Calcium Signaling Protocols pp 183-193

Part of the Methods in Molecular Biology™ book series (MIMB, volume 312)

Measurement of Phospholipase C by Monitoring Inositol Phosphates Using [3H]Inositol-Labeling Protocols in Permeabilized Cells

  • Alison Skippen
  • Philip Swigart
  • Shamshad Cockcroft

Abstract

Hormones, neurotransmitters, chemoattractants, and growth factors all elicit intracellular responses on binding to cell surface receptors by activating inositol phospholipid-specific phospholipase C (PLC). Activated PLC catalyzes the hydrolysis of phosphatidylinositol bisphosphate (PIP2), a minor membrane phospholipid, to form two second messengers, diacylglycerol (DAG) and inositol (1,4,5)trisphosphate [Ins(1,4,5,)P3]. DAG is a direct activator of protein kinase C isozymes, and Ins(1,4,5)P3 mobilizes intracellular Ca2+. G protein-coupled receptors couple to the PLC-β family via G proteins, and tyrosine kinase receptors activate PLC-γ isozymes (1,2). Regardless of the PLC isozyme activated, the product is invariantly Ins(1,4,5)P3.

References

  1. 1.
    Rhee, S. G. (2001) Regulation of phosphoinositide-specific phospholipase C. Annu. Rev. Biochem. 70, 281–312.CrossRefPubMedGoogle Scholar
  2. 2.
    Rebecchi, M. J. and Pentyala, S. N., (2000) Structure, function, and control of phosphoinositide-specific phospholipase C. Physiological Revs. 80, 1291–1335.Google Scholar
  3. 3.
    Thomas, G. M. H., Cunningham, E., Fensome, A., et al. (1993) An essential role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signalling. Cell 74, 919–928.CrossRefPubMedGoogle Scholar
  4. 4.
    Kauffmann-Zeh, A., Thomas, G. M. H., Ball, A., et al. (1995) Requirement for phosphatidylinositol transfer protein in epidermal growth factor signalling. Science 268, 1188–1190.CrossRefPubMedGoogle Scholar
  5. 5.
    Allen, V., Swigart, P., Cheung, R., Cockcroft, S., and Katan, M. (1997) Regulation of inositol lipid-specific phospholipase Cδ by changes in Ca2+ ion concentrations. Biochem. J. 327, 545–552.PubMedGoogle Scholar
  6. 6.
    Cunningham, E., Tan, S. W., Swigart, P., Hsuan, J., Bankaitis, V., and Cockcroft, S. (1996) The yeast and mammalian isoforms of phosphatidylinositol transfer protein can all restore phospholipase C-mediated inositol lipid signalling in cytosol-depleted RBL-2H3 and HL60 cells. Proc. Natl. Acad. Sci. USA 93, 6589–6593.CrossRefPubMedGoogle Scholar
  7. 7.
    Skippen. A., Jones. D. H., Morgan, C. P., Li, M., and Cockcroft, S. Mechanism of ADP Ribosylation-Factor-stimulated phosphatidylinositol 4,5-bisphosphate synthesis in HL60 cells. J. Biol. Chem. 277, 5823–5831.Google Scholar
  8. 8.
    Tatham, P. E. R. and Gomperts, B. D. (1990) Cell permeabilisation, in Peptide Hormones—A Practical Approach (Siddle, K. and Hutton, J. C., eds.), IRL, Oxford, England, pp. 257–269.Google Scholar
  9. 9.
    Bird, I. M. (1998) Phosphoinositidase C activation assay III, in Methods in Molecular Biology, vol. 105: Phospholipid Signaling Protocols, (Bird, I. M., ed.) Humana Press, Totowa, NJ, pp. 25–45.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Alison Skippen
    • 1
  • Philip Swigart
    • 1
  • Shamshad Cockcroft
    • 1
  1. 1.Department of PhysiologyUniversity College LondonLondon

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