hMRAPa specifically alters hMC4R molecular mass and N-linked complex glycosylation in HEK293 cells

Human melanocortin 2 receptor accessory protein 1(hMRAPa) is essential for human melanocortin 2 receptor (hMC2R)-regulated adrenal steroidogenesis. hMRAPa enhances hMC2R N-linked glycosylation and maturation, promotes hMC2R cell surface expression and enables ACTH to bind and activate the MC2R. However, hMRAPa is predicted to have functions beyond its critical role in hMC2R activity. It is more widely expressed than the hMC2R and it has been shown to co-immunoprecipitate with all other hMCR subtypes and other G-protein-coupled receptors, when these are co-expressed with each receptor in heterologous cells. The physiological relevance of hMRAPa interactions with these receptors is unknown. We hypothesised that hMRAPa could influence post-translational processing and maturation of these receptors, similar to its actions on the hMC2R. Here we used co-immunoprecipitation and western blotting techniques to characterise effects of hMRAPa-FLAG co-expression on the maturation of each HA-tagged hMCR subtype and the HA-tagged human calcitonin receptor-like receptor (hCL), co-expressed in HEK293 cells. While hMRAPa-FLAG interacted with all five HA-hMCR subtypes and the HA-hCL, it only altered HA-hMC4R molecular mass. This altered HA-hMC4R molecular mass was due to a change in endoglycosidase H-resistant complex N-linked glycosylation, which we observed for HA-hMC4R in both intracellular and cell surface fractions. This effect was specific to the HA-hMC4R as hMRAPa did not alter the molecular mass of any of the other receptors that we examined. In conclusion, the specific effects of hMRAPa on hMC4R molecular mass and complex N-linked glycosylation provide evidence in support of a role for MRAPα in hMC4R functions.