Jean A. Boutin

Identification of the melatonin-binding site MT3 as the quinone reductase 2.

The regulation of the circadian rhythm is relayed from the central nervous system to the periphery by melatonin, a hormone synthesized at night in the pineal gland. Besides two melatonin G-coupled receptors, mt1 and MT2, the existence of a novel putative melatonin receptor,MT3 , was hypothesized from the observation of a binding site in both central and peripheral hamster tissues with an original binding profile and a very rapid kinetics of ligand exchange compared with mt1 and MT2. In this report, we present the purification of MT3 from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR2, EC 1.6.99.2). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT3 to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR2. After transfection of CHO-K1 cells with the human QR2 gene, not only did the QR2 enzymatic activity appear, but also the melatonin-binding sites with MT3 characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data fromMT3 binding and QR2 enzymatic activity obtained from samples of Syrian hamster kidney or QR2-overexpressing Chinese hamster ovary cells, and observed an overall good correlation of the data. In summary, our results provide the identification of the melatonin-binding siteMT3 as the quinone reductase QR2and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties.

New selective ligands of human cloned melatonin MT1 and MT2 receptors

Melatonin has a key role in the circadian rhythm relay to periphery organs. Melatonin exerts its multiple roles mainly through two seven transmembrane domain, G-coupled receptors, namely MT1 or MT2 receptors. A pharmacological characterization of these human cloned melatonin hMT1 and hMT2 receptors stably expressed in HEK-293 or CHO cells is presented using a 2-[125I]-iodo-melatonin binding assay and a [35S]-GTPγS functional assay. Both reference compounds and new chemically diverse ligands were evaluated. Binding affinities at each receptor were found to be comparable on either HEK-293 or CHO cell membranes. Novel non-selective or selective hMT1 and hMT2 ligands are described. The [35S]-GTPγS functional assay was used to define the functional activity of these compounds which included partial, full agonist and/or antagonist activity. None of the compounds acted as an inverse agonist. We report new types of selective antagonists, such as S 25567 and S 26131 for MT1 and S 24601 for MT2. These studies brought other new molecular tools such as the selective MT1 agonist, S 24268, as well as the non-selective antagonist, S 22153. Finally, we also discovered S 25150, the most potent melatonin receptor agonist, so far reported in the literature.

Melatonin receptors, heterodimerization, signal transduction and binding sites: what's new?

Melatonin is a neurohormone that has been claimed to be involved in a wide range of physiological functions. Nevertheless, for most of its effects, the mechanism of action is not really known. In mammals, two melatonin receptors, MT1 and MT2, have been cloned. They belong to the G‐protein‐coupled receptor (GPCR) superfamily. They share some specific short amino‐acid sequences, which suggest that they represent a specific subfamily. Another receptor from the same subfamily, the melatonin‐related receptor has been cloned in different species including humans. This orphan receptor also named GPR50 does not bind melatonin and its endogenous ligand is still unknown. Nevertheless, this receptor has been shown to behave as an antagonist of the MT1 receptor, which opens new pharmacological perspectives for GPR50 despite the lack of endogenous or synthetic ligands. Moreover, MT1 and MT2 interact together through the formation of heterodimers at least in cells transfected with the cDNA of these two receptors. Lastly, signalling complexes associated with MT1 and MT2 receptors are starting to be deciphered. A third melatonin‐binding site has been purified and characterized as the enzyme quinone reductase 2 (QR2). Inhibition of QR2 by melatonin may explain melatonins protective effect that has been reported in different animal models and that is generally associated with its well‐documented antioxidant properties.

