The field of pharmacogenetics, the investigation of the influence of one or more sequence variants on drug response phenotypes, is a special case of pharmacogenomics, a discipline that takes a genome-wide approach. Massively parallel, next generation sequencing (NGS), has allowed pharmacogenetics to be subsumed by pharmacogenomics with respect to the identification of variants associated with responders and non-responders, optimal drug response, and adverse drug reactions. A plethora of rare and common naturally-occurring GPCR variants must be considered in the context of signals from across the genome. Many fundamentals of pharmacogenetics were established for G protein-coupled receptor (GPCR) genes because they are primary targets for a large number of therapeutic drugs. Functional studies, demonstrating likely-pathogenic and pathogenic GPCR variants, have been integral to establishing models used for in silico analysis. Variants in GPCR genes include both coding and non-coding single nucleotide variants and insertion or deletions (indels) that affect cell surface expression (trafficking, dimerization, and desensitization/downregulation), ligand binding and G protein coupling, and variants that result in alternate splicing encoding isoforms/variable expression. As the breadth of data on the GPCR genome increases, we may expect an increase in the use of drug labels that note variants that significantly impact the clinical use of GPCR-targeting agents. We discuss the implications of GPCR pharmacogenomic data derived from the genomes available from individuals who have been well-phenotyped for receptor structure and function and receptor-ligand interactions, and the potential benefits to patients of optimized drug selection. Examples discussed include the renin-angiotensin system in SARS-CoV-2 (COVID-19) infection, the probable role of chemokine receptors in the cytokine storm, and potential protease activating receptor (PAR) interventions. Resources dedicated to GPCRs, including publicly available computational tools, are also discussed.

Cysteinyl leukotrienes are proinflammatory mediators with a clinically established role in asthma and a human genetic and preclinical role in cardiovascular pathology. Given that cardiovascular disease has a critical inflammatory component, the aim of this work was to conduct an observational study to verify whether the use of a cysteinyl leukotriene antagonist, namely, montelukast, may protect asthmatic patients from a major cardiovascular event and, therefore, represent an innovative adjunct therapy to target an inflammatory component in cardiovascular disease. We performed an observational retrospective 3-year study on eight hundred adult asthmatic patients 18 years or older in Albania, equally distributed into two cohorts, exposed or nonexposed to montelukast usage, matched by age and gender according to information reported in the data collection. Patients with a previous history of myocardial infarction or ischemic stroke were excluded. In summary, 37 (4.6%) of the asthmatic patients, 32 nonexposed, and five exposed to montelukast suffered a major cardiovascular event during the 3-year observation period. All the cardiovascular events, in either group, occurred among patients with an increased cardiovascular risk. Our analyses demonstrate that, independent from gender, exposure to montelukast remained a significant protective factor for incident ischemic events (78% or 76% risk reduction depending on type of analysis). The event-free Kaplan–Meier survival curves confirmed the lower cardiovascular event incidence in patients exposed to montelukast. Our data suggest that there is a potential preventative role of montelukast for incident cardiac ischemic events in the older asthmatic population, indicating a comorbidity benefit of montelukast usage in asthmatics by targeting cysteinyl leukotriene-driven cardiac disease inflammation.

Introduction: Cysteinyl leukotrienes, namely LTC4, LTD4, and LTE4, are proinflammatory mediators of the 5-lipooxygenase (5-LO) pathway with an established role in asthma as well as genetic and preclinical evidence of a contribution to cardiovascular diseases (CVDs). Aims and Objectives: Considering that CVDs, particularly atherosclerosis, have a critical inflammatory component, the aim of this study was to delineate the potential protective role of a leukotriene antagonists (LTRA), montelukast, vs ischemic CV events. Methods: Eight hundreds asthmatic patients, 18 years or older, without a previous history of myocardial infarction or ischemic stroke were included in this three years retrospective observational study in Albania. Patients were classified in two cohorts, either exposed (10 mg daily for at least three months) or non exposed to montelukast, randomly matched by gender and age. Results: 37 (4.6%) of our asthmatic patients suffered a major CV event during the observation period. Simple and multiple Cox regression analyses revealed that despite age and ´CV risk´ are a significant independent predictor of CV events, exposure to montelukast remains a significant (HR= 0.11, p= 0.045) protective factor for incident ischemic events, independetly from gender. Event- free Kaplan Meier survival curves confirmed the lower incidence of CV events in asthmatic patients exposed to montelukast. Conclusions: The results show a potential role of LTRAs, in particular montelukast, in targeting inflammation and reducing the ischemic events in asthmatic patients.

