Melanocortin Receptor Family Research Articles

Structure elucidation of a human melanocortin-4 receptor specific orthosteric nanobody agonist

The melanocortin receptor 4 (MC4R) belongs to the melanocortin receptor family of G-protein coupled receptors and is a key switch in the leptin-melanocortin molecular axis that controls hunger and satiety. Brain-produced hormones such as α-melanocyte-stimulating hormone (agonist) and agouti-related peptide (inverse agonist) regulate the molecular communication of the MC4R axis but are promiscuous for melanocortin receptor subtypes and induce a wide array of biological effects. Here, we use a chimeric construct of conformation-selective, nanobody-based binding domain (a ConfoBody Cb80) and active state-stabilized MC4R-β2AR hybrid for efficient de novo discovery of a sequence diverse panel of MC4R-specific, potent and full agonistic nanobodies. We solve the active state MC4R structure in complex with the full agonistic nanobody pN162 at 3.4 Å resolution. The structure shows a distinct interaction with pN162 binding deeply in the orthosteric pocket. MC4R peptide agonists, such as the marketed setmelanotide, lack receptor selectivity and show off-target effects. In contrast, the agonistic nanobody is highly specific and hence can be a more suitable agent for anti-obesity therapeutic intervention via MC4R.

Activation of the melanocortin-1 receptor attenuates neuronal apoptosis after traumatic brain injury by upregulating Merlin expression

Traumatic brain injury (TBI) is a common disease worldwide with high mortality and disability rates. Besides the primary mechanical injury, the secondary injury associated with TBI can also induce numerous pathological changes, such as brain edema, nerve apoptosis, and neuroinflammation, which further aggravates neurological dysfunction and even causes the death due to the primary injury. Among them, neuronal apoptosis is a key link in the injury. Melanocortin-1 receptor (MC1R) is a G protein coupled receptor, belonging to the melanocortin receptor family. Studies have shown that activation of MC1R inhibits oxidative stress and apoptosis, and confers neuroprotective effects against various neurological diseases. Merlin is a protein product of the NF2 gene, which is widely expressed in the central nervous system (CNS) of mice, rats, and humans. Studies have indicated that Merlin is associated with MC1R. In this study, we explored the anti-apoptotic effects and potential mechanisms of MC1R. A rat model of TBI was established through controlled cortical impact. The MC1R-specific agonist Nle4-D-Phe7-α-Melanocyte (NDP-MSH) and the inhibitor MSG-606 were employed to explore the effects of MC1R and Merlin following TBI and investigated the associated mechanisms. The results showed that the expression levels of MC1R and Merlin were upregulated after TBI, and activation of MC1R promoted Merlin expression. Further, we found that MC1R activation significantly improved neurological dysfunction and reduced brain edema and neuronal apoptosis induced by TBI in rats. Mechanistically, its neuroprotective function and anti-apoptotic were partly associated with MC1R activation. In conclusion, we demonstrated that MC1R activation after TBI may inhibit apoptosis and confer neuroprotection by upregulating the expression of Merlin.

OR0302 Regulation Of Human Melanocortin-5 Receptor By Isoforms Of Melanocortin-2 Receptor Accessory Proteins

Abstract Disclosure: S. Jiang: None. Y. Tao: None. Melanocortin-5 receptor (MC5R) is ubiquitously expressed in central nervous system and peripheral tissues, has unique pharmacological properties, and physiological functions. Melanocortin-2 receptor accessory proteins (MRAPs) are regulators of melanocortin receptor family. To investigate the effects of MRAPs on trafficking, ligand binding and signaling properties of human (h) MC5R, HEK293T cells were transiently co-transfected with plasmids encoding MC5R and different isoforms of MRAPs (hMRAP1a, hMRAP1b, hMRAP2a, hMRAP2b, and hMRAP2c). The results showed that hMRAP1a and hMRAP2a increased the cell surface expression of hMC5R. All MRAPs have no effect on affinity to the superpotent analog of α-melanocyte stimulating hormone (α-MSH), NDP-MSH, while hMRAP2c significantly increased maximal binding. Additionally, α-MSH induced ERK1/2 activation. A higher basal level of ERK1/2 phosphorylation was observed when co-transfected with some MRAPs. All MRAPs had no effect on α-MSH-stimulated cAMP production (Gs-cAMP pathway). In summary, the two MRAP1s and three MRAP2s had differential effects on MC5R trafficking, binding, and signaling. These findings led to a better understanding of the regulation of MC5R by MRAP1s and MRAP2s. Presentation: Thursday, June 15, 2023

