Philippe Beauverger

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.

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.

Structure-activity relationship studies of melanin-concentrating hormone (MCH)-related peptide ligands at SLC-1, the human MCH receptor.

Melanin-concentrating hormone (MCH) is a cyclic nonadecapeptide involved in the regulation of feeding behavior, which acts through a G protein-coupled receptor (SLC-1) inhibiting adenylcyclase activity. In this study, 57 analogues of MCH were investigated on the recently cloned human MCH receptor stably expressed in HEK293 cells, on both the inhibition of forskolin-stimulated cAMP production and guanosine-5′-O-(3-[35S]thiotriphosphate ([35S]- GTPγS) binding. The dodecapeptide MCH-(6–17) (MCH ring between Cys7 and Cys16, with a single extra amino acid at the N terminus (Arg6) and at the C terminus (Trp17)) was found to be the minimal sequence required for a full and potent agonistic response on cAMP formation and [35S]- GTPγS binding. We Ala-scanned this dodecapeptide and found that only 3 of 8 amino acids of the ring, namely Met8, Arg11, and Tyr13, were essential to elicit full and potent responses in both tests. Deletions inside the ring led either to inactivity or to poor antagonists with potencies in the micromolar range. Cys7 and Cys16 were substituted by Asp and Lys or one of their analogues, in an attempt to replace the disulfide bridge by an amide bond. However, those modifications were deleterious for agonistic activity. In [35S]- GTPγS binding, these compounds behaved as weak antagonists (K B 1–4 μm). Finally, substitution in MCH-(6–17) of 6 out of 12 amino acids by non-natural residues and concomitant replacement of the disulfide bond by an amide bond led to three compounds with potent antagonistic properties (K B = 0.1–0.2 μm). Exploitation of these structure-activity relationships should open the way to the design of short and stable MCH peptide antagonists.

Binding of prostaglandins to human pparγ: Tool assessment and new natural ligands

The peroxisome proliferator-activated receptors (PPAR) form a family of nuclear receptors with a wide variety of biological roles from adipogenesis to carcinogenesis. More ligands (agonist and antagonist) are needed to explore the multiple functions of PPAR, particularly PPARgamma. In order to complete such ligand screening, a binding test should be assessed versus the classical transactivation reporter gene assay. In the present work, the full-length human PPARgamma protein as well as its ligand binding domain portion were expressed in Escherichia coli. Bacterial membrane preparations expressing those constructs were characterized using a classical binding competition assay [3H]rosiglitazone as the radioligand. When the receptor preparations were soluble, binding had to be measured with a new alternative method. The systems were assessed using a series of reference PPAR (alpha, beta and gamma) ligands. The full-length human PPARgamma fused to glutathione-S-transferase, expressed in E. coli and tested as a bacterial membrane-bound protein led to the most accurate results when compared to the literature. Furthermore, in an attempt to complete the panel of natural PPARgamma ligands, 29 commercially available prostaglandins were screened in the binding assay. Prostaglandins H(1) and H(2) were found to be modest ligands, however as potent as 15Delta(12-14 )prostaglandin J(2). These results were confirmed in the classical transactivation assay. The fact that these three prostaglandins were equally potent, suggests new pathways of PPARgamma-linked gene activation.

[125I]-S36057: a new and highly potent radioligand for the melanin-concentrating hormone receptor.

Shortened, more stable and weakly hydrophobic analogues of melanin‐concentrating hormone (MCH) were searched as candidates for radioiodination. Starting from the dodecapeptide MCH6 – 17, we found that: (1) substitution of Tyr13 by a Phe residue; (2) addition of a 3‐iodo‐Tyr residue at the N‐terminus; and (3) addition of a hydrophilic spacer 8‐amino‐3,6‐dioxyoctanoyl between the 3‐iodo‐Tyr and MCH6 – 17 (compound S36057), led to an agonist more potent than MCH itself in stimulating [35S]‐GTPγS binding at membranes from HEK293 cells stably expressing the human MCH receptor. Specific binding of [125I]‐S36057 was found in HEK293 and CHO cell lines stably expressing the human MCH receptor. This radioligand recognized a similar number of binding sites (ca. 800 fmol mg−1) than [125I]‐[3‐iodo Tyr13]‐MCH. However, the KD for [125I]‐S36057 obtained from saturation studies (0.037 nM) or from binding kinetics (0.046 nM) was at least 10 fold higher to that of [125I]‐[3‐iodo Tyr13]‐MCH (0.46 nM). Affinities determined for a series of MCH analogues were similar with both radioligands, S36057 being the most potent compound tested (Ki=0.053 nM). Finally, [125I]‐S36057 also potently labelled the MCH receptor in membranes from whole rat brain (KD 0.044 nM, Bmax=11 fmol mg−1). In conclusion, [125I]‐S36057 is a more potent and more stable radioligand than [125I]‐[3‐iodo Tyr13]‐MCH that will represent a reliable tool for binding assays in the search of novel MCH ligands. It should also provide great help for autoradiographic studies of the MCH receptor distribution in the central nervous system.