Aim: Cysteinyl-Leukotrienes (cysteinyl-LTs) are arachidonic acid metabolites that are involved in different inflammatory conditions, including CV disorders. CysLT receptor antagonists (LTRAs) are commonly used in diverse inflammatory pulmonary pathologies such as asthma, chronic obstructive pulmonary disease (COPD), or allergic rhinitis. Data have shown, however, that LTRA can prevent atherosclerosis progression, and can play a protective role after cerebral ischemia. Montelukast is a potent CysLT1 antagonist approved in patients with asthma and allergic rhinitis. The aim of our study was to evaluate the possible role of montelukast in the prevention of myocardial infarction (MI) and ischemic stroke (IS) in asthmatic patients.

Recent structural data on GPCRs using a variety of spectroscopic approaches suggest that GPCRs adopt a dynamic conformational landscape, with ligands stabilizing subsets of these states to activate one or more downstream signaling effectors. A key outstanding question posed by this emerging dynamic structural model of GPCRs is what states, active, inactive, or intermediate are captured by the numerous crystal structures of GPCRs complexed with a variety of agonists, partial agonists, and antagonists. In the early nineties the discovery of inverse agonists and constitutive activity led to the idea that the active receptor state (R⁎) is an intrinsic property of the receptor itself rather than of the RG complex, eventually leading to the formulation of the cubic ternary complex model (CTC). Here, by a careful analysis of a series of data obtained with a number of mutants of the highly conserved E/DRY motif, we show evidences for the existence of all the receptor states theorized by the CTC, four 'uncoupled (R, R⁎ and HR and HR⁎), and, consequently four 'coupled' (RG, R⁎G, HRG and HR⁎G). The E/DRY motif located at the cytosolic end of transmembrane helix III of Class A GPCRs has been widely studied and analyzed because it forms a network of interactions believed to lock receptors in the inactive conformation (R), and, thus, to play a key role in receptor activation. Our conclusions are supported by recent crystal and NMR spectra, as well as by results obtained with two prototypical GPCRs using a new FRET technology that de-couples G protein binding to the receptor from signal transduction. Thus, despite its complexity and limitations, we propose that the CTC is a useful framework to reconcile pharmacological, biochemical and structural data.

Objectives: In the 1999s, COX-2 selective inhibitors (COXIBs) entered the market to limit the gastrointestinal (GI) side effects associated to the use of traditional non steroidal anti-inflammatory drugs (NSAIDs). However, several COXIBs were withdrawn due to their potential cardiovascular (CV) toxicity. Patients with high CV risk owing to ageing and/or comorbidity (diabetes, atherosclerosis) may take advantage from a new class of NSAIDs that integrate the advantages of COXIBs with a cardioprotective component involving antagonism of thromboxane A2 receptor (TP). In addition, these new compounds can block the activity of nonenzymatic product of fatty acid oxidation such as isoprostanes, enhanced during atherosclerosis. The aim of this study was to obtain new compounds with a balanced TP antagonist and COXIB activity.

