Cysteinyl-Leukotriene Receptors and Cellular Signals

Cysteinyl-leukotrienes (cysteinyl-LTs) exert a range of proinflammatory effects, such as constriction of airways and vascular smooth muscle, increase of endothelial cell permeability leading to plasma exudation and edema, and enhanced mucus secretion. They have proved to be important mediators in asthma, allergic rhinitis, and other inflammatory conditions, including cardiovascular diseases, cancer, atopic dermatitis, and urticaria. The classification into subtypes of the cysteinyl-LT receptors (CysLTRs) was based initially on binding and functional data, obtained using the natural agonists and a wide range of antagonists. CysLTRs have proved remarkably resistant to cloning. However, in 1999 and 2000, the CysLT1R and CysLT2R were successfully cloned and both shown to be members of the G-protein coupled receptors (GPCRs) superfamily. Molecular cloning has confirmed most of the previous pharmacological characterization and identified distinct expression patterns only partially overlapping. Recombinant CysLTRs couple to the Gq/11 pathway that modulates inositol phospholipids hydrolysis and calcium mobilization, whereas in native systems, they often activate a pertussis toxin-insensitive Gi/o-protein, or are coupled promiscuously to both G-proteins. Interestingly, recent data provide evidence for the existence of an additional receptor subtype that seems to respond to both cysteinyl-LTs and uracil nucleosides, and of an intracellular pool of CysLTRs that may have roles different from those of plasma membrane receptors. Finally, a cross-talk between the cysteinyl-LT and the purine systems is being delineated. This review will summarize recent data derived from studies on the molecular and cellular pharmacology of CysLTRs

Full and Partial Agonists of Thromboxane Prostanoid Receptor Unveil Fine Tuning of Receptor Superactive Conformation and G Protein Activation

The intrahelical salt bridge between $E/D^{3.49}$ and $R^{3.50}$ within the E/DRY motif on helix 3 (H3) and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of $E^{3.49/6.30}$ in the thromboxane prostanoid (TP) receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA) with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET) indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow "resistant" to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes

Pharmacological characterization of the cysteinyl-leukotriene antagonists CGP 45715A (iralukast) and CGP 57698 in human airways in vitro

1. Cysteinyl-leukotrienes (cysteinyl-LTs) are important mediators in the pathogenesis of asthma. They cause bronchoconstriction, mucus hypersecretion, increase in microvascular permeability, plasma extravasation and eosinophil recruitment. 2. We investigated the pharmacological profile of the cysteinyl-LT antagonists CGP 45715A (iralukast), a structural analogue of LTD(4) and CGP 57698, a quinoline type antagonist, in human airways in vitro, by performing binding studies on human lung parenchyma membranes and functional studies on human isolated bronchial strips. 3. Competition curves vs [(3)H]-LTD(4) on human lung parenchyma membranes demonstrated that: (a) both antagonists were able to compete for the two sites labelled by [(3)H]-LTD(4); (b) as in all the G-protein coupled receptors, iralukast and CGP 57698 did not discriminate between the high and the low affinity states of the CysLT receptor labelled by LTD(4) (K(i1)=K(i2)=16.6 nM±36% CV and K(i1)= K(i2)=5.7 nM±19% CV, respectively); (c) iralukast, but not CGP 57698, displayed a slow binding kinetic, because preincubation (15 min) increased its antagonist potency. 4. In functional studies: (a) iralukast and CGP 57698 antagonized LTD(4)-induced contraction of human bronchi, with pA(2) values of 7.77±4.3% CV and 8.51±1.6% CV, respectively, and slopes not significantly different from unity; (b) the maximal LTD(4) response in the presence of CGP 57698 was actually increased, thus clearly deviating from apparent simple competition. 5. Both antagonists significantly inhibited antigen-induced contraction of human isolated bronchial strips in a concentration-dependent manner, lowering the upper plateau of the anti-IgE curves. 6. In conclusion, the results of the present in vitro investigation indicate that iralukast and CGP 57698 are potent antagonists of LTD(4) in human airways, with affinities in the nanomolar range, similar to those obtained for ICI 204,219 and ONO 1078, two of the most clinically advanced CysLT receptor antagonists. Thus, these compounds might be useful drugs for the therapy of asthma and other allergic diseases

