Nucleotides and cysteinyl-leukotrienes (CysLTs) are unrelated signaling molecules inducing multiple effects through separate G-protein-coupled receptors: the P2Y and the CysLT receptors. Here we show that GPR17, a Gi-coupled orphan receptor at intermediate phylogenetic position between P2Y and CysLT receptors, is specifically activated by both families of endogenous ligands, leading to both adenylyl cyclase inhibition and intracellular calcium increases. Agonist-response profile, as determined by [(35)S]GTPgammaS binding, was different from that of already known CysLT and P2Y receptors, with EC(50) values in the nanomolar and micromolar range, for CysLTs and uracil nucleotides, respectively. Both rat and human receptors are highly expressed in the organs typically undergoing ischemic damage, that is, brain, heart and kidney. In vivo inhibition of GPR17 by either CysLT/P2Y receptor antagonists or antisense technology dramatically reduced ischemic damage in a rat focal ischemia model, suggesting GPR17 as the common molecular target mediating brain damage by nucleotides and CysLTs. In conclusion, the deorphanization of GPR17 revealed a dualistic receptor for two endogenous unrelated ligand families. These findings may lead to dualistic drugs of previously unexplored therapeutic potential.

Despite extensive study of heptahelical G protein-coupled receptors (GPCRs), the precise mechanism of G protein activation is unknown. The role of one highly conserved stretch of residues, the amino acids glutamic acid/aspartic acid-argininetyrosine (i.e., the E/DRY motif), has received considerable attention with respect to regulating GPCR conformational states. In the consensus view, glutamic acid/aspartic acid maintains the receptor in its ground state, because mutations frequently induce constitutive activity (CA). This hypothesis has been confirmed by the rhodopsin ground-state crystal structure and by computational modeling approaches. However, some class A GPCRs are resistant to CA, suggesting alternative roles for the glutamic acid/aspartic acid residue and the E/DRY motif. Here, we propose two different subgroups of receptors within class A GPCRs that make different use of the E/DRY motif, independent of the G protein type (G s , G i , or G q ) to which the receptor couples. In phenotype 1 receptors, nonconservative mutations of the glutamic acid/aspartic acid-arginine residues, besides inducing CA, increase affinity for agonist binding, retain G protein coupling, and retain an agonist-induced response. In contrast, in second phenotype receptors, the E/DRY motif is more directly involved in governing receptor conformation and G protein coupling/recognition. Hence, mutations of the glutamic acid/aspartic acid residues do not induce CA. Conversely, nonconservative mutations of the arginine of the E/DRY motif always impair agonist-induced receptor responses and, generally, reduce agonist binding affinity. Thus, it is essential to look beyond the rhodopsin ground-state model of conformational activation to clarify the role of this highly conserved triplet in GPCR activation and function.

Cysteinyl-leukotrienes (cysteinyl-LTs), that is, LTC4, LTD4, and LTE4, trigger contractile and inflammatory responses through the specific interaction with G protein-coupled receptors (GPCRs) belonging to the purine receptor cluster of the rhodopsin family, and identified as CysLT receptors (CysLTRs). Cysteinyl-LTs have a clear role in pathophysiological conditions such as asthma and allergic rhinitis (AR), and have been implicated in other inflammatory conditions including cardiovascular diseases, cancer, atopic dermatitis, and urticaria. Molecular cloning of human CysLT1R and CysLT2R subtypes has confirmed most of the previous pharmacological characterization and identified distinct expression patterns only partially overlapping. Interestingly, recent data provide evidence for the immunomodulation of CysLTR expression, the existence of additional receptor subtypes, and of an intracellular pool of CysLTRs that may have roles different from those of plasma membrane receptors. Furthermore, genetic variants have been identified for the CysLTRs that may interact to confer risk for atopy. Finally, a crosstalk between the cysteinyl-LT and the purine systems is being delineated. This review will summarize and attempt to integrate recent data derived from studies on the molecular pharmacology and pharmacogenetics of CysLTRs, and will consider the therapeutic opportunities arising from the new roles suggested for cysteinyl-LTs and their receptors.

Chagas' disease is caused by infection with the parasite Trypanosoma cruzi. We report that infected, but not uninfected, human endothelial cells (ECs) released thromboxane A2 (TXA2). Physical chromatography and liquid chromatography-tandem mass spectrometry revealed that TXA2 is the predominant eicosanoid present in all life stages of T. cruzi. Parasite-derived TXA2 accounts for up to 90% of the circulating levels of TXA2 in infected wild-type mice, and perturbs host physiology. Mice in which the gene for the TXA2 receptor (TP) has been deleted, exhibited higher mortality and more severe cardiac pathology and parasitism (fourfold) than WT mice after infection. Conversely, deletion of the TXA2 synthase gene had no effect on survival or disease severity. TP expression on somatic cells, but not cells involved in either acquired or innate immunity, was the primary determinant of disease progression. The higher intracellular parasitism observed in TP-null ECs was ablated upon restoration of TP expression. We conclude that the host response to parasite-derived TXA2 in T. cruzi infection is possibly an important determinant of mortality and parasitism. A deeper understanding of the role of TXA2 may result in novel therapeutic targets for a disease with limited treatment options.

Both cysteinyl-leukotrienes and extracellular nucleotides mediate inflammatory responses via specific G-protein-coupled receptors, the CysLT and the P2Y receptors, respectively. Since these mediators accumulate at sites of inflammation, and inflammatory cells express both classes of receptors, their responses are likely to be crossregulated. We investigated the molecular basis of desensitization and trafficking of the CysLT1 receptor constitutively and transiently expressed in the human monocyte/macrophage-like U937 or COS-7 cells in response to LTD4 or nucleotides. Exposure to agonist induced a rapid homologous desensitization of the CysLT1 receptor [as measured by the reduction in the maximal agonist-induced intracellular cytosolic Ca2+ ([Ca2+]i) transient], followed by receptor internalization (as assessed by equilibrium binding and confocal microscopy). Activation of P2Y receptors with ATP or UDP induced heterologous desensitization of the CysLT1 receptor. Conversely, LTD4-induced CysLT1 receptor activation had no effect on P2Y receptor responses, which suggests that the latter have a hierarchy in producing desensitizing signals. Furthermore, ATP/UDP-induced CysLT1 receptor desensitization was unable to cause receptor internalization, induced a faster recovery of CysLT1 functionality and was dependent upon protein kinase C. By contrast, homologous desensitization, which is probably dependent upon G-protein-receptor kinase 2 activation, induced a fast receptor downregulation and, accordingly, a slower recovery of CysLT1 functionality. Hence, CysLT1 receptor desensitization and trafficking are differentially regulated by the CysLT1 cognate ligand or by extracellular nucleotides. This crosstalk may have a profound physiological implication in the regulation of responses at sites of inflammation, and may represent just an example of a feedback mechanism used by cells to fine-tune their responses.