Use of chimeric melanocortin-2 and -4 receptors to identify regions responsible for ligand specificity and dependence on melanocortin 2 receptor accessory protein

The melanocortin 2 (MC₂) receptor differs from other melanocortin family members in its pharmacological profile and reliance on an accessory protein, MC₂ receptor accessory protein (MRAP), for surface expression and signal transduction. To identify features of the MC₂ receptor responsible for these characteristics, we created chimeras between MC₂ and MC₄ receptors and expressed these in CHO cells, where MRAP is essential for trafficking and signaling by MC₂ but not MC₄ receptors. Replacing the first transmembrane segment of the MC₂ receptor with the corresponding region from the MC₄ receptor allowed some surface expression in the absence of an accessory protein, while ACTH-induced cAMP production remained entirely MRAP-dependent. On the other hand, replacing the last two transmembrane domains, third extracellular loop and C-terminal tail of the MC₄ receptor with the corresponding regions from the MC₂ receptor resulted in MRAP-dependent signaling. Surprisingly, replacing the second and third transmembrane domains and the intervening first extracellular loop of MC₂ receptors with MC ₄ sequences generated a chimera (2C2) that responded to both adrenocorticotropic hormone (ACTH) and to the potent MSH analog 4-norleucine-7-d-phenylalanine-α-melanocyte stimulating hormone (NDP-α-MSH), which does not activate native MC₂ receptors. The 2C2 chimeric receptor was able to respond to NDP-α-MSH without MRAP, but MRAP shifted the EC50 value for NDP-α-MSH to the left and caused constitutive activity. These results identify the first transmembrane domain as important for surface expression and regions from the second to third transmembrane segments of the MC₂ receptor as important for MRAP dependent-signal transduction and ligand specificity.