MOLECULAR DETERMINANTS OF THE AGONIST BINDING SITE OF HUMAN MT2 MELATONIN RECEPTOR

K. Berka^1,2, P. Mazna², I. Jelínková², A. Balík², P. Svoboda², V. Obšilová², T. Obšil^1,2, and
J. Teisinger²

¹Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, 128 43 Prague,

²Institute of Physiology, Academy of Sciences of the Czech Republic, 142 20 Prague.

The hormone melatonin is known to play an important role in the regulation of physiological and neuroendocrine functions [1,2]. To better understand the mechanism of interactions between G protein-coupled melatonin receptors and their ligands we have generated a homology model of the helical part of the human MT2 melatonin (hMT2) receptor using the structure of bovine rhodopsin as a template [3]. Molecular modeling has been combined with site-directed mutagenesis to investigate the role of the specific amino acid residues within the transmembrane domains (TM) number 3, 5, 6 and 7 of hMT2 receptor in the interaction with 2-iodomelatonin. 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 demonstrates that residues N268 (N6.52) and A275 (A6.59) in TM6 as well as residues V291 (V7.36) and L295 (L7.40) in TM7 are essential for 2-iodomelatonin binding to the hMT2 receptor, while TM3 residues M120 (M3.32), G121 (G3.33), V124 (V3.36) and I125 (I3.37) may participate in binding of other receptor agonists and/or antagonists.

This work was supported by Grants 309/02/1479, 309/04/0496, and 204/03/0714 of the Grant Agency of the Czech Republic; by Grant B5011308 of the Grant Agency of the Czech Academy of Sciences; by Research Projects 1K03020 and MSM 1131 00001 of the Ministry of Education, Youth and Sports of the Czech Republic, and by Research Project AVOZ 5011922.

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2. G.J. Maestroni, J. Pineal Res. 14 (1993) 1-10.

3. P. Mazna, V. Obsilova, I. Jelinkova, A. Balik, K. Berka, Z. Sovova, R. Ettrich, P. Svoboda, T. Obsil & J. Teisinger, J. Neurochem. 91 (2004) 836-842.