Structure/Function Studies on the Activation Motif of Two Non-Mammalian Mrap1 Orthologs, and Observations on the Phylogeny of Mrap1, Including a Novel Characterization of an Mrap1 from the Chondrostean Fish, Polyodon spathula

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; Gallus gallus) has the putative activation motif, W¹⁸D¹⁹Y²⁰I²¹, and the N-terminal domain in the neopterygian ray-finned fish Mrap1 from bowfin (bf; Amia calva) has the putative activation motif, Y¹⁸D¹⁹Y²⁰I²¹. The current study used an alanine-substitution paradigm to test the hypothesis that only the Y²⁰ 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 (Y²⁰ >> D¹⁹ = W¹⁸ > I²¹). For bfMrap1, single alanine-substitution also resulted in a gradient of inhibition of activation (Y²⁰ >> D¹⁹ > I²¹ > Y¹⁸). This study also included an analysis of Mc2r activation in an older lineage of ray-finned fish, the paddlefish (p), Polyodon spathula (subclass Chondronstei). Currently no mrap1 gene has been found in the paddlefish genome. When pmc2r was expressed alone in our CHO cell/cAMP reporter gene assay, no activation was observed following stimulation with ACTH. However, when pmc2r was co-expressed with either cmrap1 or bfmrap1 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.