The melanocortin 2 receptor accessory protein (MRAP) is essential for the functional expression of the ACTH receptor/melanocortin 2 receptor (MC2R). The pituitary hormone ACTH acts via this complex to stimulate glucocorticoid production in the adrenal cortex. Using the bioluminescence resonance energy transfer (BRET) system we investigated the formation of MC2RMRAP homo/heterodimers in living cells and the influence of ACTH on these interactions. ACTH was found to have a significant effect on the BRET signal in cells expressing MC2R and MRAP. However co-immunoprecipitation of the two proteins showed that ACTH does not increase the physical interaction between the MC2R and MRAP. Real time analysis revealed two distinct phases of the ACTH dependent BRET increase. The antagonist ACTH 1124 only showed the first rapid phase whilst unstimulated cells and cells with ACTH 113 (non binding peptide) did not show either. This led to the suggestion that the first phase may be due to receptor binding leading to a transient conformational change of the MC2R, whilst the second phase of BRET increase, which is unique to ACTH 139 stimulation may result from the signalling by the activated MC2R. This was further supported by the observation that the ACTH-induced BRET signal was significantly reduced in the presence of the pKA inhibitor KT5720 suggesting that the phosphorylation of the receptor may play a significant role in the rearrangement of the pre-existing receptor-MRAP complex.
We have also shown that the MC2R is able to form homodimers with the BRET signal being further enhanced in the presence of ACTH. MRAP was only found to exist as antiparallel homodimers. We found no BRET signal in cells expressing parallel MRAP dimers although the signal appeared enhanced in the presence of the MC2R, which suggested that the MC2R was able to homodimerise bringing the MRAP monomers to closer proximity.