Genetic variants associated with asthma pathogenesis and altered response to drug therapy are discussed. Many studies implicate polymorphisms in genes encoding the enzymes responsible for leukotriene synthesis and intracellular signaling through activation of seven transmembrane domain receptors, such as the cysteinyl leukotriene 1 (CYSLTR1) and 2 (CYSLTR2) receptors. The leukotrienes are polyunsaturated lipoxygenated eicosatetraenoic acids that exhibit a wide range of pharmacological and physiological actions. Of the three enzymes involved in the formation of the leukotrienes, arachidonate 5 lipoxygenase 5 (ALOX5), leukotriene C4 synthase (LTC4S), and leukotriene hydrolase (LTA4H) are all polymorphic. These polymorphisms often result in variable production of the CysLTs (LTC4, LTD4, and LTE4) and LTB4. Variable number tandem repeat sequences located in the Sp1-binding motif within the promotor region of the ALOX5 gene are associated with leukotriene burden and bronchoconstriction independent of asthma risk. A 444A > C SNP polymorphism in the LTC4S gene, encoding an enzyme required for the formation of a glutathione adduct at the C-6 position of the arachidonic acid backbone, is associated with severe asthma and altered response to the CYSLTR1 receptor antagonist zafirlukast. Genetic variability in the CysLT pathway may contribute additively or synergistically to altered drug responses. The 601 A > G variant of the CYSLTR2 gene, encoding the Met201Val CYSLTR2 receptor variant, is associated with atopic asthma in the general European population, where it is present at a frequency of ∼2.6%. The variant was originally found in the founder population of Tristan da Cunha, a remote island in the South Atlantic, in which the prevalence of atopy is approximately 45% and the prevalence of asthma is 36%. In vitro work showed that the atopy-associated Met201Val variant was inactivating with respect to ligand binding, Ca2+ flux and inositol phosphate generation. In addition, the CYSLTR1 gene, located at Xq13-21.1, has been associated with atopic asthma. The activating Gly300Ser CYSLTR1 variant is discussed. In addition to genetic loci, risk for asthma may be influenced by environmental factors such as smoking. The contribution of CysLT pathway gene sequence variants to atopic asthma is discussed in the context of other genes and environmental influences known to influence asthma.

Thromboxane A2 is a potent mediator of inflammation and platelet aggregation exerting its effects through the activation of a G protein-coupled receptor (GPCR), termed TP. Although the existence of dimers/oligomers in Class A GPCRs is widely accepted, their functional significance still remains controversial. Recently, we have shown that TPα and TPβ homo-/hetero-dimers interact through an interface of residues in transmembrane domain 1 (TM1) whose disruption impairs dimer formation. Here, biochemical and pharmacological characterization of this dimer deficient mutant (DDM) in living cells indicates a significant impairment in its response to agonists. Interestingly, two single loss-of-function TPα variants, namely W29C and N42S recently identified in two heterozygous patients affected by bleeding disorders, match some of the residues mutated in our DDM. These two naturally occurring variants display a reduced potency to TP agonists and are characterized by impaired dimer formation in transfected HEK-293T cells. These findings provide proofs that lack of homo-dimer formation is a crucial process for reduced TPα function in vivo, and might represent one molecular mechanism through which platelet TPα receptor dysfunction affects the patient(s) carrying these mutations.

