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Human melanocortin 2 receptor accessory protein (hMRAPa) is hypothesised to have functions beyond promoting human melanocortin 2 receptor (hMC2R) functional expression. To understand these potential functions, we exogenously co-expressed hMRAPa-FLAG with each of the five hMCR subtypes in HEK293 cells and assessed hMCR subtype coupling to adenylyl cyclase. We also co-expressed each HA-hMCR subtype with hMRAPa-FLAG to investigate their subcellular localisation. hMRAPa-FLAG enhanced α-melanocyte stimulating hormone (α-MSH)-stimulated hMC1R and hMC3R but reduced NDP-α-MSH-stimulated hMC5R, maximum coupling to adenylyl cyclase. hMRAPa-FLAG specifically increased hMC4R constitutive coupling to adenylyl cyclase despite not co-localising with the HA-hMC4R in the cell membrane. hMRAPa-FLAG co-localised with HA-hMC1R or HA-hMC3R in the perinuclear region, in cytoplasmic vesicles and at the plasma membrane, while it co-localised with HA-hMC2R, HA-hMC4R and HA-hMC5R predominantly in cytoplasmic vesicles. These diverse effects of hMRAPa indicate that hMRAPa could be an important modulator of the central and peripheral melanocortin systems if hMRAPa and any hMCR subtype co-express in the same cell.

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.

The human melanocortin-4 receptor plays a critical role regulating energy homeostasis. Studies on obesogenic hMC4R variants have not yet revealed how human melanocortin-4 receptor maintains body weight. Here, we identified a signaling profile for obesogenic constitutively active H76R and L250Q human melanocortin-4 receptor variants transfected in HEK293 cells that included constitutive activity for adenylyl cyclase, cyclic adenosine monophosphate response element-driven transcription, and calcium mobilization, but not phosphorylated extracellular signal-regulated kinase 1/2 activity. Importantly, the signaling profile included impaired α-melanocyte-stimulating-hormone-induced cyclic adenosine monophosphate response element-driven transcription, but not impaired α-melanocyte-stimulating-hormone-induced adenylyl cyclase, calcium, or phosphorylated extracellular signal-regulated kinase 1/2. This profile was not observed for transfected H158R, a constitutively active human melanocortin-4 receptor variant associated with overweight, but not obesity. We concluded that there is potential for α-melanocyte-stimulating-hormone-induced cyclic adenosine monophosphate response element-driven transcription in HEK293 cells transfected with obesogenic human melanocortin-4 receptor variants to be the key predictive tool for determining whether they exhibit loss-of-function. Furthermore, in vivo α-melanocyte-stimulating-hormone-induced human melanocortin-4 receptor cyclic adenosine monophosphate response element-driven transcription may be key for maintaining body weight.