Elsevier

Analytical Biochemistry

Volume 313, Issue 2, 15 February 2003, Pages 311-318
Analytical Biochemistry

Scintillation proximity assay of inositol phosphates in cell extracts: High-throughput measurement of G-protein-coupled receptor activation

https://doi.org/10.1016/S0003-2697(02)00630-9Get rights and content

Abstract

The phosphatidylinositol turnover assay is used widely to measure activation, and inhibition, of Gq-linked G-protein-coupled receptors. Cells expressing the receptor of interest are labeled by feeding with tritiated myo-inositol. The label is incorporated into cellular phosphatidylinositol 4,5-bisphosphate, which, upon agonist binding to the receptor, is hydrolyzed by phospholipase C to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. In the presence of Li+, dephosphorylation of IP3 to inositol is blocked, and the mass of soluble inositol phosphates is a quantitative readout of receptor activation. Current protocols for this assay all involve an anion-exchange chromatography step to separate radiolabeled inositol phosphates from radiolabeled inositol, making the assay cumbersome and difficult to automate. We now describe a scintillation proximity assay to measure soluble inositol phosphate mass in cell extracts, thus obviating the need for the standard chromatography step. The method uses positively charged yttrium silicate beads that bind inositol phosphates, but not inositol. We have used this assay to measure activation of recombinant and endogenous muscarinic acetylcholine receptors and activation of recombinant neuropeptide FF2 receptor coupled to IP3 production by coexpression of a chimeric G protein. Further, we demonstrate the use and functional validity of this assay in a semiautomated, 384-well format, by characterizing the muscarinic receptor antagonists pirenzepine and atropine.

Section snippets

Detection of radioactive Ins and InsP standards

d-Myo-[2-3H]inositol 1-phosphate ([3H]Ins-1-P; 5–20 Ci/mmol), d-myo-[2-3H]inositol 1,4-bisphosphate ([3H]Ins-1,4-P2; 2–10 Ci/mmol), and d-myo-[1-3H]inositol 1,4,5-trisphosphate ([3H]Ins-1,4,5-P3; 15–30 Ci/mmol) were from NEN and were supplied in aqueous 10 mM ammonium phosphate, pH 8.0. Myo-[1,2-3H]inositol ([3H]Ins; NEN) was supplied in water with a specific activity of 70–80 Ci/mmol. Yttrium silicate (YSi) scintillation proximity assay (SPA) beads were supplied by Amersham Biosciences as a 100 

Detection of [3H]myo-inositol 1-phosphate versus [3H]myo-inositol using YSi SPA beads

Yttrium silicate glass beads, impregnated with cerium, are marketed by Amersham Biosciences for use in a SPA for RNA. These SPA beads carry a net positive charge and so, in principle, should bind a negatively charged InsP more efficiently than neutral Ins. Indeed, we found that with YSi SPA beads, detection of [3H]Ins-1-P was approximately 20-fold more efficient than for [3H]Ins (Table 1). Addition of unlabeled Ins-1-P reduced the number of counts recorded from YSi SPA beads mixed with [3H

Acknowledgements

We acknowledge Dr. John York for valuable discussion of this work, Dr. Qingyun Liu for provision of the NPFFR2-expressing cell line, Dr. Pierre Mallorga for discussions concerning muscarinic receptor antagonists, and Drs. Lawrence Kuo and Pierre Mallorga for critical evaluation of the manuscript.

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