ELISA versus LUMINEX assay for measuring mouse metabolic hormones and cytokines: sharing the lessons I have learned.

Enzyme-linked immunosorbent assay (ELISA) has long been the standard for quantitative analysis of metabolic hormones and cytokines. LUMINEX multiplex bead array assays were developed as cost- and time-effective alternatives to ELISA, but they are the only cost- and time-effective if they provide informative data. Here, I show that using half-volume of reagents for an adiponectin single-plex LUMINEX assay and a 6-plex LUMINEX xMAP mouse metabolic bead assay, produces reliable data and increases assay cost-effectiveness. I provide direct comparisons between LUMINEX assay and ELISA for quantitation of mouse leptin and insulin, and evaluate glucagon, GLP-1, IL-6, and TNFα data obtained using the 6-plex LUMINEX assay for a high-fat diet-induced obesity study. Good correlations between assays were obtained for fasting leptin and non-fasting insulin. However, the LUMINEX assay proved unsuitable for quantitating fasting insulin. ELISA proved suitable for quantitating fasting male, but not female, insulin. The LUMINEX assay gave lower values for leptin and higher values for insulin, compared with ELISA. The mouse metabolic LUMINEX assay proved unsuitable for quantitating glucagon, GLP-1, IL-6, and TNFα, due to undetectable levels in most fasting and non-fasting plasma. Overall, quantitative leptin levels were the only reliable data obtained from the mouse metabolic LUMINEX assay.

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.

RAMP and MRAP accessory proteins have selective effects on expression and signalling of the CB1 , CB2 , GPR18 and GPR55 cannabinoid receptors.

BACKGROUND AND PURPOSE: Receptor activity-modifying proteins (RAMPs) and melanocortin receptor accessory proteins (MRAPs) modulate expression and signalling of calcitonin and melanocortin GPCRs. Interactions with other GPCRs have also been reported. The cannabinoid receptors, CB1 and CB2 , and two putative cannabinoid receptors, GPR18 and GPR55, exhibit substantial intracellular expression and there are discrepancies in ligand responsiveness between studies. We investigated whether interactions with RAMPs or MRAPs could explain these phenomena. EXPERIMENTAL APPROACH: Receptors and accessory proteins were co-expressed in HEK-293 cells. Selected receptors were studied at basal expression levels and also with enhanced expression produced by incorporation of a preprolactin signal sequence/peptide (pplss). Cell surface and total expression of receptors and accessory proteins were quantified using immunocytochemistry. Signalling was measured using cAMP (CAMYEL) and G protein dissociation (TRUPATH Gα13 ) biosensors. KEY RESULTS: MRAP2 enhanced surface and total expression of GPR18. Pplss-GPR18 increased detection of cell surface MRAP2. MRAP1α and MRAP2 reduced GPR55 surface and total expression, correlating with reduced constitutive, but not agonist-induced, signalling. GPR55, pplss-CB1 and CB2 reduced detection of MRAP1α at the cell surface. Pplss-CB1 agonist potency was reduced by MRAP2 in Gα13 but not cAMP assays, consistent with MRAP2 reducing pplss-CB1 expression. Some cannabinoid receptors increased RAMP2 or RAMP3 total expression without influencing surface expression. CONCLUSIONS AND IMPLICATIONS: Mutual influences on expression and/or function for specific accessory protein-receptor pairings raises the strong potential for physiological and disease-relevant consequences. Sequestration and/or hetero-oligomerisation of cannabinoid receptors with accessory proteins is a possible novel mechanism for receptor crosstalk.

Weight loss effects of quaternary salts of 5-amino-1-(chloromethyl)-1,2-dihydro-3H-benz[e]indoles; structure-activity relationships.

Quaternary salt analogues based on the DNA minor groove binder and adenine N3 alkylating agent 5-amino-1-(chloromethyl)-1,2-dihydro-3H-benz[e]indole (aminoCBI) show remarkable effects on the body weight of mice (a long-term failure to gain weight relative to matched controls with no loss of appetite or perceptible deterioration in health) following administration of a single (non-toxic) dose between about 0.5-5 µmol/kg. The nature of the quaternizing group was not important, but a related hydroxyCBI analogue was much less effective. Compounds where the chloro group was replaced by a hydrogen or hydroxy group (thus abrogating DNA alkylating capability) showed no weight control activity. It is speculated, based on other studies, that the marked long-term weight control effect is due to inhibition of bile flow into the intestine and reduced absorption of triglycerides, together with accelerated cell death in spleen and white adipose tissues due to drug accumulation there. This class of compound may serve as interesting tools for further study of these phenomena.

Functions for pro-opiomelanocortin-derived peptides in obesity and diabetes.

Melanocortin peptides, derived from POMC (pro-opiomelanocortin) are produced in the ARH (arcuate nucleus of the hypothalamus) neurons and the neurons in the commissural NTS (nucleus of the solitary tract) of the brainstem, in anterior and intermediate lobes of the pituitary, skin and a wide range of peripheral tissues, including reproductive organs. A hypothetical model for functional roles of melanocortin receptors in maintaining energy balance was proposed in 1997. Since this time, there has been an extraordinary amount of knowledge gained about POMC-derived peptides in relation to energy homoeostasis. Development of a Pomc-null mouse provided definitive proof that POMC-derived peptides are critical for the regulation of energy homoeostasis. The melanocortin system consists of endogenous agonists and antagonists, five melanocortin receptor subtypes and receptor accessory proteins. The melanocortin system, as is now known, is far more complex than most of us could have imagined in 1997, and, similarly, the importance of this system for regulating energy homoeostasis in the general human population is much greater than we would have predicted. Of the known factors that can cause human obesity, or protect against it, the melanocortin system is by far the most significant. The present review is a discussion of the current understanding of the roles and mechanism of action of POMC, melanocortin receptors and AgRP (agouti-related peptide) in obesity and Type 2 diabetes and how the central and/or peripheral melanocortin systems mediate nutrient, leptin, insulin, gut hormone and cytokine regulation of energy homoeostasis.

Serotonin reciprocally regulates melanocortin neurons to modulate food intake.

The neural pathways through which central serotonergic systems regulate food intake and body weight remain to be fully elucidated. We report that serotonin, via action at serotonin1B receptors (5-HT1BRs), modulates the endogenous release of both agonists and antagonists of the melanocortin receptors, which are a core component of the central circuitry controlling body weight homeostasis. We also show that serotonin-induced hypophagia requires downstream activation of melanocortin 4, but not melanocortin 3, receptors. These results identify a primary mechanism underlying the serotonergic regulation of energy balance and provide an example of a centrally derived signal that reciprocally regulates melanocortin receptor agonists and antagonists in a similar manner to peripheral adiposity signals.

Distribution and function of melanocortin receptors within the brain.

Biological responses to pro-opiomelanocortin (POMC)-derived peptides administered in the brain were documented in the 1950s but their molecular mechanisms of action only began to be resolved with the mapping of melanocortin receptor subtypes to specific brain regions in the 1990s. Out of the five melanocortin receptor subtypes, MC3R and MC4R are widely recognised as 'neural' melanocortin receptors. In situ hybridization anatomical mapping of these receptor subtypes to distinct hypothalamic nuclei first indicated their roles in energy homeostasis, roles that were later confirmed with the obese phenotypes exhibited by Mc3R and Mc4R knockout mice. It is perhaps less well known however, that all five melanocortin receptor subtypes have been detected in developing and/or adult brains of various species. This chapter provides a comprehensive summary of the detection and mapping of each melanocortin receptor subtype in mammalian, chicken and fish brains and relates the sites of expression to functions that are either known or proposed for each receptor subtype.

New Zealand Ginger mouse: novel model that associates the tyrp1b pigmentation gene locus with regulation of lean body mass.

The study of spontaneous mutations in mice over the last century has been fundamental to our understanding of normal physiology and mechanisms of disease. Here we studied the phenotype and genotype of a novel mouse model we have called the New Zealand Ginger (NZG/Kgm) mouse. NZG/Kgm mice are very large, rapidly growing, ginger-colored mice with pink eyes. Breeding NZG/Kgm mice with CAST/Ei or C57BL/6J mice showed that the ginger coat colour is a recessive trait, while the excessive body weight and large body size exhibit a semidominant pattern of inheritance. Backcrossing F1 (NZG/Kgm x CAST/Ei) to NZG/Kgm mice to produce the N2 generation determined that the NZG/Kgm mouse has two recessive pigmentation variant genes (oca2(p) and tyrp-1(b)) and that the tyrp-1(b) gene locus associates with large body size. Three coat colors appeared in the N2 generation; ginger, brown, and dark. Strikingly, N2 male coat colour associated with body weight; the brown-colored mice weighed the most followed by ginger and then dark. The male brown coat-colored offspring reached adult body weights indistinguishable from NZG/Kgm males. The large NZG/Kgm mouse body size is a result of excessive lean body mass since these mice are not obese or diabetic. NZG/Kgm mice exhibit an unusual pattern of fat distribution; compared with other mouse strains they have disproportionately higher amounts of subcutaneous and gonadal fat. These mice are susceptible to high-fat diet-induced obesity but are resistant to high-fat diet-induced diabetes. We propose NZG/Kgm mice as a novel model to delineate gene(s) that regulate 1) growth and metabolism, 2) resistance to Type 2 diabetes, and 3) preferential fat deposition in the subcutaneous and gonadal areas.

Chronic High-Fat Diet Exacerbates Sexually Dimorphic Pomctm1/tm1 Mouse Obesity.

Mice with a targeted mutation in the pro-opiomelanocortin (Pomc) gene (Pomctm1/tm1 mice) are unable to synthesize desacetyl-α-MSH and α-MSH and they develop obesity when fed chow diet. In this study, we hypothesized that a chronic high-fat (HF) diet exacerbates Pomctm1/tm1 mouse obesity. Male and female Pomcwt/wt and Pomctm1/tm1 mice were fed low-fat (LF) (10 kcal percent fat) or HF (45 kcal percent fat) diets from weaning for 23 weeks. We show that Pomctm1/tm1 mouse obesity is sexually dimorphic and exacerbated by an HF diet. Male Pomctm1/tm1 mice develop obesity because they are hyperphagic compared with Pomcwt/wt mice when fed an LF or HF diet. Female Pomctm1/tm1 mice develop obesity when feeding on an LF or HF diet because they exhibit signs of reduced energy expenditure (no change in feed efficiency; body weight gained exceeding energy intake) compared with Pomcwt/wt mice. A chronic HF diet exacerbates male Pomctm1/tm1 and Pomcwt/wt mouse obesity, and the increased energy intake fully accounts for increased weight gain. In contrast, female Pomcwt/wt mice are protected from chronic HF diet-induced obesity because they reduce the amount of HF diet eaten, and they appear to increase their energy expenditure (no change in feed efficiency but energy intake exceeding body weight gained). A chronic HF diet exacerbates female Pomctm1/tm1 mouse obesity due to impaired ability to reduce the amount of HF diet eaten and apparent impaired HF diet-induced adaptive thermogenesis. Our data show that desacetyl-α-MSH and α-MSH are required for sexually dimorphic HF diet-induced C57BL/6J obesity. In conclusion, desacetyl-α-MSH and α-MSH play salutary roles in sexually dimorphic melanocortin obesity and sexually dimorphic HF diet-induced C57BL/6J obesity.

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.

1 kb of 5' flanking sequence from mouse MC4R gene is sufficient for tissue specific expression in a transgenic mouse.

The melanocortin 4 receptor (MC4R) plays a critical role in the regulation of energy homeostasis, and the MC4R knockout mouse and humans with MC4R defective mutations in only one allele indicate that there is a gene dosage effect. Alterations in gene expression levels for MC4R could, therefore, have significant effects on energy homeostasis. To begin to develop a mouse model for studies on MC4R promoter in situ we used approximately 1 kb mouse MC4R promoter together with 426 bp MC4R 5' UTR, previously shown to support basal expression of reporter gene transcription in cell lines with endogenous MC4R mRNA, and fused this DNA to a nuclear localized LacZ reporter gene. The construct was injected into pronuclei from FVB mice. Five transgenic lines were identified as carrying autosomal transgene insertions; three of these had significant beta-galactosidase staining in brain and in a few cells in the heart but not in kidney, liver, lung, gonadal fat or testis. The pattern of transgene expression in the brain differed markedly for the three lines, and in one of these lines was remarkably similar to endogenous MC4R mRNA expression observed using in situ hybridisation. In conclusion, approximately 1 kb mouse MC4R promoter is sufficient to direct gene expression to the brain including regions that express endogenous MC4R mRNA.

Evidence for direct actions of melanocortin peptides on bone metabolism.

Expression of melanocortin-4 receptor (MC4R) mRNA in developing rat limb buds, teeth, and skull bone first indicated a possible role for MC4R in bone metabolism. We therefore investigated whether MC4R mRNA was expressed in the rat osteosarcoma UMR106.06 cell line and in primary rat osteoblast cells. Reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot analysis, and ribonuclease protection assay (RPA) were used to demonstrate MC4R mRNA expression in UMR106.06 and primary osteoblast cells. MC4R mRNA was found to be localized to the periosteum of mouse bone using in situ hybridization. We also used RT-PCR and rat specific MC2R and MC5R oligonucleotides to amplify the correct size DNA fragments for these melanocortin receptors from rat primary osteoblasts. In conclusion, melanocortin receptor expression in mouse periosteum and rat osteoblasts suggests a direct role for POMC derived peptides in bone development and bone metabolism.

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.

Melanocortin-4 receptor messenger ribonucleic acid expression in rat cardiorespiratory, musculoskeletal, and integumentary systems.

We determined melanocortin-4 receptor (MC4-R) mRNA ontogeny in the rat using in situ hybridization and a rat MC4-R riboprobe and showed numerous peripheral sites of expression for MC4-R. The developing heart showed MC4-R mRNA expression as early as embryonic day (E) 14. In the lungs of E16-E20 fetuses, the cells surrounding developing bronchi expressed relatively strong in situ signal. Muscles associated with the respiratory system such as diaphragm and intercostal muscle expressed MC4-R mRNA as early as E14. Occipital and tongue muscles, in particular the genioglossus, showed diffuse signal at E15-E20. In the eye, a discrete signal was detected in an outer neuroblastic layer which may correspond to retina or extraocular muscle. Developing limb buds expressed relatively strong signal at E14, whereas skull bone and joint capsules of the paw of the forelimb showed signal at E18-E20. Using RT-PCR and ribonuclease protection assays, we determined that MC4-R mRNA is also expressed in adult rat heart, lung, kidney, and testis. The expression of the MC4-R in cardiorespiratory, musculoskeletal, and integumentary systems supports functional roles for the MC4-R in addition to its roles in appetite, weight control, and regulation of linear growth.

Expression of melanocortin 4 receptor mRNA in the central nervous system of the rat.

The melanocortin 4 receptor (MC4-R) plays a pivotal role in maintaining energy homeostasis in rodents and humans. For example, MC4-R deletion or mutation results in obesity, hyperphagia, and insulin resistance. Additionally, subsets of leptin-induced autonomic responses can be blocked by melanocortin receptor antagonism, suggesting that MC4-R-expressing neurons are downstream targets of leptin. However, the critical autonomic control sites expressing MC4-Rs are still unclear. In the present study, we systematically examined the distribution of MC4-R mRNA in the adult rat central nervous system, including the spinal cord, by using in situ hybridization histochemistry (ISHH) with a novel cRNA probe. Autonomic control sites expressing MC4-R mRNA in the hypothalamus included the anteroventral periventricular, ventromedial preoptic, median preoptic, paraventricular, dorsomedial, and arcuate nuclei. The subfornical organ, dorsal hypothalamic, perifornical, and posterior hypothalamic areas were also observed to express MC4-R mRNA. Within extrahypothalamic autonomic control sites, MC4-R-specific hybridization was evident in the infralimbic and insular cortices, bed nucleus of the stria terminalis, central nucleus of the amygdala, periaqueductal gray, lateral parabrachial nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus (DMV), and intermediolateral nucleus of the spinal cord (IML). By using dual-label ISHH, we confirmed that the cells expressing MC4-R mRNA in the IML and DMV were autonomic preganglionic neurons as cells in both sites coexpressed choline acetyltransferase mRNA. The distribution of MC4-R mRNA is consistent with the proposed roles of central melanocortin systems in feeding and autonomic regulation.

alpha-MSH and desacetyl-alpha-MSH signaling through melanocortin receptors.

The functional significance of N-terminal acetylation of ACTH[1-13]NH(2) is unknown. N-terminal acetylation of ACTH[1-13]NH(2) (known as desacetyl-alpha-MSH) to produce alpha-MSH enhances some activities of ACTH[1-13]NH(2) and virtually eliminates others. To determine whether alpha-MSH and desacetyl-alpha-MSH diverge in their coupling to melanocortin receptors in vitro, we measured the sensitivity of MC1, MC3, MC4, and MC5 receptors stably expressed in HEK293 cells to these peptides, functionally coupling them to adenylyl cyclase and a calcium signaling pathway. alpha-MSH and desacetyl-alpha-MSH similarly coupled these overexpressed receptors to both signaling pathways. In contrast, we discovered that alpha-MSH significantly increased primary rat osteoblast proliferation while for desacetyl-alpha-MSH there was only a trend to do the same. Osteoblast cells expressing very low levels of endogenous melanocortin receptors, in contrast with transfected HEK293 cells overexpressing a single melanocortin receptor, may provide an in vitro model for differentiating between alpha-MSH and desacetyl-alpha-MSH signaling.

Melanocortin-3 receptor gene variants in a Maori kindred with obesity and early onset type 2 diabetes.

Genetic studies suggest a diabetes susceptibility locus on human chromosome 20, near the melanocortin receptor-3 (MC3-R) gene. We examined the MC3-R as a candidate gene for type 2 diabetes in 12 members of a large Maori kindred with multiple affected members. The coding region of the MC3-R gene was sequenced for both diabetic and non-diabetic individuals. Two separate single base pair substitutions were found in the MC3-R coding sequence and these resulted in amino acid changes, Lysine6Threonine and Isoleucine81Valine. Neither of these MC3-R variants tracked with the presence of diabetes. Furthermore, the variant and wild type MC3-R showed similar functional coupling to adenylyl cyclase. A polymorphic marker (D20S32e) close to the human MC3-R (hMC3-R) coding sequence was investigated in a 60-member pedigree for association with diabetes and other metabolic parameters. There was an association between D20S32e genotype and fasting insulin (P=0.0085) and the insulin resistance index, HOMA-R (P=0.0042). An association was also found between genotype and HDL levels during oral glucose tolerance testing (P=0.0037). These associations were independent of BMI, age, gender and diabetes. Our data do not support a role for variations in the coding region of the hMC3-R in the development of type 2 diabetes in this Maori kindred, but suggest that a locus on chromosome 20 q, close to D20S32e, may contribute to both insulin secretion and action in the Maori kindred.

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.