Evolution of Xenopus Tropicalis MC2R: Expression and Activation

The structure and functional relationship between the melanocortin-2 receptor (MC2R) and adrenocorticotropic hormone (ACTH) is the most complex of the melanocortin gene family. Prior studies had been done on amniote tetrapod MC2Rs (e.g., mammals); this study analyzed the expression and activation of MC2R by an anamniote tetrapod, Xenopus tropicalis (xtMC2R). An immunofluorescence approach, done on the expression of xtMC2R in Chinese Hamster Ovary cells (CHO cells), indicated that the trafficking of xtMC2R to the plasma membrane required co-expression with a tetrapod MRAP1(melanocortin-1 receptor accessory protein). A cAMP-reporter assay was used to show that xtMC2R can activated by human ACTH(1-24, but not by α-MSH. These two properties are also observed for human MC2R, and are common for tetrapod MC2Rs in general. Alanine-substitution analogs of hACTH(1-24) were used to deduce a possible mechanism for the activation of xtMC2R. These studies showed that alanine substitutions to the HFRW motif in hACTH(1-24) eliminated activation of the receptor. Furthermore, the alanine-substitution analysis revealed that positions 15 and 16 in the KKRRP motif are more important for the activation of xtMC2R than positions, 17 through 19. Finally, the alanine-substitution assays coupled with analysis of internally truncated analogs of the GKPVG motif resulted in decreased or complete elimination of xtMC2R activation. These data were used to construct a proposed three step model for the activation of MC2R. The final goal of this thesis was to identify the region of the receptor involved in the docking of the KKRRP motif of ACTH. Based on a model of MC2R, these experiments used alanine substitution site-directed mutagenesis to analyze the transmembrane 4 (TM4), extracellular loop 2 (EL2), and transmembrane 5 (TM5) region of xtMC2R. These experiments revealed that the following mutations had the greatest effect on the sensitivity (EC50 value) of xtMC2R: I/A175, F/A178, and I/A184. These results were compared to site-directed mutagenesis studies done on human and rainbow trout MC2Rs. Collectively, these analyses revealed that all three MC2Rs have docking sites for the KKRRP motif of ACTH that are similar in general location, and mostly similar in 3-dimensional structure, but that are not identical. The evolutionary implications of these observations are discussed