Abstract
Phospholipase C β (PLC-β)-coupled G protein-coupled receptor (GPCR) activities traditionally are assessed by measuring Ca 2+ triggered by d- myo-inositol 1,4,5-trisphosphate (IP 3), a PLC-β hydrolysis product, or by measuring the production of inositol phosphate using cumbersome radioactive assays. A specific detection of IP 3 production was also established using IP 3 binding proteins. The short lifetime of IP 3 makes this detection very challenging in measuring GPCR responses. Indeed, this IP 3 rapidly enters the metabolic inositol phosphate cascade. It has been known for decades that lithium chloride (LiCl) leads to d- myo-inositol 1-phosphate accumulation on GPCR activation by inhibiting inositol monophosphatase, the final enzyme of the IP 3 metabolic cascade. We show here that IP 1 can be used as a surrogate of IP 3 to monitor GPCR activation. We developed a novel homogeneous time-resolved fluorescence (HTRF) assay that correlates perfectly with existing methods and is easily amenable to high-throughput screening. The IP–One assay was validated on various GPCR models. It has the advantage over the traditional Ca 2+ assay of allowing the measurement of inverse agonist activity as well as the analysis of PLC-β activity in any nontransfected primary cultures. Finally, the high assay specificity for d- myo-inositol 1 monophosphate (IP 1(1)) opens new possibilities in developing selective assays to study the functional roles of the various isoforms of inositol phosphates.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have