A role for inflammatory mediators in heterologous desensitization of CysLT1 receptor in human monocytes

Cysteinyl-leukotrienes (cysteinyl-LT) are rapidly generated at sites of inflammation and, in addition to their role in asthma, rhinitis, and other immune disorders, are increasingly regarded as significant inflammatory factors in cancer, gastrointestinal, cardiovascular diseases. We recently demonstrated that in monocyte/macrophage–like U937 cells, extracellular nucleotides heterologously desensitize CysLT1 receptor (CysLT1R)-induced Ca2+ transients. Given that monocytes express a number of other inflammatory and chemoattractant receptors, this study was aimed at characterizing transregulation between these different stimuli. We demonstrate that in U937 cells and in primary human monocytes, a series of inflammatory mediators activating Gi-coupled receptor (FPR1, BLT1) desensitize CysLT1R-induced Ca2+ response unidirectionally through activation of PKC. Conversely, PAF-R, exclusively coupled to Gq, cross-desensitizes CysLT1R without the apparent involvement of any kinase. Interestingly, Gs-coupled receptors (β2AR, H1/2R, EP2/4R) are also able to desensitize CysLT1R response through activation of PKA. Heterologous desensitization seems to affect mostly the Gi-mediated signaling of the CysLT1R. The hierarchy of desensitization among agonists may be important for leukocyte signal processing at the site of inflammation. Considering that monocytes/macrophages are likely to be the major source of cysteinyl-LT in many immunological and inflammatory processes, shedding light on how their receptors are regulated will certainly help to better understand the role of these cells in orchestrating this complex network of integrated signals

Agonist-induced total IP accumulation in HEK293 cells transiently expressing equal amounts of the WT (A) or E129V mutant (B) of human TP receptor.

<p>Total IP accumulation was measured after incubation in the absence (basal) or presence of increasing concentrations of the indicated agonists for 30 min. Data are expressed as dpm/well. Error bars represent mean±SE of at least three independent experiments each performed in duplicates or triplicates (For the sake of clarity, in panel B, error bar direction of U46619 and I-BOP data is above and below, respectively). Curves are computer generated from the simultaneous analysis of at least three independent experiments. Values for EC₅₀ and significant differences from WT are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060475#pone-0060475-t002" target="_blank">Table 2</a>.</p

BRET² measurement of Gα_qβ₁γ₂ complex activation in HEK293 living cells expressing equal amounts of the WT of human TP receptor or its E129V mutant.

<p>A. BRET² was measured between the donor Rluc8 and the acceptor GFP¹⁰ introduced at the residue 97 of the Gα_q subunit and the N-terminal domain of the Gγ₂ subunit, respectively. Agonist-induced coupling of TP receptor and Gq protein distances Gα_q-Rluc8 and GFP¹⁰-Gγ₂ giving rise to a decrease in the BRET signal. B. Protein expression levels of the constructs used for BRET experiments were set to be constant and able to assure the same level of basal BRET ratio in the presence of WT and E129V mutant of the human TP receptors. Total Gα_q-Rluc8 luminescence was evaluated in HEK293 cells co-expressing Gα_q-Rluc8 together with GFP¹⁰-Gγ₂ and Gβ₁ in the presence of WT or E129V mutant of the human TP receptor measuring the light emission in aliquots of the transfected cells incubated with 5 µM coelenterazine for 8 min. In the same cells stimulated with PBS, basal BRET ratio was calculated as the ratio of the light emitted by GFP¹⁰ (510–540 nm) over the light emitted by Rluc8 (370–450 nm). C. BRET was measured in HEK293 cells co-expressing Gα_q-Rluc8 together with GFP¹⁰-Gγ₂ and Gβ₁ in the presence of WT (left) or E129V (right) mutant of the human TP receptor and stimulated with increasing concentrations of the indicated full and partial agonists. Results are the differences in the BRET signal measured in the presence and the absence of agonists, and are expressed as the mean value±SE of at least two independent determinations.</p