Mutagenesis studies of the human MT2 melatonin receptor.

Melatonin mediates its physiological effects through activation of high affinity G protein-coupled receptors. The vertebrate MT(1), MT(2) and Mel(1c) melatonin receptors are molecularly and pharmacologically distinct. Three molecular models of melatonin recognition for the MT(1) and/or Mel(1c) melatonin receptors have been proposed. To determine if these models applied to the MT(2) melatonin receptor, we mutated seven conserved residues to alanine in the hMT(2) melatonin receptor and expressed the receptors in HEK-293 cells. Competition of melatonin for 2-[125I]-iodomelatonin binding revealed that mutation of Asn 16 in TM4 or His 7 in TM5 of the hMT(2) melatonin receptor significantly decreased the binding affinity for melatonin when compared with wild-type. In addition, competition of 4P-ADOT, N-acetyltryptamine, luzindole, and 5-methoxytryptophol for 2-[125I]-iodomelatonin binding suggested Asn 16 in TM4 may facilitate binding of the 5-methoxy group of the melatonin molecule to the hMT(2) melatonin receptor. Trp 13 or Phe 6 in TM6 while not critical for melatonin binding, may interact with aromatic regions of luzindole and 4P-ADOT. Mutation of Ser 8 or Ser 12 in TM3, or Ser 6 in TM7 did not affect the affinity of melatonin for competition with 2-[125I]-iodomelatonin to the hMT(2) melatonin receptor, although equivalent serines (Ser 8 and Ser 12 in TM3) were reported to be critical for melatonin binding to the hMT(1) melatonin receptor. Thus these results are the first to identify residues within the transmembrane regions of the hMT(2) melatonin receptor critical for melatonin binding, highlighting potential structural differences between the MT(1) and MT(2) melatonin receptor binding pockets.

[1]  J. Jansen,et al.  The high affinity melatonin binding site probed with conformationally restricted ligands--II. Homology modeling of the receptor. , 1996, Bioorganic & medicinal chemistry.

[2]  R. Osman,et al.  Minireview: Insights into G Protein-Coupled Receptor Function Using Molecular Models* * The work from our laboratories was supported by USPHS Grant DK-43036. , 2001, Endocrinology.

[3]  L. F. Kolakowski,et al.  Melatonin receptors are for the birds: Molecular analysis of two receptor subtypes differentially expressed in chick brain , 1995, Neuron.

[4]  S. Reppert,et al.  Expression cloning of a high-affinity melatonin receptor from Xenopus dermal melanophores. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[5]  F. Mseeh,et al.  Identification of cysteines involved in ligand binding to the human melatonin MT(2) receptor. , 2002, European journal of pharmacology.

[6]  D. Lewis,et al.  Structural requirements at the melatonin receptor , 1995, British journal of pharmacology.

[7]  J. Gusella,et al.  Molecular characterization of a second melatonin receptor expressed in human retina and brain: the Mel1b melatonin receptor. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Y. Cheng,et al.  Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. , 1973, Biochemical pharmacology.

[9]  A. Gubitz,et al.  Chimeric and point-mutated receptors reveal that a single glycine residue in transmembrane domain 6 is critical for high affinity melatonin binding. , 2000, Endocrinology.

[10]  A. Poso,et al.  A rhodopsin-based model for melatonin recognition at its G protein-coupled receptor. , 1996, European journal of pharmacology.

[11]  P. Morgan,et al.  Chimeric Melatonin mt1 and Melatonin-related Receptors , 2000, The Journal of Biological Chemistry.

[12]  M. Dubocovich,et al.  Melatonin receptor antagonists that differentiate between the human Mel1a and Mel1b recombinant subtypes are used to assess the pharmacological profile of the rabbit retina ML1 presynaptic heteroreceptor , 1997, Naunyn-Schmiedeberg's Archives of Pharmacology.

[13]  M. Dubocovich,et al.  Melatonin Receptor Signaling: Finding the Path Through the Dark , 2001, Science's STKE.

[14]  P. Morgan,et al.  The roles of valine 208 and histidine 211 in ligand binding and receptor function of the ovine Mel1a beta melatonin receptor. , 1997, Biochemical and biophysical research communications.

[15]  P. Morgan,et al.  Serine residues 110 and 114 are required for agonist binding but not antagonist binding to the melatonin MT(1) receptor. , 2001, Biochemical and biophysical research communications.

[16]  A. Brzezinski.  Melatonin in humans. , 1997, The New England journal of medicine.

[17]  J. Ballesteros,et al.  Structural mimicry in G protein-coupled receptors: implications of the high-resolution structure of rhodopsin for structure-function analysis of rhodopsin-like receptors. , 2001, Molecular pharmacology.

[18]  K. Palczewski,et al.  Crystal Structure of Rhodopsin: A G‐Protein‐Coupled Receptor , 2002, Chembiochem : a European journal of chemical biology.

[19]  E. Pebay-Peyroula,et al.  X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases. , 1997, Science.

[20]  S. Reppert,et al.  Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses , 1994, Neuron.