Promiscuous melanocortin receptors in hagfish indicate that receptor function preceded peptide specialization

Hagfishes are representatives of the phylogenetically important, early-branching vertebrate lineage Agnatha, and many endocrine signaling systems in this group remain poorly understood. In this study, we provide the first molecular and functional characterization of melanocortin receptors (Mcrs) and their accessory protein (Mrap) in hagfishes. Using genomic and transcriptomic resources from inshore hagfish (Eptatretus burgeri), Pacific hagfish (Eptatretus stoutii), and Atlantic hagfish (Myxine glutinosa), we identified genes encoding two hagfish melanocortin receptors, Mcar and Mcbr, and a single Mrap. When expressed in mammalian cells, both receptors responded to human ACTH(1-24) and α-MSH with similar affinities. Co-expression with Mrap reduced maximal activity of Mcbr but not Mcar and only modestly modulated the ligand sensitivity of either receptor. Gene expression analyses revealed that mcbr and mrap are prominently expressed in the slime gland, a tissue that also transcriptionally expressed the steroidogenic enzymes star and cyp11a1, whereas mcar is most prominently expressed in the brain. These findings suggest that hagfish Mcrs retain broad ligand responsiveness and relative Mrap-independence, consistent with a hypothesized ancestral mode of melanocortin signaling. Although no genes encoding known melanocortin prohormones have yet been identified in hagfish genomes, the presence of functional receptors and tissue-specific expression patterns suggest these genes may have physiological roles and that an as-yet-unidentified ligand may exist in hagfish. Together, these results provide new insight into the organization and evolution of the vertebrate melanocortin system and highlight hagfish as a key model for reconstructing the functional evolution of this essential endocrine signaling pathway.