Purpose: Patients with high cardiovascular risk due to ageing and/or comorbidity (diabetes, atherosclerosis) that require effective management of chronic pain may take advantage from new non-steroidal anti-inflammatory drugs (NSAIDs) that at clinical dosages may integrate the anti-inflammatory activity and reduced gastrointestinal side effects of selective cyclooxygenase-2 (COX-2) inhibitor (coxib) with a cardioprotective component involving antagonism of thromboxane A(2) prostanoid (TP) receptor.Methods: New compounds were obtained modulating the structure of the most potent coxib, lumiracoxib, to obtain novel multitarget NSAIDs endowed with balanced coxib and TP receptor antagonist properties. Antagonist activity at TP receptor (pA(2)) was evaluated for all compounds in human platelets and in an heterologous expression system by measuring prevention of aggregation and Gq-dependent production of intracellular inositol phosphate induced by the stable thromboxane A(2) (TXA(2)) agonist U46619. COX 1 and COX-2 inhibitory activities were assessed in human washed platelets and lympho-monocytes suspension, respectively. COX selectivity was determined from dose-response curves by calculating a ratio (COX-2/COX-1) of IC50 values.Results: The tetrazole derivative 18 and the trifluoromethan sulfonamido-isoster 20 were the more active antagonists at TP receptor, preventing human platelet aggregation and intracellular signalling, with pA(2) values statistically higher from that of lumiracoxib. Comparative data regarding COX-2/COX-1 selectivity showed that while compounds 18 and 7 were rather potent and selective COX-2 inhibitor, compound 20 was somehow less potent and selective for COX-2.Conclusion: These results indicate that compounds 18 and 20 are two novel combined TP receptor antagonists and COX-2 inhibitors characterized by a fairly balanced COX-2 inhibitor activity and TP receptor antagonism and that they may represent a first optimization of the original structure to improve their multitarget activity. (C) 2015 Elsevier Ltd. All rights reserved.

We evaluated the autocrine activities of cysteinyl leukotrienes (cysteinyl-LTs) in HUVEC and studied the signaling and the pharmacological profile of the CysLT2 receptor (CysLT2R) expressed by ECs, finally assessing the role of the CysLT2R in permeability alterations in a model of isolated brain. Cysteinyl-LTs and their precursor LTA4 contracted HUVEC and increased permeability to macromolecules, increasing the formation of stress fibers through the phosphorylation of myosin light-chain (MLC) following Rho and PKC activation. Accordingly, in an organ model of cerebral vasculature with an intact intima, neutrophils challenge leaded to significant formation of cysteinyl-LTs and edema. Pretreatment with a selective CysLT2R antagonist prevented cytoskeleton rearrangement and HUVEC contraction, along with edema formation in the brain preparation, while leaving the synthesis of cysteinyl-LTs unaffected. We also demonstrate here that the CysLT1R antagonist zafirlukast, pranlukast, pobilukast and iralukast also possess CysLT2R antagonistic activity, which could help in reconsidering previous data on the role of cysteinyl-LTs in the cardiovascular system. The results obtained are further supporting a potential role for CysLT2R in cardiovascular disease.

Pharmacogenetics investigates the influence of genetic variants on physiological phenotypes related to drug response and disease, while pharmacogenomics takes a genome-wide approach to advancing this knowledge. Both play an important role in identifying responders and nonresponders to medication, avoiding adverse drug reactions, and optimizing drug dose for the individual. G protein-coupled receptors (GPCRs) are the primary target of therapeutic drugs and have been the focus of these studies. With the advance of genomic technologies, there has been a substantial increase in the inventory of naturally occurring rare and common GPCR variants. These variants include single-nucleotide polymorphisms and insertion or deletions that have potential to alter GPCR expression of function. In vivo and in vitro studies have determined functional roles for many GPCR variants, but genetic association studies that define the physiological impact of the majority of these common variants are still limited. Despite the breadth of pharmacogenetic data available, GPCR variants have not been included in drug labeling and are only occasionally considered in optimizing clinical use of GPCR-targeted agents. In this chapter, pharmacogenetic and genomic studies on GPCR variants are reviewed with respect to a subset of GPCR systems, including the adrenergic, calcium sensing, cysteinyl leukotriene, cannabinoid CB1 and CB2 receptors, and the de-orphanized receptors such as GPR55. The nature of the disruption to receptor function is discussed with respect to regulation of gene expression, expression on the cell surface (affected by receptor trafficking, dimerization, desensitization/downregulation), or perturbation of receptor function (altered ligand binding, G protein coupling, constitutive activity). The large body of experimental data generated on structure and function relationships and receptor-ligand interactions are being harnessed for the in silico functional prediction of naturally occurring GPCR variants. We provide information on online resources dedicated to GPCRs and present applications of publically available computational tools for pharmacogenetic studies of GPCRs. As the breadth of GPCR pharmacogenomic data becomes clearer, the opportunity for routine assessment of GPCR variants to predict disease risk, drug response, and potential adverse drug effects will become possible.

