Functional characterization of two melanocortin (MC) receptors in lamprey showing orthology to the MC1 and MC4 receptor subtypes

<p>Abstract</p> <p>Background</p> <p>The melanocortin (MC) receptors have a key role in regulating body weight and pigmentation. They belong to the rhodopsin family of G protein-coupled receptors (GPCRs). The purpose of this study was to identify ancestral MC receptors in agnathan, river lamprey.</p> <p>Results</p> <p>We report cloning of two MC receptors from river lamprey. The lamprey receptors, designated MCa and MCb, showed orthology to the MC1 and MC4 receptor subtypes, respectively. The molecular clock analysis suggested that lamprey MC receptor genes were not duplicated recently and diverged from each other more than 400 MYR ago. Expression and pharmacological characterization showed that the lamprey MCa receptor was able to bind and be activated by both lamprey and human MSH peptides. The lamprey MCa receptor had relatively high affinity for ACTH derived peptides similarly to the fish MC receptors. We found that both of the lamprey MC receptors were expressed in skin, while the MCb receptor was also found in liver, heart and skeletal muscle.</p> <p>Conclusion</p> <p>This study shows presence of MC receptors in agnathans indicating early signs of specific functions of melanocortin receptor subtypes.</p

Generation of cAMP in response to α-MSH (filled circle), ACTH(1–24) (filled square), MSH-A (open circle), MSH-B (filled triangle) and ACTH (1–31) (open square) for the lamprey MCa receptors expressed in HEK-293 EBNA cells

<p><b>Copyright information:</b></p><p>Taken from "Functional characterization of two melanocortin (MC) receptors in lamprey showing orthology to the MC1 and MC4 receptor subtypes"</p><p>http://www.biomedcentral.com/1471-2148/7/101</p><p>BMC Evolutionary Biology 2007;7():101-101.</p><p>Published online 29 Jun 2007</p><p>PMCID:PMC1925065.</p><p></p> Untransfected cells showed no adenylate cyclase activity in response to ligands (data not shown). The cAMP assay was performed in duplicate and repeated two times with each ligand

Expression of lamprey MCa (a) and MCb (b) receptor mRNA as determined by RT-PCR

<p><b>Copyright information:</b></p><p>Taken from "Functional characterization of two melanocortin (MC) receptors in lamprey showing orthology to the MC1 and MC4 receptor subtypes"</p><p>http://www.biomedcentral.com/1471-2148/7/101</p><p>BMC Evolutionary Biology 2007;7():101-101.</p><p>Published online 29 Jun 2007</p><p>PMCID:PMC1925065.</p><p></p> The tissues, controls and expected sizes of the PCR products are denoted at the top of each figure. Ethidium bromide stained agarose gels are presented on top and autoradiographs of Southern blots, hybridised with gene specific probes, are shown below. The PCR and the hybridization were performed three times with qualitatively similar results

Phylogenetic analysis of full-length amino acid sequences of MC receptors from human, chicken, zebrafish and lamprey

<p><b>Copyright information:</b></p><p>Taken from "Functional characterization of two melanocortin (MC) receptors in lamprey showing orthology to the MC1 and MC4 receptor subtypes"</p><p>http://www.biomedcentral.com/1471-2148/7/101</p><p>BMC Evolutionary Biology 2007;7():101-101.</p><p>Published online 29 Jun 2007</p><p>PMCID:PMC1925065.</p><p></p> The consensus tree was generated by Neighbor-Joining analysis (Phylip 3.6a3). The numbers above the nodes indicate bootstrap replicates. The abbreviations used: Hsa, human; Gga, chicken; Dre, zebrafish and Lfl, lamprey. The accession numbers are listed in "Methods"

Saturation curves with Scatchard plot and competition curves for the lamprey MCa receptors expressed in HEK-293 EBNA cells

<p><b>Copyright information:</b></p><p>Taken from "Functional characterization of two melanocortin (MC) receptors in lamprey showing orthology to the MC1 and MC4 receptor subtypes"</p><p>http://www.biomedcentral.com/1471-2148/7/101</p><p>BMC Evolutionary Biology 2007;7():101-101.</p><p>Published online 29 Jun 2007</p><p>PMCID:PMC1925065.</p><p></p> The saturation curves (left) were obtained with I-labelled NDP-MSH and the figure shows total binding (filled square) and binding in the presence of 2 μM cold NDP-MSH (filled triangle). The lines represent the computer-modelled best fit of the data assuming that ligands bound to one site. The competition curves (right) for NDP-MSH (filled triangle, pointing up), α-MSH (filled square), β-MSH (open circle), γ-MSH (open diamond), ACTH(1–17) (open square), ACTH(1–24) (filled circle), ACTH(1–39) (x), MTII (filled diamond), HS024 (open triangle, pointing down), MSH-A (filled triangle, pointing down), MSH-B (open triangle, pointing up) and ACTH (1–31) (asterisk) were obtained by using a fixed concentration of approx. 0.6 nM I-labelled NDP-MSH and varying concentrations of the non-labelled competing peptide

The molecular clock analysis of MC1 (open circle), MC2 (filled triangle), MC3 (open square), MC4 (asterisk) and MC5 (filled diamond) receptor subtypes

<p><b>Copyright information:</b></p><p>Taken from "Functional characterization of two melanocortin (MC) receptors in lamprey showing orthology to the MC1 and MC4 receptor subtypes"</p><p>http://www.biomedcentral.com/1471-2148/7/101</p><p>BMC Evolutionary Biology 2007;7():101-101.</p><p>Published online 29 Jun 2007</p><p>PMCID:PMC1925065.</p><p></p> Full-length amino acid sequences were used for the calculations. The distances were calculated with Tree-Puzzle v 5.2. Lamprey MCa (open circle) and MCb (asterisk) are plotted at 564 Myr ago. We used the following divergence times from human: mouse – 90(70), chicken – 310(310), zebrafish – 450(420), dogfish – 528 (420) and lamprey – 564 (525) Myr ago. The corresponding calculated distances were plotted against molecular and fossil data for each receptor subtype separately. The lines are linear regression lines

Amino acid sequence alignment of MC receptors constructed using ClustalW 1

<p><b>Copyright information:</b></p><p>Taken from "Functional characterization of two melanocortin (MC) receptors in lamprey showing orthology to the MC1 and MC4 receptor subtypes"</p><p>http://www.biomedcentral.com/1471-2148/7/101</p><p>BMC Evolutionary Biology 2007;7():101-101.</p><p>Published online 29 Jun 2007</p><p>PMCID:PMC1925065.</p><p></p>8. Putative transmembrane (TM) regions are marked with lines. Black and grey boxes mark conserved and similar amino acid positions, respectively. The abbreviations used: Hsa, human; Gga, chicken; Dre, zebrafish; Lfl, lamprey and Mgl, hagfish. Mgl MCc sequence is partial (119 aa). The accession numbers are listed in "Methods"