Abstract
BackgroundGPR88 is an orphan G protein-coupled receptor highly expressed in the striatum and is implicated in basal ganglia-associated disorders. However, the receptor functions of GPR88 are still largely unknown due to the lack of potent and selective ligands appropriate for central nervous system investigation. Development of a high-throughput screening assay for GPR88 should facilitate the discovery of novel ligands to probe GPR88 functions.MethodsIn this paper, we describe the development of a CHO-Gαqi5-GPR88 cell-based calcium mobilization assay. The assay takes advantage of functional coupling of GPR88 with the promiscuous Gαqi5 protein and consequent mobilization of intracellular calcium, which can be measured in a 384-well format with a Fluorescent Imaging Plate Reader.ResultsThe CHO-Gαqi5-GPR88 cell-based calcium mobilization assay was validated by the structure-activity relationship study of known GPR88 agonist (1R,2R)-2-PCCA analogues. The assay was automated and miniaturized to a 384-well format, and was deemed robust and reproducible with a Z’-factor of 0.72 and tolerated dimethyl sulfoxide to a final concentration of 2%. Screening a pilot neurotransmitter library consisting of 228 compounds yielded 10 hits, but none of the hits were confirmed as GPR88 agonists in follow-up assays.ConclusionsWe have developed a high-throughput calcium mobilization assay for the orphan receptor GPR88. This calcium mobilization assay can be used to identify several different types of GPR88 ligands including agonists, competitive and noncompetitive antagonists, inverse agonists, and allosteric modulators. These ligands will serve as valuable tools to probe signaling mechanisms and in vivo functions of GPR88, and could expedite development of novel therapies for diseases potentially mediated by GPR88.
Highlights
GPR88 is an orphan G protein-coupled receptor highly expressed in the striatum and is implicated in basal ganglia-associated disorders
With these factors in mind, a heterologous system was designed in which GPR88 could couple to the promiscuous Gαq subunit in Chinese hamster ovary (CHO) cells to induce intracellular calcium mobilization
In the Gαqi5-GPR88 transients, (1R,2R)-2-PCCA produced a 2.3-fold specific response compared to the Gαqi5 mock cells, suggesting that this engineered system is suitable for stable cell line creation to monitor
Summary
GPR88 is an orphan G protein-coupled receptor highly expressed in the striatum and is implicated in basal ganglia-associated disorders. The receptor functions of GPR88 are still largely unknown due to the lack of potent and selective ligands appropriate for central nervous system investigation. Dopaminergic and glutamatergic systems are often used as drug targets to treat basal ganglia-associated disorders [2, 3], considerable efforts have focused on identifying alternative mechanisms and new candidate proteins that regulate basal ganglia functions, which may lead to new drug discovery opportunities [4, 5]. The orphan G protein-coupled receptor (GPCR) GPR88 is a potential candidate for such efforts. GPR88 is expressed in other brain regions including the cerebral cortex, amygdala, and hypothalamus [9,10,11]. Multiple lines of evidence obtained from GPR88knockout studies suggest that GPR88 plays an important
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