Advertisement

High-Throughput Assays to Measure Intracellular Ca2+ Mobilization in Cells that Express Recombinant S1P Receptor Subtypes

  • William J. ValentineEmail author
  • Gabor Tigyi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 874)

Abstract

Intracellular Ca2+ mobilization is a useful readout to screen for agonists or antagonists of G-protein ­coupled receptors (GPCRs). Here, we describe methods to conduct high-throughput screening of stably or transiently transfected HTC4 cells expressing the individual S1P1–5 receptor subtypes. The cells are grown in 96-well plates and loaded with the cell permeable fluorescent Ca2+ indicator dye Fura-2-AM. Changes in intracellular Ca2+ levels in response to S1P or test compounds are detected using a FlexStation II scanning fluorometer with integrated fluidics transfer capabilities.

Key words

Calcium assay G-protein coupled receptor Sphingosine-1-phosphate Lysophospholipid FlexStation EDG receptor 

Notes

Acknowledgments

We thank Dr. Bruce Conklin (University of California, San Francisco) for generously providing chimeric G-protein expression plasmids and Dr. Edward Goetzl (University of California, San Francisco) for the stable S1P receptor cell lines. This work was supported by NIH grant CA-092160.

References

  1. 1.
    Hopkins AL, Groom CR (2002) The druggable genome. Nat Rev Drug Discov 1:727–730PubMedCrossRefGoogle Scholar
  2. 2.
    Offermanns S, Simon MI (1998) Genetic analysis of mammalian G-protein signalling. Oncogene 17:1375–1381PubMedCrossRefGoogle Scholar
  3. 3.
    Singer WD, Brown HA, Sternweis PC (1997) Regulation of eukaryotic phosphatidylinositol-specific phospholipase C and phospholipase D. Annu Rev Biochem 66:475–509PubMedCrossRefGoogle Scholar
  4. 4.
    Kohno M, Momoi M, Oo ML, Paik JH, Lee YM, Venkataraman K, Ai Y, Ristimaki AP, Fyrst H, Sano H, Rosenberg D, Saba JD, Proia RL, Hla T (2006) Intracellular role for sphingosine kinase 1 in intestinal adenoma cell proliferation. Mol Cell Biol 26:7211–7223PubMedCrossRefGoogle Scholar
  5. 5.
    Alvarez SE, Harikumar KB, Hait NC, Allegood J, Strub GM, Kim EY, Maceyka M, Jiang H, Luo C, Kordula T, Milstien S, Spiegel S (2010) Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2. Nature 465:1084–1088PubMedCrossRefGoogle Scholar
  6. 6.
    Strub GM, Maceyka M, Hait NC, Milstien S, Spiegel S (2010) Extracellular and intracellular actions of sphingosine-1-phosphate. Adv Exp Med Biol 688:141–155PubMedCrossRefGoogle Scholar
  7. 7.
    Hla T, Brinkmann V (2011) Sphingosine 1-phosphate (S1P): Physiology and the effects of S1P receptor modulation. Neurology 76:S3–S8PubMedCrossRefGoogle Scholar
  8. 8.
    Brinkmann V (2007) Sphingosine 1-phosphate receptors in health and disease: mechanistic insights from gene deletion studies and reverse pharmacology. Pharmacol Ther 115:84–105PubMedCrossRefGoogle Scholar
  9. 9.
    An S, Bleu T, Zheng Y (1999) Transduction of intracellular calcium signals through G ­protein-mediated activation of phospholipase C by recombinant sphingosine 1-phosphate receptors. Mol Pharmacol 55:787–794PubMedGoogle Scholar
  10. 10.
    Offermanns S, Simon MI (1995) G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C. J Biol Chem 270:15175–15180PubMedCrossRefGoogle Scholar
  11. 11.
    Coward P, Chan SD, Wada HG, Humphries GM, Conklin BR (1999) Chimeric G proteins allow a high-throughput signaling assay of Gi-coupled receptors. Anal Biochem 270:242–248PubMedCrossRefGoogle Scholar
  12. 12.
    Conklin BR, Farfel Z, Lustig KD, Julius D, Bourne HR (1993) Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha. Nature 363:274–276PubMedCrossRefGoogle Scholar
  13. 13.
    Conklin BR, Herzmark P, Ishida S, Voyno-Yasenetskaya TA, Sun Y, Farfel Z, Bourne HR (1996) Carboxyl-terminal mutations of Gq alpha and Gs alpha that alter the fidelity of receptor activation. Mol Pharmacol 50:885–890PubMedGoogle Scholar
  14. 14.
    Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450PubMedGoogle Scholar
  15. 15.
    Graler MH, Goetzl EJ (2004) The immunosuppressant FTY720 down-regulates sphingosine 1-phosphate G-protein-coupled receptors. FASEB J 18:551–553PubMedGoogle Scholar
  16. 16.
    Koide Y, Uemoto K, Hasegawa T, Sada T, Murakami A, Takasugi H, Sakurai A, Mochizuki N, Takahashi A, Nishida A (2007) Pharmacophore-based design of sphingosine 1-phosphate-3 receptor antagonists that include a 3,4-dialkoxybenzophenone scaffold. J Med Chem 50:442–454PubMedCrossRefGoogle Scholar
  17. 17.
    Murakami A, Takasugi H, Ohnuma S, Koide Y, Sakurai A, Takeda S, Hasegawa T, Sasamori J, Konno T, Hayashi K, Watanabe Y, Mori K, Sato Y, Takahashi A, Mochizuki N, Takakura N (2010) Sphingosine 1-phosphate (S1P) regulates vascular contraction via S1P3 receptor: investigation based on a new S1P3 receptor antagonist. Mol Pharmacol 77:704–713PubMedCrossRefGoogle Scholar
  18. 18.
    Yamazaki Y, Kon J, Sato K, Tomura H, Sato M, Yoneya T, Okazaki H, Okajima F, Ohta H (2000) Edg-6 as a putative sphingosine 1-­phosphate receptor coupling to Ca(2+) signaling pathway. Biochem Biophys Res Commun 268:583–589PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of PhysiologyThe University of Tennessee Health Science CenterMemphisUSA
  2. 2.Department of PhysiologyUniversity of Tennessee Health Science CenterMemphisUSA

Personalised recommendations