Structure/function Studies on the Activation of the Rainbow Trout Melanocortin-2 Receptor

Functional expression of the rainbow trout (rt) melanocortin-2 receptor (MC2R) in CHO cells requires co-expression with a teleost melanocortin-2 receptor accessory protein (MRAP) such as zebrafish (zf) MRAP. Transiently transfected rtMC2R/zfMRAP1 CHO cells were used to evaluate the efficacy of alanine substituted analogs of hACTH(1-24) in three motifs in the ligand: H6F7R8W9, G10K11P12V13G14, and K15K16R17R18P19. Alanine substitution at all positions in each motif either completely blocked activation of the receptor (H6F7R8W9 and K15K16R17R18P19) or resulted in just over 400 fold increase in EC50 value (G10K11P12V13G14). Single alanine substitutions in the H6F7R8W9 motif indicated that substitution at either W9 or R8 resulted in a much larger increase in EC50 values as compared to substitutions at either F7 or W9. Alanine substitution at either K15K16 or R17R18P19 in the K15K16R17R18P19 motif resulted in a statistically equivalent increase in EC50 value of at least 600 fold. Finally, alanine substitutions in the G10K11P12V13G14 motif resulted in increases in EC50 values presumably as a result of altering the secondary structure of the ligand. However, truncated analogs of hACTH(1-24) in which either G10G14 (ACTH(1-22), or K11P12V13 (ACTH(1-21) were removed had no stimulatory activity. Finally, some of the hACTH(1-24) analogs were tested using rainbow trout head kidney pieces in vitro to confirm whether the response to analogs seen with the transient transfected rtMC2R CHO cells was similar to that of trout interrenal cells. The results of these alanine substitution analog studies are used to construct a multistep hypothetical model for the activation of teleost and tetrapod MC2Rs to account for the unique ligand selectivity of this receptor