Endocrine Abstracts

Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disease due in ∼25% of cases to defects in the ACTH receptor (melcanocortin 2 receptor -MC2R). Slow progress in characterization of these mutations has been made in view of the difficulty in establishing a functional heterologous cell transfection system for the MC2R, and the best available models relate to the mouse Y6/OS3 cell lines. However we have shown previously that the melanocortin 2 receptor accessory protein (MRAP) acts as an essential accessory protein for the MC2R enabling it to traffic to the cell surface. In this study we have generated clonal CHO and HEK293 stable cell lines expressing human MRAP α or β and these have been used for transient transfection of triple HA-tagged MC2R containing each of the 25 identified naturally occurring mutations of this receptor. Cell surface expression and receptor function can then be assessed. Cell surface expression is readily screened using immunostaining with anti-HA antibody in intact or permeabalised cells followed by quantitation with the LICOR Odysessy plate reader. MC2R-wt traffics efficiently to the cell surface in both MRAPα and MRAPβ stable cell lines, but not in wild type CHO or HEK 293 cells. Some missense mutations such as D20N also seem to be expressed efficiently at the cell surface membrane, whilst the majority of other mutations (e.g. I44M, S74I, D107N and H139Y) are retained within the cell, suggesting that these mutation may have caused a misfolding defect of the receptor or possibly disrupt the MRAP binding site and therefore traffic inefficiently to the cell surface. This model will provide interesting insights into the function of the MC2R and its disruption by mutations found in FGD.