Today COXIBs are used in the treatment of arthritis and many other painful conditions in selected patients with high gastrointestinal risk and low cardiovascular (CV) risk. Previously, we have identified an unexpected mechanism of action of a traditional non-steroidal anti-inflammatory drug (NSAID) (diclofenac) and a specific inhibitor of cyclooxygenase-2 (COXIB) (lumiracoxib) demonstrating that they possess weak competitive antagonism at the thromboxane receptor (TP). We hypothesize that modifying the structure of a known COXIB so that it becomes also a more potent TP antagonist will preserve the anti-inflammatory and gastrointestinal safety typical of COXIBs and prevent the CV risk associated with long term therapy. Keywords—Cyclooxygenase, inflammation, lumiracoxib, thromboxane A2.

Platelets play a central role not only in physiological hemostasis and repairing vascular damage, but also within the atherosclerotic process as well as in vascular occlusion and thrombosis, crucial events in myocardial infarction and stroke. They interact with activated endothelium, undergo chemotaxis, release adhesive proteins, vasoactive substances and pro-inflammatory mediators, activate other inflammatory cells, and exert phagocytosis 1. Platelets express a wide range of receptors and proteins, and among them P2Y1 and P2Y12, receptors for adenosine diphosphate (ADP), are necessary for platelet activation, aggregation and secretion 2. In particular, the P2Y12 receptor, a member of the rhodopsin family (class A, purine receptor cluster within the δ group) of the G protein-coupled receptor (GPCR) gene superfamily, has attracted a significant amount of attention. In this issue of the Journal of Thrombosis and Haemostasis, Patel and colleagues have identified a patient with a lifelong history of chronic bleeding disorder expressing a homozygous P2RY12 gene mutation, resulting in an Arg to Cys substitution (R122C) in the P2Y12 receptor sequence accompanied by a single nucleotide polymorphism (SNP) of the F2R gene encoding the PAR-1 receptor for thrombin. Because this latter intronic polymorphism has been previously associated with decreased receptor expression and reduced PAR-1 activity 3, it has not been studied further. Platelets from this patient (P1) showed lack of ADP- and PAR-1-induced aggregation responses, as well as ADP-induced pVASP phosphorylation, whereas platelets from her two sons (P2 and P3), heterozygous for the same missense mutation in the P2R12 gene, showed a reduced response, particularly at low ADP concentrations. Interestingly, binding studies in platelets from these patients showed a decrease in affinity for the P2Y12 agonist 2-MeSADP and in surface receptor expression compared with healthy donors (HD) 4. Accordingly, in vitro studies with cells stably expressing the R122C receptor variant confirmed significantly impairment of receptor activity compared with WT P2Y12, when cells with similar levels of receptor expression were skillfully selected, and no increase in basal activity. These features point to a ‘loss of function’ receptor variant likely due to an impairment of G protein coupling. It is worth noticing here that the R122 of the P2Y12 receptor, despite not being the only Arg associated with impairment of P2Y12 receptor function 5, corresponds to the central Arg (i.e. R3.50, using Ballesteros notation) of the highly conserved Glu/Asp-Arg triplet located at the boundary between transmembrane domain (TM) III and intracellular loop (ICL) 2 of class A GPCRs, the so called E/DRY motif, that has been shown to be involved in GPCR activation and/or G protein coupling 6. To date, only two other class A GPCRs have been reported to have different natural occurring mutations in the central Arg of this motif and to be linked to clinically identified syndromes, the vasopressin V2 7, 8 and the gonadotropin-releasing hormone receptors GnRH 9, 10. While both mutations of the latter receptor (R139H/C) result in hypogonadotropic hypogonadism due to lack of agonist-induced response, the V2 receptor situation is somehow more complicated, leading to two different diseases with opposite clinical outcome: R137H leading to nephrogenic diabetes insipidus (NDI) 7, while R137C/L leads to nephrogenic syndrome of inappropriate antidiuresis (NSIAD) 8. Interestingly, while NDI is likely to be due to an elevated R137H endocytosis, R137C/L