The recognition that individual GPCRs can activate multiple signaling pathways has raised the possibility of developing drugs selectively targeting therapeutically relevant ones. This requires tools to determine which G proteins and barrestins are activated by a given receptor. Here, we present a set of BRET sensors monitoring the activation of the 12 G protein subtypes based on the translocation of their effectors to the plasma membrane (EMTA). Unlike most of the existing detection systems, EMTA does not require modification of receptors or G proteins (except for Gs). EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. Profiling of 100 therapeutically relevant human GPCRs resulted in 1,500 pathway-specific concentration-response curves and revealed a great diversity of coupling profiles ranging from exquisite selectivity to broad promiscuity. Overall, this work describes unique resources for studying the complexities underlying GPCR signaling and pharmacology.

The p38 MAPK mediates transcriptional and posttranscriptional control of cyclooxygenase-2 (COX-2) mRNA following interleukin-1(IL-1)/lipopolysaccharide cellular activation. We explored a positive feedback, prostaglandin E 2 (PGE 2 )-dependent stabilization of COX-2 mRNA mediated by the p38 MAPK cascade in IL-1␤-stimulated human synovial fibroblasts. We observed a rapid (5 min), massive (>30-fold), and sustained (>48 h) increase in COX-2 mRNA, protein, and PGE 2 release following a recombinant human (rh) IL-1␤ signal that was inhibited by NS-398, a COX-2 inhibitor, and SB202190, a selective, cell-permeable p38 MAPK inhibitor. PGE 2 completely reversed NS-398-mediated inhibition but not SB202190-dependent inhibition. The eicosanoid didn't potentiate IL-1␤-induced COX-2 expression nor did it activate COX-2 gene expression in quiescent cells. Transfection experiments with a human COX-2 promoter construct revealed a minor element of p38 MAPK-dependent transcriptional control after IL-1␤ stimulation. p38 MAPK synergized with the cAMP/cAMP-dependent protein kinase cascade to transactivate the COX-2 promoter. When human synovial fibroblasts were activated with rhIL-1␤ for 3-4 h (steady state) followed by washout, the elevated levels of COX-2 mRNA declined rapidly (<2 h) to control levels. If PGE 2 , unlike EP2/3 agonists butaprost and sulprostone, was added to fresh medium, COX-2 mRNA levels remained elevated for up to 16 h. SB202190 or anti-PGE 2 monoclonal antibody compromised the stabilization of COX-2 mRNA by PGE 2 . Deletion analysis using transfected chimeric luciferase-COX-2 mRNA 3-untranslated region reporter constructs revealed that IL-1␤ increased reporter gene mRNA stability and translation via AU-containing distal regions of the untranslated region. This response was mediated entirely by a PGE 2 /p38 MAPK-dependent process. We conclude that the magnitude and duration of the induction of COX-2 mRNA, protein, and PGE 2 release by rhIL-1␤ is primarily the result of PGE 2 -dependent stabilization of COX-2 mRNA and stimulation of translation, a process involving a positive feedback loop mediated by the EP4 receptor and the downstream kinases p38 MAPK and, perhaps, cAMP-dependent protein kinase.

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The free fatty acid receptor GPR40 has been proposed as a potential target for type 2 diabetes (T2D) pharmacotherapy. This idea has been validated in both preclinical and clinical studies, in which activation of GPR40 was shown to improve glycaemic control by stimulating glucose-dependent insulin secretion; however, the recent termination of phase III clinical trials using the GPR40 agonist TAK-875 (fasiglifam) has raised important questions regarding the long-term safety and viability of targeting GPR40 and, more specifically, about our understanding of this receptor's basic biology. In the present review, we provide a summary of established and novel concepts related to GPR40's pharmacobiology and discuss the current status and future outlook for GPR40-based drug development for the treatment of T2D.