دانلود رایگان مقاله سیگنالینگ مغرضانه لیپید و اقدامات آلوستریک مولکول مصنوعی

Abstract

GPR119 is a Gαs-coupled lipid-sensor in the gut, where it mediates release of incretin hormones from the enteroendocrine cells and in pancreatic α-cells, where it releases insulin. Naturally occurring lipids such as monoacylglycerols (MAGs) and N-acylethanolamines (NAEs), like oleoylethanolamide (OEA), activate GPR119, and multiple synthetic ligands have been described. Here, we extend the GPR119 signaling profile to Gαq and Gαi in addition to β-arrestin recruitment and the downstream transcription factors CRE (cAMP response element), SRE (serum response element) and NFAT (nuclear factor of activated T cells). The endogenous OEA and the synthetic AR231453 were full agonists in all pathways except for NFAT, where no ligand-modulation was observed. The potency of AR231453 varied <16-fold (EC50 from 6 to 95 nM) across the different signaling pathways, whereas that of OEA varied >175-fold (from 85 nM to 15 μM) indicating a biased signaling for OEA. The degree of constitutive activity was 1–10%, 10–30% and 30–70% of OEA-induced Emax in Gαi, Gαq and Gαs-driven pathways, respectively. This coincided with the lowest and highest OEA potency observed in Gαi and Gαs-driven pathways, respectively. Incubation for 2 h with the 2-MAG-lipase inhibitor JZL84 doubled the constitutive activity, indicating that endogenous lipids contribute to the apparent constitutive activity. Finally, besides being an agonist, AR231453 acted as a positive allosteric modulator of OEA and increased its potency by 54-fold at 100 nM AR231453. Our studies uncovering broad and biased signaling, masked constitutive activity by endogenous MAGs, and ago-allosteric properties of synthetic ligands may explain why many GPR119 drug-discovery programs have failed so far.

4. Discussion

In the present study, we extend the signaling profile of GPR119 to include not only Gas, but also other G proteins, transcription factors as well as b-arrestin recruitment. The endogenous OEA and the synthetic AR231453 were full agonists through most pathways, and whereas the potency of AR231453 was rather constant, that of OEA varied in a pathway-dependent manner, that paralleled the basal receptor activity. This establishes OEA as a biased ligand. Together with the observation that endogenous MAGs influence the basal activity of GPR119, this indicates that the basal activity of GPR119 is – in part – mediated by endogenous lipids. Finally, AR231453 was shown to act as a positive allosteric modulator of OEA activity.