Characterization of the Mel1c melatoninergic receptor in platypus (Ornithorhynchus anatinus).

Melatonin is a neurohormone produced in both animals and plants. It binds at least three G-protein-coupled receptors: MT1 and MT2, and Mel1cGPR. Mammalian GPR50 evolved from the reptilian/avian Mel1c and lost its capacity to bind melatonin in all the therian mammal species that have been tested. In order to determine if binding is lost in the oldest surviving mammalian lineage of monotremes we investigated whether the melatonin receptor has the ability to bind melatonin in the platypus (Ornithorhynchus anatinus), and evaluated its pharmacological profile. Sequence and phylogenetic analysis showed that platypus has in fact retained the ancestral Mel1c and has the capacity to bind melatonin similar to other mammalian melatonin receptors (MT1 and MT2), with an affinity in the 1 nM range. We also investigated the binding of a set of melatoninergic ligands used previously to characterize the molecular pharmacology of the melatonin receptors from sheep, rats, mice, and humans and found that the general profiles of these compounds make Mel1c resemble human MT1 more than MT2. This work shows that the loss of GPR50 binding evolved after the divergence of monotremes less than 190MYA in therian mammals

Molecular pharmacology of Mel1c receptors in platypus and xenopus compared to the human MT₁ and MT₂ melatonin receptors.

<p>Molecular pharmacology of Mel1c receptors in platypus and xenopus compared to the human MT₁ and MT₂ melatonin receptors.</p

Saturation binding experiments for 2-[125I]-iodomelatonin.

<p>Membranes from COS7 cells transfected with platypus Mel1c (A), clawed frog Mel1c (B), and chicken Mel1c (C) were used to measure the binding at Mel1c receptors. Red line represents specific binding, black line represents total binding and dotted line represents non-specific binding.</p

Comparison of the molecular pharmacology of platypus, xenopus and chicken melatonin Mel1c receptors.

<p>Comparison of the molecular pharmacology of platypus, xenopus and chicken melatonin Mel1c receptors.</p

Therian mammals used for phylogenetic analysis.

<p>Therian mammals used for phylogenetic analysis.</p

Characterization of the Mel1c melatoninergic receptor in platypus (<i>Ornithorhynchus anatinus</i>) - Fig 5

<p><b>Molecular pharmacology of the Mel1c receptors from platypus (A) and clawed frog (B).</b> The ligand was 2-[¹²⁵I]iodomelatonin. Independent experiments were performed at least twice using different batches of membranes from stably transfected CHO cells and each point was obtained in triplicate. SD6, N-[2-(5-methoxy-1H-indol-3-yl)ethyl]iodoacetamide; 2IMLT, 2-iodomelatonin; 6-Cl- Chloro MLT, 6-chloromelatonin; Luzindole, N-acetyl-2-Br-MLT; 2-bromomelatonin, benzyltryptamine; 4P-P-DOT, N-[(2S,4S)-4-phenyl-1,2,3,4-tetrahydronaphthalen-2-yl]propanamide; Agomelatin^® (S20098), N-(2-(7-methoxynaphthalen-1-yl)ethyl)acetamide; Ramelteon^® (FLN68), (S)-N-(2-(1,6,7,8-tetrahydro-2H-indeno-(5,4)furan-8-yl)ethyl)propionamide; D600, methoxyverapamil; 5HT, 5-hydroxytryptamine; S20928, (N-[2-(1-naphthyl)ethyl] cyclobutanecarboxamide); S21278, N-[2-(6-methoxybenzimidazol-1-yl)ethyl]acetamide; S22153, N-[2-(5-ethylbenzothiophen-3-yl)ethyl]acetamide; S27128-1, N-[2-(2-iodo-5-methoxy-6-nitro-1H-indol-3-yl)ethyl]acetamide; S73893, N-[3-methoxy-2-(7-methoxy-1-naphthyl)propyl]acetamide; S75436, 2-fluoro-N-[3-hydroxy-2-(7-methoxy-1-naphthyl) propyl]acetamide; S77834S27128, N-[(8-[2-(2-iodo-5-methoxy-10,11-dihydro-5H-dibenzo[a,d][<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191904#pone.0191904.ref007" target="_blank">7</a>] annulen-106-nitro-1H-indol-3-yl)methylethyl]acetamide; S77840, 1-[(8-methoxy-10,11-dihydro-5H-dibenzo[a,d][<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191904#pone.0191904.ref007" target="_blank">7</a>]annulen-101H-indol-3-yl)methyl]urea ethyl]iodoacetamide; SD1881, N-[2-(6-iodo-5-methoxy-1H-indol-3-yl)ethyl]acetamideiodomelatonin; SD1882, N-[2-(4-iodo-5-methoxy-1H-indol-3-yl)ethyl]acetamide; SD1918, N-[7-iodomelatonin; Div 880, 2-(7-iodo-5-methoxy-1H-indol-3-yl)ethyl]acetamide2-[(2-iodo-4,5-dimethoxyphenyl)methyl]-4,5-dimethoxy phenyl; S70254, 2-iodo-N-2-[5-methoxy-2-(naphthalen-1-yl)-1H-pyrrolo[3,2-b]pyridine-3-yl])acetamide. Concentration isotherms were obtained using 10 concentrations of each product from 10⁻¹³ to 10⁻⁴ M.</p

Comparison of Mel1c sequences in platypus, xenopus and chicken to the human GPR50.

<p>Comparison of Mel1c sequences in platypus, xenopus and chicken to the human GPR50.</p

Cloning of Mel1c from platypus brain and comparison of the actual and predicted sequences.

<p>The cloning was performed using the predicted sequence XM_001512887. A, lane 1: Exon 1 amplicon, 181 bp; lane 2: Exon 2 amplicon, 875 bp. B, Comparison of the obtained sequence. In red: ATG starting codon and TAA stop codon. At position 44, a single mutation from the predicted sequence changes one amino acid in the protein sequence at position 15 (L>P).</p

Platypus has retained ancestral Mel1c showing that the ortholog GPR50 evolved after the divergence of monotremes from therian mammals.

<p>A) Protein alignment of human (<i>Homo sapiens</i>) GPR50 with platypus (<i>Ornithorhynchus anatinus</i>), chicken (<i>Gallus gallus</i>) and the clawed frog (<i>Xenopus laevis</i>) Mel1c. Each amino acid is depicted by its single letter symbol and an associated colour. Identity bar above alignment denotes the similarity at each amino acid position between the four species: green = all homologous; yellow = three species homologous; red = two species homologous; no bar = no homology. Every 50 amino acid positions are labeled. B) Neighbour-Joining phylogenetic tree showing platypus clustering together with chicken and the clawed frog Mel1c while marsupial GPR50 and eutherian GPR50 form their own distinct clusters. Bootstrap support values are shown for each node. The four species from protein alignment in panel A are highlighted in yellow. C) Amino acid sequence of the second extracellular loop (E2) of Mel1c and GPR50 for all species used in the phylogenetic tree from panel B. Each amino acid is depicted by its single letter symbol and an associated colour. Identity bar above alignment denotes the similarity at each amino acid position between all species: green = all homologous; yellow = some species homologous. Every 10 amino acid positions are labeled.</p

Primer sequences used in this study.

<p>Primer sequences used in this study.</p