Agonists of the Orphan Human G2A Receptor Identified from Inducible G2A Expression and β-Lactamase Reporter Screen

Abstract

The G protein-coupled receptor (GPCR) G2A (for G2 accumulation) was identified as a stress-inducible antiproliferative cell cycle regulator. Targeted G2A gene deletion in mice resulted in systemic lupus erythematosus-like and atherosclerotic lesion phenotypes. These findings suggested that G2A may be a therapeutic target for cancers and autoimmune and cardiovascular diseases. The G2A receptor is cytotoxic upon ectopic expression, and its cognate ligand has not been identified, making it difficult to generate a cell line for screening using a conventional approach. The function of human G2A remains obscure. Here we show that by using an inducible T-REx (Invitrogen, Carlsbad, CA) expression system an inducible G2A functional cell-based beta-lactamase reporter assay could be developed using the constitutive activity of the receptor. Furthermore, G2A expression levels can be controlled under this inducible system to avoid the expression artifacts of conventional approaches using constitutive expression vectors. This stable cell line expressing the human G2A receptor was screened against a chemical library containing 740,000 compounds, and small molecules showing selective agonistic activity on G2A were identified. We believe the strategy employed here for G2A should be applicable to other "intractable" GPCRs where target gene expression results in cytotoxic and/or high constitutive activities.

Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic ‘eat-me’ signals: cleavage and inhibition of phagocytosis by Lp-PLA2

“…Indeed, Lp-PLA 2 appears to be well placed to modulate macrophage accumulation and function due to its ability to cleave oxidized phospholipids generating lysoPC, lysoPS and various oxidized FFAs including 9-and 13-HODE. 18,49 Although still somewhat controversial, 50 all three lipid products have been described as ligands for the G-protein-coupled receptor G2A, 51,52 which is highly expressed in peripheral blood leukocytes and has been proposed to not only attract phagocytes to apoptotic cells but to facilitate their removal. Interestingly, lyso-PS can be generated by two distinct pathways to influence phagocytosis; by cleavage of externalized PSox, as described herein by Lp-PLA 2 , or generated intracellularly via a NADPH oxidasedependent pathway by neutrophils.…”

Mouse and Human Eosinophils Degranulate in Response to Platelet-Activating Factor (PAF) and LysoPAF via a PAF-Receptor–Independent Mechanism: Evidence for a Novel Receptor

“…PAFR is the only known receptor that responds to PAF, although the family of lipid receptors is large, and there are quite a few orphan receptors that remain unexplored and incompletely characterized (reviewed in (53)). The G2A receptor was initially characterized as responding to lysoPC, but this finding has recently been reconsidered (56). Likewise, several groups have reported that the actions of lysoPC are dependent on the presence of the receptor GPR4 (57, 58) yet the receptor itself has been characterized alternatively as ligand-independent (59) or proton sensing (60).…”

Strategy for the Identification of GPR23/LPA4 Receptor Agonists and Inverse Agonists

“…The fact that Compound 3 only inhibited doxycycline-induced constitutive bla activity in GPR23-expressing cells and not in another cell line expressing the G2A receptor under the same T-REx™ regulation system indicating the effect is GPR23-specific. 30 These results suggest that Compound 3 binds the GPR23 receptor and modulates its constitutive activity at a domain other than the LPA-binding site.…”