Structural insights into ligand recognition and subtype selectivity of the human melanocortin-3 and melanocortin-5 receptors

Members of the melanocortin receptor (MCR) family that recognize different melanocortin peptides mediate a broad spectrum of cellular processes including energy homeostasis, inflammation and skin pigmentation through five MCR subtypes (MC1R–MC5R). The structural basis of subtype selectivity of the endogenous agonist γ-MSH and non-selectivity of agonist α-MSH remains elusive, as the two agonists are highly similar with a conserved HFRW motif. Here, we report three cryo-electron microscopy structures of MC3R–Gs in complex with γ-MSH and MC5R–Gs in the presence of α-MSH or a potent synthetic agonist PG-901. The structures reveal that α-MSH and γ-MSH adopt a “U-shape” conformation, penetrate into the wide-open orthosteric pocket and form massive common contacts with MCRs via the HFRW motif. The C-terminus of γ-MSH occupies an MC3R-specific complementary binding groove likely conferring subtype selectivity, whereas that of α-MSH distances itself from the receptor with neglectable contacts. PG-901 achieves the same potency as α-MSH with a shorter length by rebalancing the recognition site and mimicking the intra-peptide salt bridge in α-MSH by cyclization. Solid density confirmed the calcium ion binding in MC3R and MC5R, and the distinct modulation effects of divalent ions were demonstrated. Our results provide insights into ligand recognition and subtype selectivity among MCRs, and expand the knowledge of signal transduction among MCR family members.

Reversion of MRAP2 Protein Sequence Generates a Functional Novel Pharmacological Modulator for MC4R Signaling.

Simple SummaryReversion of the wild-type protein sequences of single transmembrane melanocortin accessory protein families (MRAP2) in mice and zebrafish created novel functional pharmacological modulators for regulating melanocortin 4 receptor (MC4R) signaling. All of the brand new reversed MRAP2 (rMRAP2) proteins could form proper dimeric topology on the plasma membrane and interact with and affect the ligand-stimulated pharmacological profiles of zebrafish and mouse MC4R signaling in vitro.As a member of the melanocortin receptor family, melanocortin 4 receptor (MC4R) plays a critical role in regulating energy homeostasis and feeding behavior, and has been proven as a promising therapeutic target for treating severe obesity syndrome. Numerous studies have demonstrated that central MC4R signaling is significantly affected by melanocortin receptor accessory protein 2 (MRAP2) in humans, mice and zebrafish. MRAP2 proteins exist as parallel or antiparallel dimers on the plasma membrane, but the structural insight of dual orientations with the pharmacological profiles has not yet been fully studied. Investigation and optimization of the conformational topology of MRAP2 are critical for the development of transmembrane allosteric modulators to treat MC4R-associated disorders. In this study, we synthesized a brand new single transmembrane protein by reversing wild-type mouse and zebrafish MRAP2 sequences and examined their dimerization, interaction and pharmacological activities on mouse and zebrafish MC4R signaling. We showed that the reversed zebrafish MRAPa exhibited an opposite function on modulating zMC4R signaling and the reversed mouse MRAP2 lost the capability for regulating MC4R trafficking but exhibited a novel function for cAMP cascades, despite proper expression and folding. Taken together, our results provided new biochemical insights on the oligomeric states and membrane orientations of MRAP2 proteins, as well as its pharmacological assistance for modulating MC4R signaling.