variants are characterized by an elevated basal agonist-independent signal 11. Indeed, the consensus picture derived mostly from the studies on α1B and β2 adrenergic (AR) receptors is that the basic Arg forms a stabilizing intrahelical salt-bridge with the neighboring Asp or Glu (E/D3.49), and interhelical hydrogen bonds with another charged residue on helix 6 (E6.30), thereby constraining GPCRs in the inactive (R) conformation 12, 13. Receptor activation is therefore anticipated to disrupt this network by unlashing constraints imposed on the two helices. Accordingly, non-conservative mutation of the E/D3.49 or of the R3.50 of the E/DRY motif (and/or of the E6.30 in TM VI) resulted in the ability of some GPCRs to adopt an active (R*) conformation in the absence of agonist stimulation (i.e. to become constitutively active) 14. However, for a number of class A GPCRs, including the GnRH receptor 15, 16, mutations in the E/D3.49 residues do not induce an increase in constitutive activity (CA), while mutations of the R3.50 resulted in a loss of function phenotype likely due to a loss of G protein coupling 6. Therefore, disruption of this network might be necessary but not sufficient to achieve full receptor activation, leading us to classify these receptors as Constitutively Inactive Mutant (CIM) 6. These assumptions have been recently confirmed by the crystal structure of opsin in its G-protein-interacting conformation 17, clearly demonstrating the importance of R3.50 in the formation of the receptor-G protein complex. This structure also clearly shows a significant rearrangement of other receptor regions beyond the disruption of the E/DRY network, suggesting that its neutralization might be only one of multiple constraints that must be ‘unlashed’ to achieve full receptor activation. Interestingly, R122C P2Y12 is the second platelet receptor described to share this behavior, the first one being the thromboxane prostanoid (TP) receptor, another class A GPCR coupled to several G proteins (promiscuous receptor), including Gq 18. The R130V mutant TP receptor was characterized by a significantly blunted agonist response, no increase in basal activity, loss of high affinity agonist binding and uncoupling from its cognate G protein 19. Accordingly, substitutions in position E3.49/E6.30 have been shown not to induce a surge in CA 20, 21. Therefore, even though a careful analysis of the signaling effects of non-conservative substitutions in position D3.49 will be necessary to fully characterize R122C P2Y12 as a CIM, it appears to possess many of their characteristics. It is thus conceivable that both P2Y12 4 and TP receptors 19-21 have developed a large activation energy barrier, consistent with their relevance in the homeostasis of the cardiovascular system 18, 22, while other receptors (such as β2-AR) might have a low energy barrier between the R and R* states 14. It is tempting to speculate here that this behavior, common to many other GPCRs, is the result of evolutionary forces that may have selected regulatory mechanisms against more highly active phenotypes that might not be compatible with a physiological milieu. The authors state that they have no conflict of interests.

The activation of thromboxane prostanoid (TP) receptor on platelets, monocytes/macrophages, endothelial cells, and vascular smooth muscle cells (SMC) plays important roles in regulating platelet activation andvascular tone and in the pathogenesis of thrombosis and vascular inflammation. Oxidative stress and vascular inflammation increase the formation of TP receptor agonists, which promote initiation and progression of atherogenesis and thrombosis. Furthermore, TP receptor activation promotes angiogenesis and vessel wall constriction. Besides thromboxane A(2) and its endoperoxide precursors, prostaglandin G(2) and H-2, isoprostanes, and 20-hydroxyeicosatetraenoic acid also activate TP receptor as autocrine or paracrine ligands. These additional TP activators play a role in pathological conditions such as diabetes, obesity, and hypertension, and their biosynthesis is not inhibited by aspirin, at variance with that of thromboxane A(2). The understanding of TP receptor function increased our current knowledge of the pathogenesis of atherosclerosis and thrombosis, highlighting the great impact that this receptor has in cardiovascular disorders.

