NanoLuc (NLuc) complementation assay elucidates role of specific G‐proteins in GPR88 signaling

Abstract

GPR88 is an orphan G protein-coupled receptor (GPCR) expressed at high levels in the striatum and is a target for the development of therapeutics to treat multiple neuropsychiatric disorders. While a few small-molecule synthetic agonists have been reported, no synthetic antagonists or endogenous ligands have been identified. Typical 2nd messenger, cell-based assays for agonist and antagonist identification and signaling pathway elucidation rely on complex downstream events, with interpretation of results complicated from effects of transfected receptor overexpression and interference from endogenous receptors and proteins. The NLuc complementation assay, based on an engineered luciferase from the deep-sea shrimp, Oplophorus gracilirostris, can monitor receptor-G protein interaction in live cells and profile compounds according to the G proteins they activate. The synthetic agonists, 2-PCCA [(1R,2R)-2-(pyridin-2-yl)cyclopropane carboxylic acid], and 2-OMPP [(2R)-N-(2-hydroxy-1-(4-((2-methylpentyl)oxy)phenyl)ethyl)-2-phenylpropanamide] are suspected to activate GPR88 via a Gαi-coupled signaling pathway. In this study we show the activation of GPR88 by 2-PCCA and 2-OMPP inhibited isoproterenol induced cAMP production in transiently transfected GPR88 HEK-293T cells with relative EC50 values of 250 nM and 1.0 μM, respectively, consistent with prior reports. The EC50 value for PCCA in HEK-293T/GPR88 transients is five times that in RH-7777 cells with COS-7 cells showing no cAMP response. These results highlight the importance of the cellular background in engineered systems. Additionally, the NLuc complementation assay shows PCCA receptor binding triggers activation of several G-proteins including Gαi1(EC50= 760 nM), Gαi0 (EC50=3.3μM) and GαS (EC50=4.5 nM) indicating the effects of varying G-protein expression in different cell lines. In combination with our computational endeavors, 2nd messenger and the NLuc complementation assays are valuable tools in our research as we develop high-throughput screening processes and probe signaling pathways in search of GPCR based therapeutics.