Recombinant Human Melatonin Receptor MT1 Isolated in Mixed Detergents Shows Pharmacology Similar to That in Mammalian Cell Membranes

Abstract

The human melatonin MT1 receptor—belonging to the large family of G protein-coupled receptors (GPCRs)—plays a key role in circadian rhythm regulation and is notably involved in sleep disorders and depression. Structural and functional information at the molecular level are highly desired for fine characterization of this receptor; however, adequate techniques for isolating soluble MT1 material suitable for biochemical and biophysical studies remain lacking. Here we describe the evaluation of a panel of constructs and host systems for the production of recombinant human MT1 receptors, and the screening of different conditions for their solubilization and purification. Our findings resulted in the establishment of an original strategy using a mixture of Fos14 and CHAPS detergents to extract and purify a recombinant human MT1 from Pichia pastoris membranes. This procedure enabled the recovery of relatively pure, monomeric and ligand-binding active MT1 receptor in the near-milligram range. A comparative study based on extensive ligand-binding characterization highlighted a very close correlation between the pharmacological profiles of MT1 purified from yeast and the same receptor present in mammalian cell membranes. The high quality of the purified MT1 was further confirmed by its ability to activate its cognate Gαi protein partner when reconstituted in lipid discs, thus opening novel paths to investigate this receptor by biochemical and biophysical approaches.

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

“…This compound is one of the rare selective antagonists described at MT 1 . A powerful anti‐calcium compound derived from verapamil, D600 is completely nonspecific (toward nonmelatoninergic targets) but had unique selectivity toward MT 1 , as demonstrated previously . Despite an affinity for MT 1 in the low micromolar range (pK i = 7), D600 acted as a clear antagonist in the cAMP production and GTPγS binding tests with pEC50s in the micromolar range, as expected.…”

Alternative Radioligands for Investigating the Molecular Pharmacology of Melatonin Receptors

“…Furthermore, we established the molecular pharmacology of MT 2 using those specific ligands, and compared it with the profiles obtained with other, nonspecific ligands (i.e., 2-[ 125 I]-MLT and [ 3 H]-MLT). This study contributes to the knowledge in this field, to help develop as many tools as possible to determine the characteristics of the purified receptors (Logez et al, 2014) as well as specific ligand(s) amenable for autoradiography in native tissues. MLT,serotonin, from SigmaAldrich (St. Louis, MO); 4P-PDOT (4-phenyl-2-propionamidotetraline) and luzindole (2-benzyl-N-acetyltryptamine) from Tocris (Bristol, UK); and 2-bromomelatonin from Toronto Research Chemicals, Inc. (North York, Canada).…”

The five dimensions of receptor pharmacology exemplified by melatonin receptors: An opinion

“…MT 1 and MT 2 have shown almost no difference in pharmacology. Despite the synthesis of several hundreds of agonist ligands, only a handful are specific for MT 2 over MT 1 , and only a single ligand is specific for MT 1 over MT 2 . Several excellent reviews have detailed the pharmacology of these receptors .…”

“…It might be somewhat easier to study these systems with purified receptors that maintain their signaling pathway(s) and a specialized approach, as described for the ghrelin receptor . Furthermore, despite many difficulties, essentially due to poor MT 1 stability, we obtained pure receptors in the active form, and they could couple with a G‐protein. Although, we are gaining quite a lot of knowledge about the concept of dimerization, the ability to predict appears to remain far in the future …”