NPY receptor subtype in the rabbit isolated ileum

The purpose of this work was to verify the hypothesis that the rabbit ileum is a selective preparation for the NPY Y5 receptor by using new selective antagonists recently synthesized. Spontaneous contractions of the rabbit isolated ileum were recorded and binding experiments were performed in cells expressing the human NPY Y1, Y2, Y4 or Y5 receptor subtype. NPY analogues produced a concentration‐dependent transient inhibition of the spontaneous contractions of the rabbit ileum with the following order of potency hPP>rPP>PYY[Leu31,Pro34]‐NPY>NPY>>NPY13–36. Pre‐exposure to rPP, PYY, [Leu31,Pro34]‐NPY or NPY (but not NPY13–36) inhibited the effect of subsequent administration of hPP suggesting cross‐desensitization of the preparation. The apparent affinity of the various agonists studied was correlated to the affinity reported for the human Y4 receptor subtype (and to a lesser extent for the rat Y4 subtype) but not to the affinity for the Y5 receptor subtype. BIBO 3304, a selective NPY Y1 receptor antagonist, and CGP 71683A, a selective NPY Y5 receptor antagonist, did not affect the response to hPP. JCF 109, another NPY Y5 receptor antagonist, produced an inhibition of the response to hPP but only at the highest dose tested (10 μM) which also, by itself, produced intrinsic inhibitory effects. 1229U91, a non‐selective ligand for Y1, Y2, Y4 and Y5 receptors with high affinity toward the Y1 and Y4 receptor subtypes, produced a concentration‐dependent transient inhibition of the spontaneous contractions of the rabbit ileum and a dose‐dependent inhibition of the response to hPP (apparent pKB: 7.2). These results suggest that in the rabbit ileum, the NPY receptor involved in the inhibition of the spontaneous contractile activity is a NPY Y4 receptor subtype.

Ligand modulation of [35S]GTPγS binding at human α2A, α2B and α2C adrenoceptors

Abstract Affinities and efficacies of chemically diverse ligands—some of them used as clinical agents—were examined, employing [3H]RX821,002 and [35S]GTPγS binding assays, respectively, at human (h) cloned, hα2A, hα2B and hα2C adrenoceptors (AR) expressed in Chinese hamster ovary (CHO) cells. As compared to noradrenaline (NA, efficacy defined as 100%), the majority of the 13 agonists tested generally behaved as partial agonists. Amongst 18 antagonists, pKB and pKi values, which were highly correlated for each α2-AR subtype, failed to reveal any strikingly selective agents. Inverse agonist properties were not detected for any antagonist, consistent with a lack of constitutive activity suggested by the monophasic inhibition of [35S]GTPγS binding by GTPγS. These data should facilitate interpretation of experimental and clinical actions of adrenergic agonists. Moreover, they emphasize the continuing need for α2-AR subtype-selective antagonists in order to define further the roles and therapeutic relevance of hα2A-, hα2B-, and hα2C-AR.

SVK14 cells express an MCH binding site different from the MCH1 or MCH2 receptor

Melanin-concentrating hormone (MCH) is a cyclic peptide, mainly involved in the regulation of skin pigmentation in teleosts and feeding behavior in mammals. The human keratinocyte SVK14 cell line has been previously shown to express binding sites for the MCH analog [125I]-[Phe13,3-iodo-Tyr19]MCH. We report here that: (1) this binding site similarly recognized [125I]-[3-iodo-Tyr13]MCH; (2) its pharmacological profile clearly differed from those observed at the two human MCH receptor subtypes, MCH1-R and MCH2-R; (3) MCH did not induce any effect on second messenger systems (including cAMP, calcium, and MAP kinase signaling pathways), and (4) no mRNAs corresponding to the MCH receptors were found. In conclusion, the binding site characterized in the SVK14 cell line is distinct from the MCH1 and MCH2 receptors and deserves therefore further investigation.