Autotaxin Is Released from Adipocytes, Catalyzes Lysophosphatidic Acid Synthesis, and Activates Preadipocyte Proliferation UP-REGULATED EXPRESSION WITH ADIPOCYTE DIFFERENTIATION AND OBESITY

Our group has recently demonstrated (Gesta, S., Simon, M., Rey, A., Sibrac, D., Girard, A., Lafontan, M., Valet, P., and Saulnier-Blache, J. S. (2002) J. Lipid Res. 43, 904–910) the presence, in adipocyte conditioned-medium, of a soluble lysophospholipased-activity (LPLDact) involved in synthesis of the bioactive phospholipid lysophosphatidic acid (LPA). In the present report, LPLDact was purified from 3T3F442A adipocyte-conditioned medium and identified as the type II ecto-nucleotide pyrophosphatase phosphodiesterase, autotaxin (ATX). A unique ATX cDNA was cloned from 3T3F442A adipocytes, and its recombinant expression in COS-7 cells led to extracellular release of LPLDact. ATX mRNA expression was highly up-regulated during adipocyte differentiation of 3T3F442A-preadipocytes. This up-regulation was paralleled by the ability of newly differentiated adipocytes to release LPLDact and LPA. Differentiation-dependent up-regulation of ATX expression was also observed in a primary culture of mouse preadipocytes. Treatment of 3T3F442A-preadipocytes with concentrated conditioned medium from ATX-expressing COS-7 cells led to an increase in cell number as compared with concentrated conditioned medium from ATX non-expressing COS-7 cells. The specific effect of ATX on preadipocyte proliferation was completely suppressed by co-treatment with a LPA-hydrolyzing phospholipase, phospholipase B. Finally, ATX expression was found in mature adipocytes isolated from mouse adipose tissue and was substantially increased in genetically obese-diabeticdb/db mice when compared with their lean siblings. In conclusion, the present work shows that ATX is responsible for the LPLDact released by adipocytes and exerts a paracrine control on preadipocyte growth via an LPA-dependent mechanism. Up-regulations of ATX expression with adipocyte differentiation and genetic obesity suggest a possible involvement of this released protein in the development of adipose tissue and obesity-associated pathologies.

Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis.

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC 2.3.1.87). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN → RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the Km for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.

Comparative pharmacological studies of melatonin receptors: MT1, MT2 and MT3/QR2. Tissue distribution of MT3/QR2 .

The neurohormone melatonin is the central switch of the circadian rhythm and presumably exerts its activities through a series of receptors among which MT1 and MT2 have been widely studied. The third binding site of melatonin, MT3, has been recently characterized as a melatonin-sensitive form of the quinone reductase 2 (QR2, EC 1.6.99.2). In the present work, we showed that the binding of melatonin at MT3/QR2 was better described with 2-[125I]-iodomethoxy-carbonylamino-N-acetyltryptamine (2-[125I]-I-MCA-NAT) and, most importantly, that it was measurable at 20 degrees while it has been initially described and thoroughly studied using 2-[125I]-iodomelatonin at 4 degrees. Under these novel conditions, binding to MT3 could be traced without cross-reactivity with MT1 and MT2 receptors and, moreover, under conditions similar to those used to measure MT3/QR2 catalytic activity. The pharmacology established here on hamster kidney samples using the reference compounds remained essentially as already described using other experimental conditions. A new series of compounds with nanomolar affinity for the MT3 binding site and a high MT3 selectivity versus MT1 and MT2 is reported. In addition, we further document the MT3/QR2 binding site by demonstrating that it was widely distributed among mammals, although inter-species and inter-tissues differences exist. The present report details new experimental conditions for the pharmacological study of melatonin-sensitive QR2 isoforms, and suggests that, in addition to an already demonstrated inter-species difference, inter-tissues differences in QR2 sensitivity to melatonin may exist in primates and, therefore, represent an original and interesting route of investigation on the effect of melatonin on MT3/QR2.

