The ACTH receptor (melanocortin 2 receptor-MC2R) gene only produces a functional receptor when transfected in cells of adrenocortical origin, implying that it may require an adrenal-specific accessory factor. Recently we showed that the melanocortin 2 receptor accessory protein (MRAP) is essential for the cell surface expression of the MC2R in such models. Using RNAi technology we have further explored the action of MRAP in the functioning of the MC2R in Y1 mouse adrenocortical cells that endogenously express MRAP and MC2R. We created stable cell lines expressing mouse MRAP shRNAs by transfecting cells with an expression vector containing the MRAP siRNA sequence. The knockdown of MRAP resulted in a reduction in the MC2R signalling. The over-expression of a mouse MRAP-Flag construct did not restore the expression of MRAP due to its degradation by the mouse shRNAs. The introduction of human MRAP that is resistant to silencing by mouse MRAP shRNAs resulted in the rescue of the MC2R signalling. MRAP protein migrates on SDS PAGE with markedly lower mobility than predicted for a 14.1kDa protein. Co-immunoprecipitation and mass spectroscopy suggests that MRAP exists as a homodimer that is resistant to dissociation by SDS and reducing agents.