Table 1.
Primer sequences used in this study.
Table 2.
Therian mammals used for phylogenetic analysis.
Fig 1.
Cloning of Mel1c from platypus brain and comparison of the actual and predicted sequences.
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).
Table 3.
Comparison of Mel1c sequences in platypus, xenopus and chicken to the human GPR50.
Fig 2.
Platypus has retained ancestral Mel1c showing that the ortholog GPR50 evolved after the divergence of monotremes from therian mammals.
A) Protein alignment of human (Homo sapiens) GPR50 with platypus (Ornithorhynchus anatinus), chicken (Gallus gallus) and the clawed frog (Xenopus laevis) 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.
Fig 3.
Expression of Mel1c receptors in COS7 cells.
Subcellular localization of HA epitope-tagged Mel1c receptors. Immunofluorescence studies were performed with transfected COS7cells. COS7 cells and COS7 expressing 3HA-platypus Mel1C or 3HA-X. laevis Mel1C or 3HA-G. gallus Mel1C receptors were probed with mouse monoclonal anti-HA antibody directed against the N-terminal epitope tag present on recombinant receptor. Experiments were carried out with paraformaldehyde-fixed and non-permeabilized cells. The fluorescence images were obtained by using Alexa 488-conjugated goat anti- mouse IgG secondary antibody. COS7 cell nuclei were stained with 4′, 6′-diamidino-2-phenylindole. Native COS7 cells were used as a negative control.
Fig 4.
Saturation binding experiments for 2-[125I]-iodomelatonin.
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.
Table 4.
Comparison of the molecular pharmacology of platypus, xenopus and chicken melatonin Mel1c receptors.
Fig 5.
Molecular pharmacology of the Mel1c receptors from platypus (A) and clawed frog (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][7] annulen-106-nitro-1H-indol-3-yl)methylethyl]acetamide; S77840, 1-[(8-methoxy-10,11-dihydro-5H-dibenzo[a,d][7]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.
Table 5.
Molecular pharmacology of Mel1c receptors in platypus and xenopus compared to the human MT1 and MT2 melatonin receptors.
Fig 6.
Correlation between the data obtained from platypus Mel1c and those obtained with clawed frog Mel1c.
Data were obtained in a binding assay with 2-[125I]-iodomelatonin as the radioligand. See Fig 5.
Fig 7.
Comparison of the correlations between the molecular pharmacology of platypus Mel1c (A) or clawed frog Mel1c (B) and human melatonin receptors MT1 and MT2. The correlation in red is between hMT1 and platypus Mel1c; in blue, between hMT2 and Platypus Mel1c (upper panel). The correlation in red is between hMT1 and clawed frog Mel1c; and in green between hMT2 and clawed frog Mel1c (lower panel.) See Fig 5 for data.