The melanocortin-2-receptor accessory protein (MRAP) is a type I integral transmembrane protein required for the functional expression of the melanocortin-2-receptor (MC2R).
Here we have investigated the expression and biochemical properties of mouse MRAP. Initially, mouse MRAP and MC2R tissue expression were determined by RT-PCR, being only present in the adrenal gland and fat tissue. Then, by in situ hybridisation studies using a full-length MRAP digoxigenin (DIG) labelled riboprobe, MRAP expression was highly detected within the adrenal gland, specifically in the adrenal cortex (zona glomerulosa). Furthermore, MRAP protein was characterise by Western blot analysis using specific anti-MRAP polyclonal antibodies (produced in house & commercially). These antibodies detected MRAP as being a 32 kDa protein in extracts from adrenal tissue or Y1 mouse adrenocortical cells, contrasting with the in silico prediction of 14.1 kDa MW protein. To understand/explain this discrepancy, hypothetical MRAP glycosylation and/or homodimerisation were investigated. First, Y1 cells lysates were treated with N-Glycosidase F enzyme, but the protein size was unaffected, suggesting that MRAP is a non glycosylated protein. Then, CHO cells were transiently co-transfected with MRAP-FLAG and MRAP-HA tagged DNA constructs and this lysate was used in co-immunoprecipitation experiments. In these, FLAG antibodies were able to pull down HA tagged MRAP proteins and the HA antibodies were able to pull down FLAG tagged MRAP proteins, showing MRAP-MRAP intracellular interaction which supports its hypothetical dimerisation.
In conclusion, our data indicates that in vivo MRAP transcript is specifically expressed in the adrenal gland cortex and fat tissue and that it is a non-glycosylated protein with the ability to form oligomeric structures.