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.
Highlights
The neurohormone melatonin is produced by the pineal gland at night in all mammals, whether diurnal or nocturnal [1]
We further show that the purified receptor displays a pharmacological profile that closely resembles that of the membrane-bound human MT1 receptor expressed in a mammalian cell line, and that it exhibits a specific agonist-dependent G protein activation when reconstituted in lipid nanodiscs
In the case of membrane proteins and G protein-coupled receptors (GPCRs) in particular, this systematically implies the use of recombinant systems that efficiently overexpress the gene of interest
Summary
The neurohormone melatonin is produced by the pineal gland at night in all mammals, whether diurnal or nocturnal [1]. Despite the immense interest in such approaches and the huge efforts put towards the study of other GPCRs, references and procedures describing the successful production and purification of active receptors remain rather limited This lack of data directly relates to the fact that it continues to be highly challenging to obtain significant amounts of these membrane proteins in the purest form and retaining characteristics resembling the native proteins. We further show that the purified receptor displays a pharmacological profile that closely resembles that of the membrane-bound human MT1 receptor expressed in a mammalian cell line, and that it exhibits a specific agonist-dependent G protein activation when reconstituted in lipid nanodiscs To our knowledge, this is the first report of the purification of a functional melatonin receptor in amounts compatible with a number of protein-based analytical methodologies. This work forges a path towards improving the structural and functional characterization of MT1 at the molecular level, including the investigation of its interactions with specific ligands and protein partners
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