ABSTRACT
G-protein-coupled receptors (GPCRs) are one of the largest classes of drug discovery targets [1,2]. GPCR ligands regulate cellular and physiological pathways by signaling through several second messengers, including cyclic AMP, inositol phospholipids, and calcium [3]. Quantitation of second messengers is frequently used as a means to screen and pharmacologically characterize GPCR ligands [4]. The GPCR signaling process occurs by two major pathways. GPCRs coupling to G
α
and G
α
proteins activate or inhibit, respectively, adenylate cyclase and subsequently change intracellular cAMP levels. GPCRs coupling to G
α
or G
α
proteins activate phosphoinositol phospholipase C
β
, which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP
) forming
sn
1,2diacylglycerol and inositol 1,4,5-trisphosphate (Ins P
) [5]. Ins P
binds and opens an endoplasmic Ins P
gated calcium channel, causing release of bound calcium into the cytosol [6]. Several metabolic products of Ins P
also modulate cellular function, including inositol 1,3,4,5-P
(Ins P
), which acts to facilitate Ins P
-mediated calcium release synergistically [7]. There are several HTS assay systems to measure intracellular cyclic AMP as a marker of G
- and G
-coupled GPCRs [8]. In contrast, there are few assays available to selectively measure Ins P
to monitor Gq-coupled GPCR activation, particularly those suitable for automated HTS. Consequently, many HTS laboratories measure changes in intracellular calcium to assay G
-coupled GPCRs using a fluorescent calcium-sensitive dye, loaded into intact cells as a cell-permeable ester. Real-time changes in the GPCR-induced signal are then determined in a microtiter plate using imaging instruments, such as a fluorescent imaging plate reader system (FLIPR, Molecular Devices Corp) [9].
