Ligand binding to the human MT2 melatonin receptor: The role of residues in transmembrane domains 3, 6, and 7 Petr Mazna a , Karel Berka b , Irena Jelinkova a , Ales Balik a , Petr Svoboda a , Veronika Obsilova a , Tomas Obsil b , Jan Teisinger a, * a Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague, Czech Republic b Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 2030/8, 128 43 Prague, Czech Republic Received 28 April 2005 Available online 12 May 2005 Abstract To better understand the mechanism of interactions between G-protein-coupled melatonin receptors and their ligands, our pre- viously reported homology model of human MT2 receptor with docked 2-iodomelatonin was further refined and used to select res- idues within TM3, TM6, and TM7 potentially important for receptor–ligand interactions. Selected residues were mutated and radioligand-binding assay was used to test the binding affinities of hMT2 receptors transiently expressed in HEK293 cells. Our data demonstrate that residues N268 and A275 in TM6 as well as residues V291 and L295 in TM7 are essential for 2-iodomelatonin bind- ing to the hMT2 receptor, while TM3 residues M120, G121, V124, and I125 may participate in binding of other receptor agonists and/or antagonists. Presented data also hint at possible specific interaction between the side-chain of Y188 in second extracellular loop and N-acetyl group of 2-iodomelatonin. Ó 2005 Elsevier Inc. All rights reserved. Keywords: MT2 melatonin receptor; Site-directed mutagenesis; Homology modeling; G-protein-coupled receptor Melatonin (N-acetyl-5-methoxytryptamine) is the main hormone synthesized by the vertebrate pineal gland. In mammals, the melatonin is known to play an important role in the regulation of physiological and neuroendocrine functions, such as synchronization of seasonal reproductive rhythms [1], and entrainment of circadian cycles (see [2] for review). In addition to its chronobiotic role, melatonin influences a number of other physiologically diverse processes including tumor suppression [3] and modulation of the free radical levels [4]. It has also been reported that melatonin reduces tis- sue destruction during inflammatory reactions [5] and shows immunotherapeutic potential (reviewed in [6]). To date, three mammalian melatonin receptors have been described: MT1 [7], MT2 [8], and MT3 [9]. First two are G-protein-coupled receptors (GPCRs) and their activations modulate a wide range of intracellular mes- sengers, e.g., cAMP, cGMP or [Ca 2+ ] i (reviewed in [10,11]). The MT3-binding site has been identified as quinone reductase protein and its physiological signifi- cance remains to be clarified. In mammals, both MT1 and MT2 receptor subtypes are expressed in a wide vari- ety of tissues including specific brain structures and peripheral organs [10]. All GPCRs are thought to share the basic structural organization characterized by a bundle of seven putative transmembrane domains (TMs), that form a ligand-bind- ing site, connected by three extracellular (ECLs) and three intracellular loops [12]. Extracellular loops may partici- pate in ligand recognition by the receptor whereas intra- cellular loops are believed to directly activate 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.05.017 * Corresponding author. Fax: +420 2 4106 2488. E-mail address: teisingr@biomed.cas.cz (J. Teisinger). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 332 (2005) 726–734 BBRC