Trends in the evolution of the elasmobranch melanocortin-2 receptor: Insights from structure/function studies on the...

To understand the mechanism for activation of the melanocortin-2 receptor (Mc2r) of the elasmobranch, Rhincodon typus (whale shark; ws), wsmc2r was co-expressed with wsmrap1 in CHO cells, and the transfected cells were stimulated with alanine-substituted analogs of ACTH(1-24) at the "message" motif (H⁶F⁷R⁸W⁹) and the "address" motif (K¹⁵K¹⁶R¹⁷R¹⁸P¹⁹). Complete alanine substitution of the H⁶F⁷R⁸W⁹ motif blocked activation, whereas single alanine substitution at this motif indicated the following hierarchy of position importance for activation: W⁹ > R⁸, and substitution at F⁷ and H⁶ had no effect on activation. The same analysis was done on a representative bony vertebrate Mc2r ortholog (Amia calva; bowfin; bf) and the order of position importance for activation was W⁹ > R⁸ = F⁷, (alanine substitution at H⁶ was negligible). Complete alanine substitution at the K¹⁵K¹⁶R¹⁷R¹⁸P¹⁹ motif resulted in distinct outcomes for wsMc2r and bfMc2r. For bfMc2r, this analog blocked activation-an outcome typical for bony vertebrate Mc2r orthologs. For wsMc2r, this analog resulted in a shift in sensitivity to stimulation of the analog as compared to ACTH(1-24) by two orders of magnitude, but the dose response curve did reach saturation. To evaluate whether the EC2 domain of wsMc2r plays a role in activation, a chimeric wsMc2r was made in which the EC2 domain was replaced with the EC2 domain from a melanocortin receptor that does not interact with Mrap1 (i.e., Xenopus tropicalis Mc1r). This substitution did not negatively impact the activation of the chimeric receptor. In addition, alanine substitution at a putative activation motif in the N-terminal of wsMrap1 did not affect the sensitivity of wsMc2r to stimulation by ACTH(1-24). Collectively, these observations suggest that wsMc2r may only have a HFRW binding site for melanocortin-related ligand which would explain how wsMc2r could be activated by either ACTH or MSH-sized ligands.