A unique melanocortin-4-receptor signaling profile for obesity-associated constitutively active variants.

The melanocortin-4 receptor (MC4R) plays a critical role in regulating energy homeostasis. Studies on obesogenic human MC4R (hMC4R) variants have not yet revealed how hMC4R maintains body weight. Here, we identified a signaling profile for obesogenic constitutively active H76R and L250Q hMC4R variants transfected in HEK293 cells that included constitutive activity for adenylyl cyclase (AC), cyclic adenosine monophosphate (cAMP) response element (CRE)-driven transcription, and calcium mobilization but not phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) activity. Importantly, the signaling profile included impaired α-melanocyte-stimulating hormone-induced CRE-driven transcription but not impaired α-melanocyte-stimulating hormone-induced AC, calcium, or pERK1/2. This profile was not observed for transfected H158R, a constitutively active hMC4R variant associated with overweight but not obesity. We concluded that there is potential for α-melanocyte-stimulating hormone-induced CRE-driven transcription in HEK293 cells transfected with obesogenic hMC4R variants to be the key predictive tool for determining whether they exhibit loss of function. Furthermore, in vivo, α-melanocyte-stimulating hormone-induced hMC4R CRE-driven transcription may be key for maintaining body weight.

Desacetyl-α-melanocyte stimulating hormone and α-melanocyte stimulating hormone are required to regulate energy balance.

OBJECTIVE: Regulation of energy balance depends on pro-opiomelanocortin (POMC)-derived peptides and melanocortin-4 receptor (MC4R). Alpha-melanocyte stimulating hormone (α-MSH) is the predicted natural POMC-derived peptide that regulates energy balance. Desacetyl-α-MSH, the precursor for α-MSH, is present in brain and blood. Desacetyl-α-MSH is considered to be unimportant for regulating energy balance despite being more potent (compared with α-MSH) at activating the appetite-regulating MC4R in vitro. Thus, the physiological role for desacetyl-α-MSH is still unclear. METHODS: We created a novel mouse model to determine whether desacetyl-α-MSH plays a role in regulating energy balance. We engineered a knock in targeted QKQR mutation in the POMC protein cleavage site that blocks the production of both desacetyl-α-MSH and α-MSH from adrenocorticotropin (ACTH1-39). RESULTS: The mutant ACTH1-39 (ACTHQKQR) functions similar to native ACTH1-39 (ACTHKKRR) at the melanocortin 2 receptor (MC2R) in vivo and MC4R in vitro. Male and female homozygous mutant ACTH1-39 (Pomctm1/tm1) mice develop the characteristic melanocortin obesity phenotype. Replacement of either desacetyl-α-MSH or α-MSH over 14 days into Pomctm1/tm1 mouse brain significantly reverses excess body weight and fat mass gained compared to wild type (WT) (Pomcwt/wt) mice. Here, we identify both desacetyl-α-MSH and α-MSH peptides as regulators of energy balance and highlight a previously unappreciated physiological role for desacetyl-α-MSH. CONCLUSIONS: Based on these data we propose that there is potential to exploit the naturally occurring POMC-derived peptides to treat obesity but this relies on first understanding the specific function(s) for desacetyl-α-MSH and α-MSH.

hMRAPα, but Not hMRAP2, Enhances hMC4R Constitutive Activity in HEK293 Cells and This Is Not Dependent on hMRAPα Induced Changes in hMC4R Complex N-linked Glycosylation.

MRAP1 but not MRAP2, is essential for melanocortin receptor 2 functional expression. Human MRAP1 splice variant (hMRAPα) and human MRAP2 (hMRAP2) also interact with the other melanocortin receptor subtypes in vitro, although the physiological significance of these interactions is unknown. Previously we showed that HA-hMC4R co-expression with hMRAPα, but not hMRAP2, specifically alters HA-hMC4R complex N-linked glycosylation. hMRAPα-FLAG also enhances hMC4R constitutive activity in vitro. Here we directly compare hMRAPα and hMRAP2 effects on hMC4R constitutive activity in HEK293 cells. In contrast to hMRAPα, co-expression with hMRAP2 had no effect on HA-hMC4R or untagged hMC4R constitutive coupling to adenylyl cyclase. We used fixed and live cell imaging of HA-hMC4R and hMC4R-eGFP respectively, to further characterise effects of hMRAPα on hMC4R subcellular trafficking. hMRAPα-FLAG co-expression did not alter the partitioning of either HA-hMC4R or hMC4R-eGFP into either the ER or the Golgi apparatus, therefore the hMRAPα effect on hMC4R complex N-linked glycosylation is probably not due to hMC4R retention in the ER. We also observed that unlike HA-hMC4R, hMC4R-eGFP lacks complex glycosylation both in the presence and absence of hMRAPα, although both HA-hMC4R and hMC4R-eGFP exhibited increased constitutive coupling to adenylyl cyclase following co-expression with hMRAPα. We conclude that hMRAPα and not hMRAP2 modulates hMC4R constitutive activity. Furthermore, hMRAPα does not increase hMC4R constitutive activity by altering hMC4R complex N-linked glycosylation. Instead we hypothesise that hMRAPα alters hMC4R conformational states leading to increased hMC4R constitutive activity.

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.

hMRAPa increases αMSH-induced hMC1R and hMC3R functional coupling and hMC4R constitutive activity.

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.