Familial Glucocorticoid Deficiency type 2 (patients with normal MC2R) is associated with mutations in the MRAP (Melanocortin Receptor Accessory Protein) gene. In order to investigate the function of this novel single transmembrane domain protein, CHO and SKN-SH cells were transfected with MRAP-FLAG and/or MC2R-GFP constructs and imaged using confocal microscopy. Although the MC2R-GFP failed to be expressed at the cell surface when transfected alone, it was found to be expressed at the cell surface in the presence of MRAP implying that MRAP may be involved in the trafficking or folding and processing of the MC2R. (L A Metherell, et al. (2005) Nature Genetics 37, 166 170) Co-immunoprecipitation studies revealed an interaction between the MC2R and MRAP. Co-transfection of MC2R and MRAP generated a functional cell surface receptor as indicated by the enhanced cAMP response to ACTH in SKN-SH and CHO cells and to a limited extent in HEK293 and Hela cells. RNA interference studies were carried out using MRAP siRNA transiently transfected into Y1 adrenocortical cells, which endogenously express both a functional MC2R and MRAP. The knockdown of MRAP expression in this cell line resulted in the reduction of the MC2R mediated signalling as determined by the use of a CRE-luciferase reporter assay. The production of cAMP through MC1R, MC3R, MC4R or MC5R was not enhanced in the presence of MRAP when stimulated with NDP-MSH. However, co-immunoprecipitation studies showed an interaction between MRAP and MC1R. In summary MRAP plays a role in the cell surface expression of a functional MC2R, and may also interact with the other melanocortin receptors.