The PAR2 inhibitor I-287 selectively targets Gαq and Gα12/13 signaling and has anti-inflammatory effects.

Protease-activated receptor-2 (PAR2) is involved in inflammatory responses and pain, therefore representing a promising therapeutic target for the treatment of immune-mediated inflammatory diseases. However, as for other GPCRs, PAR2 can activate multiple signaling pathways and those involved in inflammatory responses remain poorly defined. Here, we describe a new selective and potent PAR2 inhibitor (I-287) that shows functional selectivity by acting as a negative allosteric regulator on Gαq and Gα12/13 activity and their downstream effectors, while having no effect on Gi/o signaling and ßarrestin2 engagement. Such selective inhibition of only a subset of the pathways engaged by PAR2 was found to be sufficient to block inflammation in vivo. In addition to unraveling the PAR2 signaling pathways involved in the pro-inflammatory response, our study opens the path toward the development of new functionally selective drugs with reduced liabilities that could arise from blocking all the signaling activities controlled by the receptor.

A role for K268 in V2R folding.

The V2 vasopressin receptor, a member of the rhodopsin subfamily of GPCRs, mediates arginine vasopressin control of water reabsorption in the kidney by activating Gs. Requirement of the third intracellular loop of the V2R for G(s) activation was identified by introducing V2R segments into the Gq coupled V1aR [Liu, J. and Wess, J. (1996) J. Biol. Chem. 271, 8772-8778]; the same approach recognized glutamate 231 and glutamine 225 at the amino terminus of loop 3i as being needed for signal transduction. Site-directed mutagenesis of the V2R confirmed their observations. Recently, we found that a positively charged amino acid at codon 268 is essential for V2R expression, although a double-mutant bearing lysine at position 231 and glutamic acid at position 268 was expressed at higher levels than the wild type V2R and displayed unchanged ligand-binding affinity. Ligand-induced internalization and phosphorylation of the double-mutant receptor was indistinguishable from that observed with the wild type protein but signaling activity was greatly diminished. The data suggested these two amino acids might interact with each other and might play a role in promoting GDP/GTP exchange.

CysLT1 receptor engagement induces activator protein-1- and NF-kappaB-dependent IL-8 expression.

Because cysteinyl-leukotrienes (cysLTs) are major protagonists in the pathophysiology of human asthma, and because neutrophils are involved in the more severe form of asthma, we studied the potential for leukotriene (LT) D(4) to induce synthesis of the chemokine IL-8 through activation of the CysLT1 receptor. We found LTD(4) to induce IL-8 gene expression in monocytic THP-1 cells and human dendritic cells with complete abrogation by selective CysLT1 antagonists. Human embryonic kidney-293 cells stably transfected with CysLT1 were used to better study the transcriptional regulation of the IL-8 promoter. Stimulation of the cells with graded concentrations of LTD(4) resulted in a time- and concentration-dependent induction of IL-8 transcription and protein synthesis. Use of IL-8 promoter mutants with substitutions in their NF-kappaB, activator protein (AP)-1, and NF-IL-6 binding elements revealed a requirement for NF-kappaB and AP-1, but not NF-IL-6, in LTD(4)-induced activation of the IL-8 promoter. Overexpression of dominant-negative IkappaBalpha inhibited the IL-8 transactivation induced by LTD(4). NF-kappaB DNA binding activity was induced by LTD(4), as determined by electrophoretic mobility shift assays, and could be supershifted by antibodies against p50 and p65. Supershift assays after LTD(4) stimulation also indicated the formation of a c-Jun/c-Fos complex. Moreover, our results demonstrate that LTD(4) upregulates the expression of c-fos and c-jun at the mRNA level. Our data show for the first time that LTD(4), via the CysLT1 receptor, can transcriptionally activate IL-8 production, with involvement of the transcription factors p50, p65, Fos, and Jun. These findings provide mechanistic and potentially therapeutic elements for modulation of the inflammatory component of asthma.