Analyzing the Interactions Between Xenopus tropicalis MC2R, MC5R, and the MRAPs: Modeling the Regulation of Frog Interrenal Cells

The role of Melanocortin 2 Receptor (MC2R) in adrenal/interrenal glucocorticoid secretion has been well documented in many organisms. Studies in mammals have shown that in the adrenal gland two melanocortin receptors and two melanocortin receptor accessory proteins are expressed: MC2R, MC5R, MRAP, and MRAP2. The MRAPs have an opposite effect on the cell surface expression of MC2R and MC5R. In mammals, MRAP aids MC2R but inhibits MC5R cell surface expression. This thesis aims to explore the functional relationship between MC2R, MC5R, MRAP, and MRAP2 in Xenopus tropicalis to determine if the MRAPs have a similar effect on amphibian MC2R and MC5R when the receptors are stimulated with ACTH, or -MSH. Expression of these four genes in the frog interrenal was verified through RT-PCR. Then CHO-K1 cells were transfected with plasmid constructs containing X. tropicalis MC2R, MC5R, MRAP2, and a mouse MRAP, and stimulated with ACTH or -MSH. Levels of receptor activation were measured using a CRE-luciferase reporter gene. The findings presented here indicate that in the frog the MRAPs do not have a negative effect on MC5R, since activation of MC5R was not inhibited in the presence of MRAPs. The sensitivity of MC5R for ACTH increased from an EC50 of 1.2 x 10 -9 M to an EC50 of 8.7 x 10 -11 M in the presence of MRAP (p<0.05) and to an EC50 of 1.3 x 10 -10 M in the presence of MRAP2 (p<0.01).