Background: The concept of permanent narrowing of the airways resulting from chronic inflammation and fibrosis is called remodeling and is a common feature of asthma and chronic obstructive pulmonary disease (COPD). The eicosanoid contractile agents thromboxane A(2) (TXA(2)) and cysteinyl-leukotriene D-4 (LTD4) are among the recognized mitogens for human airway smooth muscle (ASM) cells. Statins are known to possess anti-inflammatory and immunomodulatory properties that are independent on their cholesterol-lowering effects and may result in clinical lung benefits. Rosuvastatin is the last agent of the lipid-lowering drugs to be introduced and experimental evidence indicates that it possess favorable pleiotropic effects in the cardiovascular and nervous systems. Yet, no data is available in the literature regarding its effects on human airway remodeling. The present study was aimed at examining the effect of rosuvastatin and the involvement of prenylated proteins in the response of human ASM cells to serum, epidermal growth factor (EGF) and eicosanoid contractile mitogens that activate TxA(2) prostanoid and LTD4 receptors.Methods: Cell growth was assessed by nuclear incorporation of [H-3]thymidine in human ASM cells serum-starved and then stimulated for 48 h in MEM plus 0.1% BSA containing mitogens in the absence and presence of modulators of the mevalonate and prenylation pathways.Results: We found that rosuvastatin dose-dependently inhibited serum-, EGF-, the TxA(2) stable analog U46619-, and LTD4-induced human ASM cells growth. All these effects were prevented by pretreatment with mevalonate. Addition of the prenylation substrates farnesol and geranylgeraniol reversed the effect of rosuvastatin on EGF and U46619, respectively. Interestingly, only mevalonate showed restoration of cell growth following rosuvastatin treatment in LTD4 and LTD4 plus EGF treated cells, suggesting a possible involvement of both farnesylated and geranylgeranylated proteins in the cysteinyl-LT-induced cell growth.Conclusions: The hydrophilic statin rosuvastatin exerts direct effects on human ASM cells mitogenic response in vitro by inhibiting prenylation of signaling proteins, likely small G proteins. These findings are consistent with previous observed involvement of small GTPase signaling in EGF- and U46619-induced human airway proliferation and corroborate the recent interest in the potential clinical benefits of statins in asthma/COPD. (C) 2013 Elsevier Ltd. All rights reserved.

The intrahelical salt bridge between E/D3.49 and R3.50 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 E3.49/6.30 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.

Eicosanoids are biologically active lipids in both physiologic and pathophysiologic situations. These mediators rapidly generate at sites of inflammation and act through specific receptors that following the generation of a signal transduction cascade, lead to coordinated cellular responses to specific stimuli. Prostanoids, that is, prostaglandins and thromboxane A(2), are active products of the cyclooxygenase pathway, while leukotrienes and lipoxins derive from the lipoxygenase pathway. In addition, a complex family of prostaglandin isomers called isoprostanes is derived as free-radical products of oxidative metabolism. While there is a wide consensus on the importance of the balance between proaggregating (thromboxane A(2)) and antiaggregating (prostacyclin) cyclooxygenase products in cardiovascular homeostasis, an increasing body of evidence suggests a key role also for other eicosanoids generated by lipoxygenases, epoxygenases, and nonenzymatic pathways in cardiovascular diseases. This intricate network of lipid mediators is unique considering that from a single precursor, arachidonic acid, may derive an array of bioproducts that interact within each other synergizing or, more often, behaving as functional antagonists.