Agonist and antagonist actions of yohimbine as compared to fluparoxan at ?2-adrenergic receptors (AR)s, serotonin (5-HT)1A, 5-HT1B, 5-HT1D and dopamine D2 and D3 receptors. Significance for the modulation of frontocortical monoaminergic transmission and depressive states

Herein, we evaluate the interaction of the α2‐AR antagonist, yohimbine, as compared to fluparoxan, at multiple monoaminergic receptors and examine their roles in the modulation of adrenergic, dopaminergic and serotonergic transmission in freely‐moving rats. Yohimbine displays marked affinity at human (h)α2A‐, hα2B‐ and hα2C‐ARs, significant affinity for h5‐HT1A, h5‐HT1B, h5‐HT1D, and hD2 receptors and weak affinity for hD3 receptors. In [35S]GTPγS binding protocols, yohimbine exerts antagonist actions at hα2A‐AR, h5‐HT1B, h5‐HT1D, and hD2 sites, yet partial agonist actions at h5‐HT1A sites. In vivo, agonist actions of yohimbine at 5‐HT1A sites are revealed by WAY100,635‐reversible induction of hypothermia in the rat. In guinea pigs, antagonist actions of yohimbine at 5‐HT1B receptors are revealed by blockade of hypothermia evoked by the 5‐HT1B agonist, GR46,611. In distinction to yohimbine, fluparoxan shows only modest partial agonist actions at h5‐HT1A sites versus marked antagonist actions at hα2‐ARs. While fluparoxan selectively enhances hippocampal noradrenaline (NAD) turnover, yohimbine also enhances striatal dopamine (DA) turnover and suppresses striatal turnover of 5‐HT. Further, yohimbine decreases firing of serotonergic neurones in raphe nuclei, an action reversed by WAY100,635. Fluparoxan increases extracellular levels of DA and NAD, but not 5‐HT, in frontal cortex. In analogy, yohimbine enhances FCX levels of DA and NAD, yet suppresses those of 5‐HT, the latter effect being antagonized by WAY100,635. The induction by fluoxetine of FCX levels of 5‐HT, DA, and NAD is potentiated by fluparoxan. Yohimbine likewise facilitates the influence of fluoxetine upon DA and NAD levels, but not those of 5‐HT. In conclusion, the α2‐AR antagonist properties of yohimbine increase DA and NAD levels both alone and in association with fluoxetine. However, in contrast to the selective α2‐AR antagonist, fluparoxan, the 5‐HT1A agonist actions of yohimbine suppress 5‐HT levels alone and underlie its inability to augment the influence of fluoxetine upon 5‐HT levels. Synapse 35:79–95, 2000.

Natural ligands of PPARγ:: Are prostaglandin J2 derivatives really playing the part?

Abstract The peroxisome proliferator-activated receptor (PPAR) family was discovered from an orphan nuclear receptor approach, and thereafter, three subtypes were identified, namely PPARα, PPARβ or PPARδ and PPARγ. The two former seem to regulate lipid homeostasis, whereas the latter is involved, among others, in glucose homeostasis and adipocyte differentiation. PPARs were pharmacologically characterised first using peroxisome proliferators such as clofibrates, which demonstrate moderate affinity (efficiency at micromolar concentrations) and low PPARα/δ versus PPARγ specificity. Hence, several laboratories have started the search for potent and subtype-specific natural PPAR activators. In this respect, prostaglandin (PG)-related compounds were identified as good PPARγ agonists with varying specificity, the most notable PPARγ ligand being 15-deoxy-Δ12–14-PGJ2 (15d-PGJ2). Recently, an oxidized phosphatidylcholine was identified as a potent alternative (patho)physiological natural ligand of PPARγ. In the present review, we discuss the different PPARγ-dependent and -independent biological effects of the PG PPARγ ligands and the concern about their low potency in molecular models as compared with thiazolidinediones (TZDs), a family of potent (nanomolar) synthetic PPARγ ligands. Finally, the oxidized lipids are presented as a novel and interesting alternative for discovering potent PPARγ activators in order to understand more in details the implications of PPARγ in various pathophysiological conditions.

Murine and human autotaxin alpha, beta, and gamma isoforms: gene organization, tissue distribution, and biochemical characterization.

