 5375–5380.

    Article  CAS  Google Scholar 

  14. Warrick, H.M. and Spudich, J.A. 1988. Codon preference in Dictyostelium discoideum. Nucl. Acids Res. 16: 6617–6635.

    Article  CAS  Google Scholar 

  15. Goodloe-Holland, C.M. and Luna, E.J. 1987. Purification and characterization of Dictyostelium discoideum plasma membranes. p. 103–128. In: Purification and characterization of Dictyostelium discoideum plasma membranes. Spudich, J. A. (Ed.). Methods in Cell Biology. 28, Academic Press, Orlando

    Google Scholar 

  16. Spudich, J. 1974. Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. 2. Purification, properties and membrane association of actin from amoebae of Dictyostelium discoideum. J. Biol. Chem. 249: 6013–6020.

    CAS  PubMed  Google Scholar 

  17. Malchow, D. and Gerisch, G. 1974. Short-term binding and hydrolysis of cyclic 3′:5′-adenosine monophosphate by aggregating Dictyostelium cells. Proc. Natl. Acad. Sci. USA 71: 2423–2427.

    Article  CAS  Google Scholar 

  18. Wetterauer, B., Jacobsen, G., Morandini, P. and Macwilliams, H. 1993. Mutants of Dictyostelium discoideum with defects in the regulation of discoidin-I expression. Dev. Biol. 159: 184–195.

    Article  Google Scholar 

  19. Voith, G. and Dingermann, T. 1995. Expression of CSA-hm2 fusion in Dictyostelium discoideum under the control of the Dictyostelium ras promoter reveals functional muscarinic receptors. Pharmazie. 50: 758–762.

    CAS  PubMed  Google Scholar 

  20. Fasel, N., Begdadi-Rais, C., Bernard, M., Bron, G., Corradin, G. and Reymond, C.D. 1992. Dictyostelium discoideum as a expression host for the circumsporozoite protein of Plasmodium falciparum. Gene 111: 157–163.

    Article  CAS  Google Scholar 

  21. Dingermann, T., Troidl, E.M., Bröker, M. and Nerke, K. 1991. Expression of human antithrombin III in the cellular slime mold Dictyostelium discoideum. App. Microbiol. Biotechnol. 35: 496–503.

    Article  CAS  Google Scholar 

  22. Dittrich, W., Williams, K.L. and Slade, M.B. 1994. Production and secretion of recombinant proteins in Dictyostelium discoideum. Bio/Technology 12: 614–618.

    CAS  PubMed  Google Scholar 

  23. Zhouchou, T., Slade, M.B., Williams, K.L. and Gooley, A.A. 1995. Expression, purification and characterisation of secreted recombinant glyco-protein PsA in Dictyostelium discoideum. J. Biotechnol. 38: 137–149.

    Article  CAS  Google Scholar 

  24. Huang, H.J., Liao, C.F., Yang, B.C. and Kuo, T.T. 1992. Functional expression of rat m5 muscarinic acetylcholine receptor in yeast. Biochem. Biophys. Res. Comm. 182: 1180–1186.

    Article  CAS  Google Scholar 

  25. Vasudevan, S., Hulme, E.C., Bach, M., Haase, W., Pavia, J. and Reilander, H. 1995. Characterization of the rat m3 muscarinic acetylcholine receptor produced in insect cells infected with recombinant baculovirus. Eur. J. Biochem. 227: 466–475.

    Article  CAS  Google Scholar 

  26. Vasudevan, S., Reilander, H., Maul, G. and Michel, H. 1991. Expression and cell membrane localization of Rat M3 Muscarinic Acetylcholine Receptor produced in Sf 9 insect cells using the baculovirus system. FEBS Letters 283: 52–56.

    Article  CAS  Google Scholar 

  27. Bonner, T.L., Buckley, N.J., Young, A.C. and Brann, M.R. 1987. Identification of a family of muscarinic acetylcholine receptor genes. Science 237: 527–532.

    Article  CAS  Google Scholar 

  28. Watts, D.J. and Ashworth, J.M. 1970. Growth of myxamoebae of the cellular slime mold Dictyostelium discoideum in axenic culture. Biochem. J. 119: 171–174.

    Article  CAS  Google Scholar 

  29. Early, A. and Williams, J. 1987. Two vectors which facilitate gene manipulation and a simplified transformation procedure for Dictyostelium discoideum. Gene 59: 99–106.

    Article  CAS  Google Scholar 

  30. Nellen, W. and Firtel, R.A. 1985. High copy number transformants and co-transformation in Dictyostelium discoideum. Gene 39: 155–163.

    Article  CAS  Google Scholar 

  31. Nellen, W., Silan, C. and Firtel, R.A. 1984. DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion. Mol. Cell Biol. 4: 2890.

    Article  CAS  Google Scholar 

  32. Stratford, C.A. and Brown, S.S. 1985. Isolation of an Actin-binding Protein from Membranes of Dictyostelium discoideum. J. of Cell Biol. 100: 727–735.

    Article  CAS  Google Scholar 

  33. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.

    Article  CAS  Google Scholar 

  34. Buckley, N.J., Bonner, T.I., Buckley, C.M. and Brann, M.R. 1989. Antagonist binding properties of five cloned muscarinic receptors expressed in CHO-K1 cells. Mol. Pharmacol. 35: 469–476.

    CAS  PubMed  Google Scholar 

  35. Dingermann, T., Reindl, N., Werner, H. and Welker, D.L. 1989. Optimization and in situ detection of Escherichia coli β-galactosidase gene expression in Dictyostelium discoideum. Gene 85: 353–362.

    Article  CAS  Google Scholar 

  36. Done, R., Wess, J., Lambrecht, G., Tacke, R., Mitschler, E. and Brann, M.R. 1991. Antagonist binding of five cloned human muscarinic receptor subtypes. J. Pharm. Exp. Therap. 256: 727–733.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institut für Pharmazeutische Biologie, Johann Wolfgang Goethe Universität, Biozentrum, Marie-Curie-Str. 9, D-60439, Frankfurt, Germany

    Guido Voith & Theodor Dingermann

Authors
  1. Guido Voith
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Theodor Dingermann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Theodor Dingermann.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Voith, G., Dingermann, T. Expression of the Human Muscarinic Receptor Gene m2 in Dictyostelium discoideum. Nat Biotechnol 13, 1225–1229 (1995). https://doi.org/10.1038/nbt1195-1225

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1195-1225

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing