Melanocortin 1 receptor mutations impact differentially on signalling to the cAMP and the ERK mitogen-activated protein kinase pathways

Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor expressed in melanocytes, is a major determinant of skin pigmentation, phototype and cancer risk. MC1R activates cAMP and mitogen-activated protein kinase ERK1/ERK2 signalling. When expressed in rat pheochromocytoma cell line cells, the R151C, R160W and D294H MC1R variants associated with melanoma and impaired cAMP signalling mediated ERK activation and ERK-dependent, agonist-induced neurite outgrowth comparable with wild-type. Dose-response curves for ERK activation and cAMP production indicated higher sensitivity of the ERK response. Thus, the melanoma-associated MC1R mutations impact differently on cAMP and ERK signalling, suggesting that cAMP is not responsible for functional coupling of MC1R to the ERK cascade.     

Pivotal roles of alpha-melanocyte-stimulating hormone and the melanocortin 4 receptor in leptin stimulation of prolactin secretion in rats

Leptin, the obese gene product, was reported to stimulate prolactin (PRL) secretion, but the neuroendocrine mechanism underlying this hormonal response is largely unknown. Thus, in this study we examined the involvement of several important PRL regulators in the leptin-induced PRL secretion in male rats. Compared with the values in normally fed rats, food deprivation for 3 days significantly decreased both PRL and leptin levels in the plasma. These changes were reverted to normal by a 3-day constant infusion of 75 microg/kg/day of leptin to the fasted rats, while 225 microg/kg/day of leptin further elevated both PRL and leptin levels. These four groups of animals were used for the following experiments. Results of dopamine and serotonin turnover studies in the brain and the pituitary indicated that neither of these biogenic amines plays a primary role in mediating leptin's effects on PRL. Repeated intracerebroventricular injections over 72 h of neutralizing antibodies against vasoactive intestinal peptide, PRL-releasing peptide, or beta-endorphin, did not significantly suppress the leptin actions. However, both the blockade of the melanocortin (MC) 4 receptor (R) and the immunoquenching of brain alpha-melanocyte-stimulating hormone (alpha-MSH) completely abolished the leptin-induced PRL release, and the stimulation of the MC4-R, but not the MC3-R, significantly elevated PRL levels in the fasted rats. These results suggest that alpha-MSH, a cleaved peptide from pro-opiomelanocortin of which synthesis is stimulated by leptin, may be the pivotal neuropeptide in the brain mediating the leptin's stimulatory influence on PRL secretion. It was also suggested that the MC4-R may be the primary subtype of the MC-Rs mediating this action of alpha-MSH.     

Human melanocortin 1 receptor-mediated ubiquitination of nonvisual arrestins. Role of Mahogunin Ring Finger 1 E3 ligase

Signaling from the melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor (GPCR) crucial for melanocyte proliferation and differentiation, is regulated by cytosolic β-arrestins (ARRBs). MC1R signaling is also negatively modulated by the E3-ubiquitin ligase Mahogunin Ring Finger-1 (MGRN1), whose mutation causes hyperpigmentation, congenital heart defects and neurodegeneration in mice. We showed previously that although MC1R interacts stably with human ARRB1 or ARRB2, only ARRB2 mediates receptor desensitization and internalization. We analyzed MC1R-dependent ARRB ubiquitination, and the possible role of MGRN1. ARRB1 expressed in heterologous cells or human melanoma cells migrated in SDS-PAGE as a 55 kDa protein whereas ARRB2 migrated as two major bands of apparent molecular weight near 45 and 55 kDa, with an intermediate mobility band occasionally detected. These forms were related by post-translational modification rather than by proteolysis. Presence of MC1R favored expression of the 45 kDa protein, the form that interacted preferentially with MC1R. MC1R also mediated poly- or multimonoubiquitination of ARRB2. Ubiquitination was agonist-independent, but required a native MC1R conformation and/or normal receptor trafficking to the plasma membrane, as it was not observed for loss-of-function MC1R variants. In a heterologous expression system, MC1R-dependent ARRB ubiquitination was enhanced by overexpression of MGRN1 and was impaired by siRNA-mediated MGRN1 knockdown thus pointing to MGRN1 as the responsible E3-ligase. Co-immunoprecipitation experiments demonstrated interaction of MGRN1 and ARRBs in the presence of MC1R, suggesting a scaffolding role for the GPCR that may determine the selectivity of E3-ubiquitin ligase engagement and the functional outcome of ARRB ubiquitination.     

High diversity in functional properties of melanocortin 1 receptor (MC1R) in divergent primate species is more strongly associated with phylogeny than coat color

We have characterized the biochemical function of the melanocortin 1 receptor (MC1R), a critical regulator of melanin synthesis, from 9 phylogenetically diverse primate species with varying coat colors. There is substantial diversity in melanocyte-stimulating hormone (MSH) binding affinity and basal levels of activity in the cloned MC1Rs. MSH binding was lost independently in lemur and New World monkey lineages, whereas high basal levels of MC1R activity occur in lemurs and some New World monkeys and Old World monkeys. Highest levels of basal activity were found in the MC1R of ruffed lemurs, which have the E94K mutation that leads to constitutive activation in other species. In 3 species (2 lemurs and the howler monkey), we report the novel finding that binding and inhibition of MC1R by agouti signaling protein (ASIP) can occur when MSH binding has been lost, thus enabling continuing regulation of the melanin type via ASIP expression. Together, these findings can explain the previous paradox of a predominantly pheomelanic coat in the red ruffed lemur (Varecia rubra). The presence of a functional, MSH-responsive MC1R in orangutan demonstrates that the mechanism of red hair generation in this ape is different from the prevalent mechanism in European human populations. Overall, we have found unexpected diversity in MC1R function among primates and show that the evolution of the regulatory control of MC1R activity occurs by independent variation of 3 distinct mechanisms: basal MC1R activity, MSH binding and activation, and ASIP binding and inhibition. This diversity of function is broadly associated with primate phylogeny and does not have a simple relation to coat color phenotype within primate clades.     

Membrane orientation and oligomerization of the melanocortin receptor accessory protein 2

The melanocortin receptor accessory protein 2 (MRAP2) plays a pivotal role in the regulation of several G protein–coupled receptors that are essential for energy balance and food intake. MRAP2 loss-of-function results in obesity in mammals. MRAP2 and its homolog MRAP1 have an unusual membrane topology and are the only known eukaryotic proteins that thread into the membrane in both orientations. In this study, we demonstrate that the conserved polybasic motif that dictates the membrane topology and dimerization of MRAP1 does not control the membrane orientation and dimerization of MRAP2. We also show that MRAP2 dimerizes through its transmembrane domain and can form higher-order oligomers that arrange MRAP2 monomers in a parallel orientation. Investigating the molecular details of MRAP2 structure is essential for understanding the mechanism by which it regulates G protein–coupled receptors and will aid in elucidating the pathways involved in metabolic dysfunction.     

MTSEA prevents ligand binding to the human melanocortin-4 receptor by modification of cysteine 130 in transmembrane helix 3

We have investigated the effect of the sulfhydryl-reactive reagent, methyl thiosulfonate ethylammonium (MTSEA), on ligand binding to the human melanocortin-4 (MC4) receptor stably expressed in HEK-293 cells. MTSEA inhibited binding of the agonist, 125I-NDPalpha-MSH, and the antagonist, 125I-SHU9119, in a concentration-dependent manner. Pre-incubation of cells with either the agonist or antagonist protected from subsequent MTSEA inhibition of radioligand binding. Mutation of Cys130 in transmembrane helix 3 to alanine, whilst not affecting ligand binding, led to a complete loss of the inhibitory effect of MTSEA. Since other types of sulfhydryl-reactive reagents had no effect on ligand binding, we conclude that covalent modification of Cys130 by MTSEA disrupts ligand binding by neutralising a close-by negative charge, most likely on Asp126.     

Interactions of the Melanocortin-4 Receptor with the Peptide Agonist NDP-MSH

Melanocortin-4 receptor (MC4R) has an important regulatory role in energy homeostasis and food intake. Peptide agonists of the MC4R are characterized by the conserved sequence His₆-Phe₇-Arg₈-Trp₉, which is crucial for their interaction with the receptor. This investigation utilized the covalent attachment approach to identify receptor residues in close proximity to the bound ligand [Nle₄,d-Phe₇]melanocyte-stimulating hormone (NDP-MSH), thereby differentiating between residues directly involved in ligand binding and those mutations that compromise ligand binding by inducing conformational changes in the receptor. Also, recent X-ray structures of G-protein-coupled receptors were utilized to refine a model of human MC4R in the active state (R^?), which was used to generate a better understanding of the binding mode of the ligand NDP-MSH at the atomic level.The mutation of residues in the human MC4R—such as Leu106 of extracellular loop 1, and Asp122, Ile125, and Asp126 of transmembrane (TM) helix 3, His264 (TM6), and Met292 (TM7)—to Cys residues produced definitive indications of proximity to the side chains of residues in the core region of the peptide ligand. Of particular interest was the contact between d-Phe₇ on the ligand and Ile125 of TM3 on the MC4R. Additionally, Met292 (TM7) equivalent to Lys(7.45) (Ballesteros numbering scheme) involved in covalently attaching retinal in rhodopsin is shown to be in close proximity to Trp₉.For the first time, the interactions between the terminal regions of NDP-MSH and the receptor are described. The amino-terminus appears to be adjacent to a series of hydrophilic residues with novel interactions at Cys196 (TM5) and Asp189 (extracellular loop 2). These interactions are reminiscent of sequential ligand binding exhibited by the β₂-adrenergic receptor, with the former interaction being equivalent to the known interaction involving Ser204 of the β₂-adrenergic receptor.     

Evidence for constitutive dimerization of niacin receptor subtypes

The recently deorphanized niacin receptor subtypes NIACR1 (GPR109A) and NIACR2 (GPR109B) play an essential role in the regulation of metabolic processes and immune reactions. Both receptors belong to the G-protein-coupled receptor (GPCR) family, whose members have traditionally been treated as monomeric entities, but now appear to exist and function as both homodimers and heterodimers. In this study, a close physical interaction is shown between the highly homologous niacin receptor subtypes, NIACR1 and NIACR2, using bioluminescence resonance energy transfer (BRET²) in living cells. The extent of homo- and hetero-dimerization of the niacin receptors did not vary after activation of the receptors with selective agonists, indicating that the dimerization state of NIACR1 and NIACR2 is not regulated by ligand binding. Moreover, detection of niacin receptor dimers in both plasma membrane- and endoplasmic reticulum-enriched fractions suggests that they are formed early in the biosynthetic pathway. Taken together, these results demonstrate that niacin receptor dimerization is a constitutive process occurring early during biosynthesis.     

Diffusion of the mu opioid receptor at the surface of human neuroblastoma SH-SY5Y cells is restricted to permeable domains

Previous single-molecule studies have shown a long-term diffusion superimposed to a short-term confinement of the human mu opioid (hMOP) receptors at the surface of heterologous cells. However, additional ensemble average measurements are required to reach a complete understanding of the undergoing process. Here, we analyse, by fluorescence recovery after photobleaching measurements, the lateral diffusion of fully functional T7-EGFP-hMOP receptors in neuroblastoma SH-SY5Y cells naturally expressing a low level of the wild-type receptor. Experiments carried out at variable observation radii demonstrate the restriction of the receptors diffusion to sub-micrometer sized domains. Furthermore, consistently with the long-term single-molecule data, the domains are found permeable.     

Single amino acid of an octopamine receptor as a molecular switch for distinct G protein couplings

Octopamine (OA) is thought to be the invertebrate counterpart of noradrenaline and regulates various behavioral patterns of invertebrates by activating OA receptors. As a typical G protein-coupled receptor, BmOAR1, a Bombyx mori α-adrenergic-like OA receptor, is coupled to both G_s and G_q proteins to induce the release of the intracellular second messengers cAMP and Ca²⁺. In this study, we examined the pharmacological and functional properties of the cloned OA receptor, using OA enantiomers. The wild-type OA receptor exhibited significant stereoselectivity for OA enantiomers in cAMP production and binding affinity, but not in calcium signaling response. On the contrary, the Y412F mutant abolished the discrimination between OA enantiomers in the binding affinity and did not evoke any cAMP signaling response. This mutant exhibited levels of potency and efficacy similar to those of the wild-type receptor in the calcium assays. Taken together, these results suggest that Tyr412 might act as a molecular switch to regulate distinct G protein couplings, and a sequential activation model is proposed for such specific-residue-dependent, selective activation in receptors that are coupled to multiple G proteins.     

Functional studies on twenty novel naturally occurring melanocortin-4 receptor mutations

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor critically involved in regulating energy balance. MC4R activation results in decreased food intake and increased energy expenditure. Genetic and pharmacological studies demonstrated that the MC4R regulation of energy balance is conserved from fish to mammals. In humans, more than 150 naturally occurring mutations in the MC4R gene have been identified. Functional study of mutant MC4Rs is an important component in proving the causal link between MC4R mutation and obesity as well as the basis of personalized medicine. In this article, we studied 20 MC4R mutations that were either not characterized or not fully characterized. We showed that 11 mutants had decreased or absent cell surface expression. D126Y was defective in ligand binding. Three mutants were constitutively active but had decreased cell surface expression. Eleven mutants had decreased basal signaling, with two mutants defective only in this parameter, suggesting that impaired basal signaling might also be a cause of obesity. Five mutants had normal functions. In summary, we provided detailed functional data for further studies on identifying therapeutic approaches for personalized medicine to treat patients harboring these mutations.     

Identification of distinct c-terminal domains of the Bombyx adipokinetic hormone receptor that are essential for receptor export, phosphorylation and internalization

Neuropeptides of the adipokinetic hormone (AKH) family play important roles in insect hemolymph sugar homeostasis, larval lipolysis and storage-fat mobilization. Our previous studies have shown that the adipokinetic hormone receptor (AKHR), a Gs-coupled receptor, induces intracellular cAMP accumulation, calcium mobilization and ERK1/2 phosphorylation upon agonist stimulation. However, the underlying molecular mechanisms that regulate the internalization and desensitization of AKHR remain largely unknown. In the current study we made a construct to express AKHR fused with enhanced green fluorescent protein (EGFP) at its C-terminal end to further characterize AKHR internalization. In stable AKHR-EGFP-expressing HEK-293 cells, AKHR-EGFP was mainly localized at the plasma membrane and was rapidly internalized in a dose- and time-dependent manner via the clathrin-coated pit pathway upon agonist stimulation, and internalized receptors were slowly recovered to the cell surface after the removal of AKH peptides. The results derived from RNA interference and arrestin translocation demonstrated that G protein-coupled receptor kinase 2 and 5 (GRK2/5) and β-arrestin2 were involved in receptor phosphorylation and internalization. Furthermore, experiments using deletion and site-directed mutagenesis strategies identified the three residues (Thr356, Ser359 and Thr362) responsible for GRK-mediated phosphorylation and internalization and the C-terminal domain from residue-322 to residue-342 responsible for receptor export from ER. This is the first detailed investigation of the internalization and trafficking of insect G protein-coupled receptors.     

Significance of the melanocortin 1 receptor in the DNA damage response of human melanocytes to ultraviolet radiation

Activation of the melanocortin 1 receptor (MC1R) by α‐melanocortin (α‐MSH) stimulates eumelanin synthesis and enhances repair of ultraviolet radiation (UV)‐induced DNA damage. We report on the DNA damage response (DDR) of human melanocytes to UV and its enhancement by α‐MSH. α‐MSH up‐regulated the levels of XPC, the enzyme that recognizes DNA damage sites, enhanced the UV‐induced phosphorylation of the DNA damage sensors ataxia telangiectasia and Rad3‐related (ATR) and ataxia telangiectasia mutated (ATM) and their respect‐ive substrates checkpoint kinases 1 and 2, and increased phosphorylated H2AX (γH2AX) formation. These effects required functional MC1R and were absent in melanocytes expressing loss of function (LOF) MC1R. The levels of wild‐type p53‐induced phosphatase 1 (Wip1), which dephosphorylates γH2AX, correlated inversely with γH2AX. We propose that α‐MSH increases UV‐induced γH2AX to facilitate formation of DNA repair complexes and repair of DNA photoproducts, and LOF of MC1R compromises the DDR and genomic stability of melanocytes.     

Cloning,distribution and effects of fasting status of melanocortin 4 receptor (MC4R) in Schizothorax prenanti

Melanocortin 4 receptor (MC4R) has an important role in the regulation of energy homeostasis in both mammals and fish. In this study, MC4R was characterized in S. prenanti (Schizothorax prenanti) and designated as SpMC4R. SpMC4R cDNA is composed of 1004 nucleotides with a 978 nucleotide open reading frame encoding a protein of 326 amino acids. The SpMC4R contained predicted regions that were structural features of MCR subtypes of vertebrates. In addition, phylogenetic analyses suggested that S. prenanti MC4R was closely related to fish MC4Rs. The SpMC4R mRNA was detected in embryos at developmental stages. Further, its mRNA was detectable in unfertilized eggs. Using real-time RT-PCR, MC4R is widely expressed, with highest levels of expression in brain and ovary. An experiment was conducted to determine the expression profile of MC4R during short-term and long-term fasting of the brain. The expression level of MC4R in unfed fish was significantly increased at 6, 9 and 24 h post-fasting (hpf) and 14 days fasting than in fed fish, this suggests that MC4R is conserved peptide that might be involved in the regulation of food intake and other physiological function in S. prenanti.     

Hemodynamic actions and mechanisms of systemically administered α-MSH analogs in mice

α-Melanocyte-stimulating hormone (α-MSH) regulates important physiological functions including energy homeostasis and inflammation. Potent analogs of α-MSH, [Nle⁴, d-Phe⁷]-α-MSH (NDP-α-MSH) and melanotan-II (MT-II), are widely used in pharmacological studies, but the hemodynamic effects associated with their systemic administration have not been thoroughly examined. Therefore, we investigated the hemodynamic actions of these compounds in anesthetized and conscious C57Bl/6N mice using peripheral routes of administration. NDP-α-MSH and MT-II induced mild changes in blood pressure and heart rate in anesthetized mice compared to the effects observed in conscious mice, suggesting that anesthesia distorts the hemodynamic actions of α-MSH analogs. In conscious mice, NDP-α-MSH and MT-II increased blood pressure and heart rate in a dose-dependent manner, but the tachycardic effect was more prominent than the pressor effect. Pretreatment with the melanocortin (MC) 3/4 receptor antagonist SHU9119 abolished these hemodynamic effects. Furthermore, the blockade of β₁-adrenoceptors with metoprolol prevented the pressor effect and partly the tachycardic action of α-MSH analogs, while the ganglionic blocker hexamethonium abrogated completely the difference in heart rate between vehicle and α-MSH treatments. These findings suggest that the pressor effect is primarily caused by augmentation of cardiac sympathetic activity, but the tachycardic effect seems to involve withdrawal of vagal tone in addition to sympathetic activation. In conclusion, the present results indicate that systemic administration of α-MSH analogs elevates blood pressure and heart rate via activation of MC_3/4 receptor pathways. These effects and the consequent increase in cardiac workload should be taken into account when using α-MSH analogs via peripheral routes of administration.     

Structure-function analysis of helix 8 of human calcitonin receptor-like receptor within the adrenomedullin 1 receptor

Adrenomedullin 1 (AM₁) receptor is a heterodimer composed of calcitonin receptor-like receptor (CLR) - a family B G protein-coupled receptor (GPCR) - and receptor activity-modifying protein 2 (RAMP2). Both family A and family B GPCRs possess an eighth helix (helix 8) in the proximal portion of their C-terminal tails; however, little is known about the function of helix 8 in family B GPCRs. We therefore investigated the structure-function relationship of human (h)CLR helix 8, which extends from Glu430 to Trp439, by separately transfecting nine point mutants into HEK-293 cells stably expressing hRAMP2. Glu430, Val431, Arg437 and Trp439 are all conserved among family B GPCRs. Flow cytometric analysis revealed that Arg437Ala or Trp438Ala mutation significantly reduced cell surface expression of the receptor complex, leading to a ∼20% reduction in specific ¹²⁵I-AM binding but little change in their IC₅₀ values. Both mutants showed 6-8-fold higher EC₅₀ values for AM-induced cAMP production and ∼50% reductions in their maximum responses. Glu430Ala mutation also reduced AM signaling by ∼45%, but surface expression and ¹²⁵I-AM binding were nearly the same as with wild-type CLR. Surprisingly, Glu430Ala and Val431Ala mutations significantly enhanced AM-induced internalization of the mutant receptor complexes. Taken together, these findings suggest that within hCLR helix 8, Glu430 is crucial for Gs coupling, and Arg437 and Trp439 are involved in both cell surface expression of the hAM₁ receptor and Gs coupling. Moreover, the Glu430-Val431 sequence may participate in the negative regulation of hAM₁ receptor internalization, which is not dependent on Gs coupling.     

N-glycosylation of the human melanocortin 1 receptor: occupancy of glycosylation sequons and functional role

The melanocortin 1 receptor (MC1R), a major determinant of skin pigmentation and phototype, mediates the actions of α-melanocyte-stimulating hormone on melanocytes and is critical for melanocyte proliferation and differentiation. MC1R has two putative N-glycosylation targets, Asn15 and Asn29. It has been shown that MC1R is a glycoprotein with an unusual sensitivity to endoglycosidase H digestion. However, the occupancy and functional importance of each specific glycosylation sequon remains unknown. We demonstrate that MC1R is N-glycosylated at Asn15 and Asn29, with structurally and functionally different glycan chains. N-glycosylation is not necessary for high affinity agonist binding or functional coupling but has a strong effect on the availability of MC1R molecules on the plasma membrane, most likely by a combination of improved forward trafficking and decreased internalization. Finally, we found that MC1R variants exhibit different degrees of glycosylation which do not show a simple correlation with their functional status or intracellular trafficking.     

Functional role of the conserved proline in helix 6 of the human bradykinin B2 receptor

Pro258 in transmembrane domain (TMD) 6 of the bradykinin (BK) B₂ receptor (B₂R) is highly conserved among G-protein coupled receptors (GPCRs). Using mutagenesis, we show that Pro258 is required for normal trafficking of the receptor to the plasma membrane and that mutation of Pro258 to Ala or Leu but not Gly, enhances BK efficacy to induce receptor activation. Furthermore, P258A mutation suppresses the constitutive activity of a constitutively activated N113A-B₂R mutant but preserves the antagonist to agonist efficacy shift previously observed on the N113A single mutant. Our data suggest that Pro258 in TMD6 is required for agonist-independent activation of the B₂R and that straightening of TMD6 at the Pro-kink might favor G-protein coupling. It is also shown that Asn113 is a contact point of BK interaction and it is proposed that the release of a TMD3-TMD6 interaction involving Asn113 is crucial for the efficacy shift from antagonism toward agonism.     

Two transmembrane Cys residues are involved in 5-HT4 receptor dimerization

The 5-HT₄ receptor (5-HT₄R) belongs to the G-protein-coupled receptor (GPCR) family and is of considerable interest for the development of new drugs to treat gastrointestinal diseases and memory disorders. The 5-HT₄R exists as a constitutive dimer but its molecular determinants are still unknown. Using co-immunoprecipitation and Bioluminescence Resonance Energy Transfer (BRET) techniques, we show here that 5-HT₄R homodimerization but not 5-HT₄R-β₂ adrenergic receptor (β₂AR) heterodimerization is largely decreased under reducing conditions suggesting the participation of disulfide bonds in 5-HT₄R dimerization. Molecular modeling and protein docking experiments identified four cysteine (Cys) residues potentially involved in the dimer interface through intramolecular or intermolecular disulfide bonds. We show that disulfide bridges between Cys112 and Cys145 located within TM3 and TM4, respectively, are of critical importance for 5-HT₄R dimer formation. Our data suggest that two disulfide bridges between two transmembrane Cys residues are involved in the dimerization interface of a GPCR.