Trends in the evolution of the elasmobranch melanocortin-2 receptor: Insights from structure/function studies on the activation of whale shark Mc2r.

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 (H ⁶ F ⁷ R ⁸ W ⁹ ) and the "address" motif (K ¹⁵ K ¹⁶ R ¹⁷ R ¹⁸ P ¹⁹ ). Complete alanine substitution of the H ⁶ F ⁷ R ⁸ W ⁹ motif blocked activation, whereas single alanine substitution at this motif indicated the following hierarchy of position importance for activation: W ⁹  > R ⁸ , and substitution at F ⁷ and H ⁶ 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 W ⁹  > R ⁸  = F ⁷ , (alanine substitution at H ⁶ was negligible). Complete alanine substitution at the K ¹⁵ K ¹⁶ R ¹⁷ R ¹⁸ P ¹⁹ 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.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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