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

Abstract

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 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. 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.

Alternative Radioligands for Investigating the Molecular Pharmacology of Melatonin Receptors

“…3 H]-MLT and 2-[ 125 I]-MLT, are pure agonists, and exhibit the same affinity and almost identical pharmacological parameters for both receptors (Browning et al, 2000;Masana and Dubocovich, 2001;Legros et al, 2013Legros et al, , 2014. In the present work, we described three new radioligands that can be used for MT 1 and MT 2 receptor characterization.…”

“…The kinetics curves are presented in Fig. 1 We assessed a set of 24 compounds that were previously described in the literature [including 4-phenyl-2-propionamidotetraline (Dubocovich et al, 1997), luzindole (Dubocovich, 1988a), and ramelteon (Uchikawa et al, 2002)] or that were issued from our own medicinal chemistry programs (Depreux et al, 1994; Audinot et al, 2003Audinot et al, , 2008Mailliet et al, 2004;Devavry et al, 2012a,b;Ettaoussi et al, 2013;Legros et al, 2013Legros et al, , 2014 (Fig. 3) with a pK d 1 (site 1) of 10.35 6 0.03, a pK d 2 (site 2) of 9.25 6 0.13, a B max 1 (site 1) of 1.46 6 0.13 fmol·mg protein 21 , and a B max 2 (site 2) of 2.32 6 0.98 fmol·mg protein 21 (n 5 4).…”

“…Few diverse tools have been developed for MLT molecular pharmacology studies (Markl et al, 2011), such as subtypespecific agonists or antagonists, or biased agonists (Devavry et al, 2012b;Legros et al, 2014). We further screened our compounds (Yan et al, 2008) to find various types of ligands that are amenable to chemical conversion to iodinated and radioactive derivatives.…”

Melatonin improves non-alcoholic fatty liver disease via MAPK-JNK/P38 signaling in high-fat-diet-induced obese mice

“…Melatonin was also found to regulate glucose metabolism. There are two membrane receptors of melatonin called MT1 and MT2, which are G-protein-coupled receptors, in the periphery [31]. Both receptors are expressed in the islet of Langerhans and are involved in the regulation of glucagon secretion from α-cells and insulin secretion from β-cells [16, 32].…”

Characterization of the various functional pathways elicited by synthetic agonists or antagonists at the melatonin MT1 and MT2 receptors

“…The profile of the compound is very similar to that of melatonin itself. Only minute differences were seen between the results obtained with this ligand and the tritiated melatonin . The affinities (pEC50) were between 9 and 11 and, except for GTPγS binding, the efficiency at both receptors was close to the efficiency observed with melatonin itself.…”