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, mt 1 and MT 2 , the existence of a novel putative melatonin receptor, MT 3 , 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 mt 1 and MT 2 . In this report, we present the purification of MT 3 from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR 2 , EC 1.6.99.2). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT 3 to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR 2 . After transfection of CHO-K1 cells with the human QR 2 gene, not only did the QR 2 enzymatic activity appear, but also the melatonin-binding sites with MT 3 characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data from MT 3 binding and QR 2 enzymatic activity obtained from samples of Syrian hamster kidney or QR 2 -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 site MT 3 as the quinone reductase QR 2 and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties.Melatonin, a neurohormone produced at night in the pineal gland, is suspected to relay to the peripheral organs the circadian rhythm detected by the central nervous system. Several high affinity melatonin receptors have been identified to date, among which the mt 1 (1) and MT 2 (2) receptors have been cloned from human tissues. The pharmacology of these two receptors is well documented, and several compounds, including melatonin, are ligands with picomolar binding affinity (for review, see Ref.3). Another putative melatonin receptor was identified on pharmacological grounds, with lower melatonin affinity (nanomolar range), very rapid ligand association/dissociation kinetics, and an original pharmacological profile (4 -6). In line with mt 1 and MT 2 receptors, this putative receptor was named MT 3 , according to the nomenclature recommendations of the IUPHAR (7). So far, the known inhibitors of MT 3 hardly reach the nanomolar range and encompass an unusually large structural diversity of highly hydrophobic cyclic or polycyclic compounds (Refs. 5 and 6, and for review, see Ref.3).1 All pharmacological investigations on mt 1 , MT 2 , and MT 3 were performed using the radioligand [125 I]melatonin, a ligand with high affinity for mt 1 and MT 2 (K d ϭ 10 -200 pM) and with lower affinity for MT 3 (K d ϭ 3-9 nM). The hamster kidney, liver, and brain have been used as model tissues for MT 3 pharmacological studies, and our recent data con...

Because little comparative information is available concerning receptor profiles of antiparkinson drugs, affinities of 14 agents were determined at diverse receptors implicated in the etiology and/or treatment of Parkinson's disease: human (h)D 1 , hD 2S , hD 2L , hD 3 , hD 4 , and hD 5 receptors; human 5-hydroxytryptamine (5-HT) 1A , h5-HT 1B , h5-HT 1D , h5-HT 2A , h5-HT 2B , and h5-HT 2C receptors; h␣ 1A -, h␣ 1B -, h␣ 1D -, h␣ 2A -, h␣ 2B -, h␣ 2C -, rat ␣ 2D -, h␤ 1 -, and h␤ 2 -adrenoceptors (ARs); and native histamine 1 receptors. A correlation matrix (294 pK i values) demonstrated substantial "covariance". Correspondingly, principal components analysis revealed that axis 1, which accounted for 76% variance, was associated with the majority of receptor types: drugs displaying overall high versus modest affinities migrated at opposite extremities. Axis 2 (7% of variance) differentiated drugs with high affinity for hD 4 and H 1 receptors versus h␣ 1 -AR subtypes. Five percent of variance was attributable to axis 3, which distinguished drugs with marked affinity for h␤ 1 -and h␤ 2 -ARs versus hD 5 and 5-HT 2A receptors. Hierarchical (cluster) analysis of global homology generated a dendrogram differentiating two major groups possessing low versus high affinity, respectively, for multiple serotonergic and hD 5 receptors. Within the first group, quinpirole, quinerolane, ropinirole, and pramipexole interacted principally with hD 2 , hD 3 , and hD 4 receptors, whereas piribedil and talipexole recognized dopaminergic receptors and h␣ 2 -ARs. Within the second group, lisuride and terguride manifested high affinities for all sites, with roxindole/bromocriptine, cabergoline/pergolide, and 6,7-dihydroxy-N,N-dimethyl-2-ammotetralin (TL99)/apomorphine comprising three additional subclusters of closely related ligands. In conclusion, an innovative multivariate analysis revealed marked heterogeneity in binding profiles of antiparkinson agents. Actions at sites other than hD 2 receptors likely participate in their (contrasting) functional profiles.

N-myristoylation is an acylation process absolutely specific to the N-terminal amino acid glycine in proteins. This maturation process concerns about a hundred proteins in lower and higher eukaryotes involved in oncogenesis, in secondary cellular signalling, in infectivity of retroviruses and, marginally, of other virus types. Thy cytosolic enzyme responsible for this activity, N-myristoyltransferase (NMT), studied since 1987, has been purified from different sources. However, the studies of the specificities of the various NMTs have not progressed in detail except for those relating to the yeast cytosolic enzyme. Still to be explained are differences in species specificity and between various putative isoenzymes, also whether the data obtained from the yeast enzyme can be transposed to other NMTs. The present review discusses data on the various addressing processes subsequent to myristoylation, a patchwork of pathways that suggests myristoylation is only the first step of the mechanisms by which a protein associates with the membrane. Concerning the enzyme itself, there are evidences that NMT is also present in the endoplasmic reticulum and that its substrate specificity is different from that of the cytosolic enzyme(s). These differences have major implications for their differential inhibition and for their respective roles in several pathologies. For instance, the NMTs from mammalians are clearly different from those found in several microorganisms, which raises the question whether the NMT may be a new targets for fungicides. Finally, since myristoylation has a central role in virus maturation and oncogenesis, specific NMT inhibitors might lead to potent antivirus and anticancer agents.

The principal soy phytoestrogen genistein has an array of biological actions. It binds to estrogen receptor (ER) ␣ and ␤ and has ER-mediated estrogenic effects. In addition, it has antiestrogenic effects as well as non-ERmediated effects such as inhibition of tyrosine kinase. Because of its complex biological actions, the molecular mechanisms of action of genistein are poorly understood. Here we show that genistein dose-dependently increases estrogenic transcriptional activity in mesenchymal progenitor cells, but its biological effects on osteogenesis and adipogenesis are different. At low concentrations (<1 M), genistein acts as estrogen, stimulating osteogenesis and inhibiting adipogenesis. At high concentrations (>1 M), however, genistein acts as a ligand of PPAR␥, leading to up-regulation of adipogenesis and down-regulation of osteogenesis.Transfection experiments show that activation of PPAR␥ by genistein at the micromolar concentrations down-regulates its estrogenic transcriptional activity, while activation of ER␣ or ER␤ by genistein downregulates PPAR␥ transcriptional activity. Genistein concurrently activates two different transcriptional factors, ERs and PPAR␥, which have opposite effects on osteogenesis or adipogenesis. As a result, the balance between activated ERs and PPAR␥ determines the biological effects of genistein on osteogenesis and adipogenesis. Our findings may explain distinct effects of genistein in different tissues.