title: 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.
authors: CNRS UMR7242/Laboratoire d'excellence MEDALIS, Institut de Recherche de l'ESBS, Biotechnologie et Signalisation Cellulaire, Université de Strasbourg, Illkirch, France; Biotechnologie, Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, Croissy-sur-Seine, France.
authors: Christel
authors: C
authors: Logez
authors: Biotechnologie, Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, Croissy-sur-Seine, France.
authors: Sylvie
authors: S
authors: Berger
authors: Biotechnologie, Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, Croissy-sur-Seine, France.
authors: Céline
authors: C
authors: Legros
authors: CNRS UMR 5247, Institut des Biomolécules Max Mousseron (IBMM), Université de Montpellier 1 and Montpellier 2, Faculté de Pharmacie, Montpellier, France.
authors: Jean-Louis
authors: JL
authors: Banères
authors: Biotechnologie, Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, Croissy-sur-Seine, France.
authors: William
authors: W
authors: Cohen
authors: Unité de Recherches et Découvertes en Neurosciences, Institut de Recherche Servier, Croissy-sur-Seine, France.
authors: Philippe
authors: P
authors: Delagrange
authors: Biotechnologie, Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, Croissy-sur-Seine, France.
authors: Olivier
authors: O
authors: Nosjean
authors: Biotechnologie, Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, Croissy-sur-Seine, France.
authors: Jean A
authors: JA
authors: Boutin
authors: Biotechnologie, Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, Croissy-sur-Seine, France.
authors: Gilles
authors: G
authors: Ferry
authors: CNRS UMR7242/Laboratoire d'excellence MEDALIS, Institut de Recherche de l'ESBS, Biotechnologie et Signalisation Cellulaire, Université de Strasbourg, Illkirch, France.
authors: Frédéric
authors: F
authors: Simonin
authors: CNRS UMR7242/Laboratoire d'excellence MEDALIS, Institut de Recherche de l'ESBS, Biotechnologie et Signalisation Cellulaire, Université de Strasbourg, Illkirch, France.
authors: Renaud
authors: R
authors: Wagner
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003412:Cricetulus; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D044122:Receptor, Melatonin, MT1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
chemical_list: D003902:Detergents; D008024:Ligands; D044122:Receptor, Melatonin, MT1; D011994:Recombinant Proteins