HPA/HPI Axis: Analyzing the Co- Evolution of the Melanocortin-2 Receptor (MC2R) and the Accessory Protein MRAP1 – from Cartilaginous Fishes to Mammals

Presentation Number: MON 369
Date of Presentation: April 3rd, 2017

Robert M Dores*1, Megan Deyarmond1, Perry V Davis1, Michael R Dores2, Ayuke Iki3 and Susumu Hyodo3
1University of Denver, Denver, CO, 2Hofstra University, Hempstead, NY, 3University of Tokyo, Chiba, Japan


A striking feature of the mammalian HPA axis, is the obligatory interaction between the melanocortin-2 receptor (MC2R) and the accessory protein, MRAP1 to facilitate trafficking of the receptor from the ER to the PM of adrenal cortex cells, and for the exclusive activation of MC2R by ACTH, but not by any MSH-sized ligand (1). While the same obligatory interaction between MC2R and MRAP1 is observed for other bony vertebrates (i.e., bony fishes, amphibians, reptiles, and birds), the same cannot be said for cartilaginous fishes. Recent studies on MC2R orthologs from the elephant shark (es) and the Japanese stingray showed that these receptors could be functionally expressed in CHO cells in the absence of MRAP1, and activated by either ACTH or various cartilaginous fish MSH-related peptides (2&3). These observations led to the assumption that the mrap1 gene evolved after the divergence of the ancestral cartilaginous fishes and bony fishes. However, the recent discovery of an MRAP1 ortholog in the genome of the elephant shark indicates that this gene evolved prior to the divergence of cartilaginous and bony fishes. To test whether esMRAP1 has an effect on either ligand sensitivity or ligand selectivity, esmrap1 and esmc2r constructs were transiently transfected in CHO cells and stimulated with either cartilaginous fish ACTH (1-24) or ACTH(1-13)NH2.In these experiments, co-expression with esMRAP1 increased sensitivity for ACTH(1-24) 10 fold, and sensitivity for ACTH(1-13)NH2 4 fold. rtPCR analysis indicated that mc2r, mrap1, as well as mc3r, and mc5r mRNAs are present in the interrenal gland, the glucocorticoid-producing tissue of the elephant shark. In this regard, co-expression of esmc5r and esmrap1 in CHO cells resulted in a 100 fold increase in sensitivity for ACTH(1-24). Immunocytochemical analysis indicated that esMC2R and esMRAP1 are co-localized on the surface of the CHO cells. Alanine substitutions in the N-terminal domain esMRAP1 followed by co-expression of the mutant esMRAP1 with wild type esMC2R revealed the importance of residues E38 and Y39 for activation. These residues are located in a site analogous to the activation motif for mouse MRAP1 (4). Collectively these results indicate that the interaction between MRAP1 and MC2R arose early in the evolution of the jawed vertebrates. In addition, multiple melanocortin receptors are expressed in glucocorticoid cells of the elephant shark, and regulation of glucocorticoid release could be mediated by separate hypothalamus/anterior pituitary, and hypothalamus/intermediate pituitary axes. Finally studies on cartilaginous fish MC2R and MRAP1 orthologs provide a reference point for understanding the unique co-evolution of this receptor and this accessory protein in modern bony fishes and tetrapods.


Nothing to Disclose: RMD, MD, PVD, MRD, AI, SH