Endocrine Abstracts

MC2R is the smallest member of the GPCR superfamily and belongs to the melanocortin subfamily of receptors. The pituitary hormone ACTH acts through MC2R to induce the intracellular production cAMP and the stimulation of steroidogenesis. We have previously shown that MC2R interacts with a single-transmembrane domain protein called MRAP (melanocortin-2 receptor accessory protein) and is required for the functional expression of MC2R. Both MC2R and MRAP are mutated in the rare recessive disorder FGD (familial glucocorticoid deficiency). In an effort to characterise the role of MRAP required in the localisation and function of MC2R we have produced a series of MRAP constructs with truncations of the N- and C-terminus. Co-immunoprecipitation with these constructs and MC2R reveal the transmembrane domain of MRAP to be sufficient for interaction with MC2R. Interestingly, we have also identified the transmembrane domain as the site of MRAP homodimerisation. To address the role of MRAP in the plasma membrane expression and consequent formation of a functional receptor we have used a combination of confocal microscopy, a fluorescent cell-surface assay and cAMP reporter assay. In the presence of MRAP approximately 40% of the total MC2R protein is detected at the plasma membrane. This expression, along with the production of cAMP following ACTH stimulation is lost when part of the N-terminus is deleted. Scanning mutagenesis of the N-terminus has allowed further definition of this domain. When co-expressed with a C-terminal MRAP deletion construct 60% of MC2R is detected at the cell-surface, implying the presence of a domain involved in the negative regulation of MC2R plasma membrane expression. In summary, we have identified the MRAP transmembrane domain as the MC2R interaction domain, a cell-surface expression motif in the MRAP N-terminus and a possible role for the C-terminus in the negative regulation of cell-surface expression.