Récepteurs de la mélatonine : pharmacologie du récepteur ovin MT2, identification de leur activité constitutive et développement d'une approche par ARN interférent

Diplôme : Dr. d'Universit

Description of the constitutive activity of cloned human melatonin receptors hMT1 and hMT2 and discovery of inverse agonists

Melatonin receptors have been described to activate different G proteindependent signaling pathways, both in laboratory, heterologous, cellular models and in physiological conditions. Furthermore, the constitutive activity of G proteincoupled receptors has been shown to be key in physiological and pathological conditions. In the case of melatonin receptors, information is rather scare and concerns only MT1 receptors. In the present report, we show that the G proteincoupled melatonin receptors do have a constitutive, nonmelatonin-induced signaling activity using two cellular models of different origins, the Chinese hamster ovary cell line and Neuro2A, a neuroblastoma cell line. Furthermore, we show that this constitutive activity involves mainly Gi proteins, which is consistent with the common knowledge on the melatonin receptors. Importantly, we also describe, for the first time, inverse agonist properties for melatonin ligands. Although it is clear than more in-depth, biochemistry-based studies will be required to better understand by which pathway(s) the constitutively active melatonin receptors transfer melatonin information into intracellular biochemical events; our data open interesting perspectives for understanding the importance of the constitutive activity of melatonin receptors in physiological conditions

Melatonin MT1 and MT2 receptors display different molecular pharmacologies only in the G-protein coupled state

Melatonin receptors have been extensively characterized regarding their affinity and pharmacology, mostly using 2-[(125) I]-melatonin as a radioligand. Although [(3) H]-melatonin has the advantage of corresponding to the endogenous ligand of the receptor, its binding has not been well described. We characterized [(3) H]-melatonin binding to the hMT1 and hMT2 receptors expressed in a range of cell lines and obtained new insights into the molecular pharmacology of melatonin receptors. The binding of [(3) H]-melatonin to the hMT1 and hMT2 receptors displayed two sites on the saturation curves. These two binding sites were observed on cell membranes expressing recombinant receptors from various species as well as on whole cells. Furthermore, our GTPγS/NaCl results suggest that these sites on the saturation curves correspond to the G-protein coupled and uncoupled states of the receptors, whose pharmacology was extensively characterized. hMT1 and hMT2 receptors spontaneously exist in two states when expressed in cell lines; these states can be probed by [(3) H]-melatonin binding. Overall, our results suggest that physiological regulation of the melatonin receptors may result from complex and subtle mechanisms, a small difference in affinity between the active and inactive states of the receptor, and spontaneous coupling to G-proteins

Design, synthesis and biological evaluation of fluorescent ligands for MT1 and/or MT2 melatonin receptors

Design, synthesis and biological evaluation of fluorescent ligands for MT1 and/or MT2 melatonin receptors. 24. Conference of the Groupement des Pharmacochimistes de l'Arc Atlantique (GP2A

Molecular pharmacology of the mouse melatonin receptors MT1 and MT2

International audienc

The end of a myth: cloning and characterization of the ovine melatonin MT2 receptor

International audienceBackground and purpose: For many years, it was suspected that sheep expressed only one melatonin receptor (closely resembling MT1 from other mammal species). Here we report the cloning of another melatonin receptor, MT2, from sheep. Experimental approach: Using a thermo-resistant reverse transcriptase and polymerase chain reaction primer set homologous to the bovine MT2 mRNA sequence, we have cloned and characterized MT2 receptors from sheep retina. Key results: The ovine MT2 receptor presents 96%, 72% and 67% identity with cattle, human and rat respectively. This MT2 receptor stably expressed in CHO-K1 cells showed high-affinity 2[125I]-iodomelatonin binding (K-D = 0.04 nM). The rank order of inhibition of 2[125I]-iodomelatonin binding by melatonin, 4-phenyl-2-propionamidotetralin and luzindole was similar to that exhibited by MT2 receptors of other species (melatonin > 4-phenyl-2-propionamidotetralin > luzindole). However, its pharmacological profile was closer to that of rat, rather than human MT2 receptors. Functionally, the ovine MT2 receptors were coupled to G(i) proteins leading to inhibition of adenylyl cyclase, as the other melatonin receptors. In sheep brain, MT2 mRNA was expressed in pars tuberalis, choroid plexus and retina, and moderately in mammillary bodies. Real-time polymerase chain reaction showed that in sheep pars tuberalis, premammillary hypothalamus and mammillary bodies, the temporal pattern of expression of MT1 and MT2 mRNA was not parallel in the three tissues. Conclusion and implications: Co-expression of MT1 and MT2 receptors in all analysed sheep brain tissues suggests that MT2 receptors may participate in melatonin regulation of seasonal anovulatory activity in ewes by modulating MT1 receptor action