Autotaxin is a type II ectonucleotide pyrophosphate phosphodiesterase enzyme. It has been recently discovered that it also has a lysophospholipase D activity. This enzyme probably provides most of the extracellular lysophosphatidic acid from lysophosphatidylcholine. The cloning and tissue distribution of the three isoforms (imaginatively called α, β, and γ) from human and mouse are reported in this study, as well as their tissue distribution by PCR in the human and mouse. The fate of the α isoform from human was also studied after purification and using mass spectrometry. Indeed, this particular isoform expresses the intron 12 in which a cleavage site is present, leading to a rapid catabolism of the isoform. For the human isoform γ and the total autotaxin mRNA expression, quantitative PCR is presented in 21 tissues. The isoforms were expressed in two different hosts, insect cells and Chinese hamster ovary cells, and were highly purified. The characteristics of the six purified isoforms (pH and temperature dependence, Km and Vmax values, and their dependence on metal ions) are presented in this study. Their sensitivity to a small molecule inhibitor, hypericin, is also shown. Finally, the specificity of the isoforms toward a large family of lysophosphatidylcholines is reported. This study is the first complete description of the reported autotaxin isoforms.Autotaxin is a type II ectonucleotide pyrophosphate phosphodiesterase enzyme. It has been recently discovered that it also has a lysophospholipase D activity. This enzyme probably provides most of the extracellular lysophosphatidic acid from lysophosphatidylcholine. The cloning and tissue distribution of the three isoforms (imaginatively called alpha, beta, and gamma) from human and mouse are reported in this study, as well as their tissue distribution by PCR in the human and mouse. The fate of the alpha isoform from human was also studied after purification and using mass spectrometry. Indeed, this particular isoform expresses the intron 12 in which a cleavage site is present, leading to a rapid catabolism of the isoform. For the human isoform gamma and the total autotaxin mRNA expression, quantitative PCR is presented in 21 tissues. The isoforms were expressed in two different hosts, insect cells and Chinese hamster ovary cells, and were highly purified. The characteristics of the six purified isoforms (pH and temperature dependence, K(m) and V(max) values, and their dependence on metal ions) are presented in this study. Their sensitivity to a small molecule inhibitor, hypericin, is also shown. Finally, the specificity of the isoforms toward a large family of lysophosphatidylcholines is reported. This study is the first complete description of the reported autotaxin isoforms.

S32826, A Nanomolar Inhibitor of Autotaxin: Discovery, Synthesis and Applications as a Pharmacological Tool

Autotaxin catalyzes the transformation of lyso-phosphatidylcholine in lyso-phosphatidic acid (LPA). LPA is a phospholipid possessing a large panel of activity, in particular as a motility factor or as a growth signal, through its G-protein coupled seven transmembrane receptors. Indirect evidence strongly suggests that autotaxin is the main, if not the only source of circulating LPA. Because of its central role in pathologic conditions, such as oncology and diabetes/obesity, the biochemical properties of autotaxin has attracted a lot of attention, but confirmation of its role in pathology remains elusive. One way to validate and/or confirm its central role, is to find potent and selective inhibitors. A systematic screening of several thousand compounds using a colorimetric assay and taking advantage of the phosphodiesterase activity of autotaxin that requires the enzymatic site than for LPA generation, led to the discovery of a potent nanomolar inhibitor, [4-(tetradecanoylamino)benzyl]phosphonic acid (S32826). This compound was inhibitory toward the various autotaxin isoforms, using an assay measuring the [14C]lyso-phosphatidylcholine conversion into [14C]LPA. We also evaluated the activity of S32826 in cellular models of diabesity and oncology. Nevertheless, the poor in vivo stability and/or bioavailability of the compound did not permit to use it in animals. S32826 is the first reported inhibitor of autotaxin with an IC50 in the nanomolar range that can be used to validate the role of autotaxin in various pathologies in cellular models.