Search

Background and Purpose Melatonin receptors have been extensively characterized regarding their affinity and pharmacology, mostly using 2-[ 125I]-melatonin as a radioligand. Although [3H]-melatonin has the advantage of corresponding to the endogenous ligand of the receptor, its binding has not been well described. Experimental Approach We characterized [3H]-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. Key Results The binding of [ 3H]-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 GTPS/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. Conclusions and Implications hMT1 and hMT2 receptors spontaneously exist in two states when expressed in cell lines; these states can be probed by [3H]-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. 2013 The British Pharmacological Society

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 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.

The main melatonin receptors are two G-protein coupled receptors named MT(1) and MT(2). Having described the molecular pharmacology of the human versions of these receptors, we turned to two of the three species most useful in studying melatonin physiology: rat and sheep (a diurnal species used to understand the relationship between circadian rhythm and depression). We also employed previously used compounds to describe the mouse melatonin receptors; despite the early cloning of mouse receptors, few molecular pharmacology studies on these receptors exist. To our surprise, we detected no major differences between the data obtained from mice and those from other species.

Melatonin receptors have been described to activate different G protein-dependent signaling pathways, both in laboratory, heterologous, cellular models and in physiological conditions. Furthermore, the constitutive activity of G protein-coupled 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 protein-coupled 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.