Scintillation proximity assay of inositol phosphates in cell extracts: high-throughput measurement of G-protein-coupled receptor activation.
The phosphatidylinositol turnover assay is used widely to measure activation, and inhibition, of G(q)-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 (IP(3)) and diacylglycerol. In the presence of Li(+), dephosphorylation of IP(3) 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 IP(3) 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.
Brandish
Philip E
PE
Merck Research Laboratories, West Point, PA 19422, USA. philip_brandish@merck.com
Hill
Lorraine A
LA
Zheng
Wei
W
Scolnick
Edward M
EM
D000818:Animals; D001285:Atropine / Q000494:pharmacology; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology; D002457:Cell Extracts / Q000032:analysis*; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007294:Inositol / Q000737:chemistry / Q000378:metabolism; D007295:Inositol Phosphates / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D018021:Lithium Chloride / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D010890:Pirenzepine / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012588:Scintillation Counting / Q000379:methods; D017640:Silicates / Q000737:chemistry / Q000378:metabolism; D014316:Tritium; D015019:Yttrium / Q000737:chemistry / Q000378:metabolism; D015021:Yttrium Radioisotopes
D002457:Cell Extracts; D007295:Inositol Phosphates; D018727:Muscarinic Antagonists; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D017640:Silicates; D015021:Yttrium Radioisotopes; D014316:Tritium; C028301:yttrium silicate; D010890:Pirenzepine; D007294:Inositol; D015019:Yttrium; D001285:Atropine; D002217:Carbachol; D019204:GTP-Binding Proteins; D018021:Lithium Chloride