| Summary: | In derived bony vertebrates, activation of the melanocortin-2 receptor (Mc2r) by its ACTH ligand requires chaperoning by the Mc2r accessory protein (Mrap1). The N-terminal domain of the non-mammalian tetrapod MRAP1 from chicken (c; <i>Gallus gallus</i>) has the putative activation motif, W<sup>18</sup>D<sup>19</sup>Y<sup>20</sup>I<sup>21</sup>, and the N-terminal domain in the neopterygian ray-finned fish Mrap1 from bowfin (bf; <i>Amia calva</i>) has the putative activation motif, Y<sup>18</sup>D<sup>19</sup>Y<sup>20</sup>I<sup>21</sup>. The current study used an alanine-substitution paradigm to test the hypothesis that only the Y<sup>20</sup> position in the Mrap1 ortholog of these non-mammalian vertebrates is required for activation of the respective Mc2r ortholog. Instead, we found that for cMRAP1, single alanine-substitution resulted in a gradient of inhibition of activation (Y<sup>20</sup> >> D<sup>19</sup> = W<sup>18</sup> > I<sup>21</sup>). For bfMrap1, single alanine-substitution also resulted in a gradient of inhibition of activation (Y<sup>20</sup> >> D<sup>19</sup> > I<sup>21</sup> > Y<sup>18</sup>). This study also included an analysis of Mc2r activation in an older lineage of ray-finned fish, the paddlefish (p), <i>Polyodon spathula</i> (subclass Chondronstei). Currently no <i>mrap1</i> gene has been found in the paddlefish genome. When <i>pmc2r</i> was expressed alone in our CHO cell/cAMP reporter gene assay, no activation was observed following stimulation with ACTH. However, when <i>pmc2r</i> was co-expressed with either <i>cmrap1</i> or <i>bfmrap1</i> robust dose response curves were generated. These results indicate that the formation of an Mc2r/Mrap1 heterodimer emerged early in the radiation of the bony vertebrates.
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