Structure/Function studies on the MRAP1 of Amia calva

Previous studies of the N-terminal domain of mammalian MRAP1 indicated that the amino acid motifs LDYI (i.e, the activation motif), and YEYY (i.e., the secondary activation motif) are essential for facilitating the activation of mammalian MC2R, a critical component of the mammalian hypothalamus/pituitary/adrenal axis. This study tested the hypothesis that the corresponding motifs (i.e., Y18D19Y20I21, Y14 E15Y16 F17) in the N-terminal of the MRAP1 ortholog of the neopterygian fish, Amia calva (i.e., the bowfin) are essential for facilitating the activation of bowfin MC2R, a critical component of the bowfin hypothalamus/pituitary/interrenal axis. To test this hypothesis a series of alanine-substituted mutants of bfMRAP1 were made. Alanine substitution at every position in the Y18D19Y20I21 motif completely blocked activation of bfMC2R. Single alanine substitution in this motif indicated a gradient in the inhibition of activation: Y18 > D19 > I21 > Y20 (percent inhibition 80% > 52% > 25% > 10%). These results confirm that the Y18D19Y20I21 motif is the activation motif for bfMRAP1. Surprisingly, single alanine substitution at the Y14E15Y16 F17 motif also completely blocked activation. Perhaps this motif is required for the structural integrity of the N-terminal of bfMRAP1. In any event, this study supports the hypothesis that bony vertebrate MRAP1 orthologs must have a δDYδ motif in the N-terminal domain to facilitate the activation of bony vertebrate MC2R orthologs. Hence, the MC2R/MRAP1 heterodimer is a critical component of the hypothalamus/pituitary/adrenal – interrenal axis.