Melanocortin receptor activation alleviates amyloid pathology and glial reactivity in an Alzheimer\u2019s disease transgenic mouse model

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder with no disease-modifying treatment. AD progression is characterized by cognitive decline, neuroinflammation, and accumulation of amyloid-beta (Aβ) and neurofibrillary tangles in the brain, leading to neuronal and glial dysfunctions. Neuropeptides govern diverse pathophysiological processes and represent key players in AD pathogenesis, regulating synaptic plasticity, glial cell functions and amyloid pathology. Activation of the pro-opiomelanocortin (POMC)-derived neuropeptide and its receptor from the melanocortin receptor (MCR) family have previously been shown to rescue the impairment in hippocampus-dependent synaptic plasticity in the APP/PS1 mouse model of AD. However, the functional roles of MCR signaling in AD conditions, particularly in glial functions, are largely unknown. In this study, we investigated the potential benefits of MCR activation in AD. In APP/PS1 transgenic mice, we demonstrate that MCR activation mediated by the central administration of its agonist D-Tyr MTII substantially reduces Aβ accumulation, while alleviating global inflammation and astrocytic activation, particularly in the hippocampus. MCR activation prominently reduces the A1 subtype of reactive astrocytes, which is considered a key source of astrocytic neurotoxicity in AD. Concordantly, MCR activation suppresses microglial activation, while enhancing their association with amyloid plaques. The blunted activation of microglia may contribute to the reduction in the neurotoxic phenotypes of astrocytes. Importantly, transcriptome analysis reveals that MCR activation restores the impaired homeostatic processes and microglial reactivity in the hippocampus in APP/PS1 mice. Collectively, our findings demonstrate the potential of MCR signaling as therapeutic target for AD.

Amino acid residue L112 in the ACTH receptor plays a key role in ACTH or α-MSH selectivity

The adrenocorticotropic hormone (ACTH) receptor, known as the melanocortin-2 receptor (MC2R), plays a key role in regulating adrenocortical function. MC2R is a subtype of the melanocortin receptor family and ACTH is only agonist for MC2R. Our previous result indicates that ACTH1-17 is the minimal peptide required for MC2R activation but DPhe7-ACTH1-17 has no activity at MC2R. In this study, we examined the molecular basis of the MC2R responsible for ligand selectivity using ACTH analogues and MC2R mutagenesis. Our results indicate that substitution of the 3TM of the MC2R with the corresponding region of the MC3R switches DPhe-ACTH1-17 from no activity to agonist. Further experiment indicates that substitution of the amino acid residue leucine to isoleucine in 112 (L112I) of the 3TM of the MC2R changes both DPhe-ACTH1-17 and ACTH1-15 from no activity to agonists. Surprisingly, mutation L112I switches α-MSH from no activity to agonist, suggesting that this residue plays a key role at MC2R for ligand ACTH or α-MSH selectivity.

Click-Chemistry-Mediated Synthesis of Selective Melanocortin Receptor 4 Agonists

The melanocortin receptor 4 (MC4R) subtype of the melanocortin receptor family is a target for therapeutics to ameliorate metabolic dysfunction. Endogenous MC4R agonists possess a critical pharmacophore (HFRW), and cyclization of peptide agonists often enhances potency. Thus, 17 cyclized peptides were synthesized by solid phase click chemistry to develop novel, potent, selective MC4R agonists. Using cAMP measurements and a transcriptional reporter assay, we observed that several constrained agonists generated by a cycloaddition reaction displayed high selectivity (223- to 467-fold) toward MC4R over MC3R and MC5R receptor subtypes without compromising agonist potency. Significant variation was also observed between the EC50 values for the two assays, with robust levels of reporter expression measured at lower concentrations than those effecting appreciable increases in cAMP levels for the majority of the compounds tested. Collectively, we characterized significant elements that modulate the activity of the core pharmacophore for MC4R and provide a rationale for careful assay selection for agonist screening.

Key amino acid residue in Melanocortin-1 receptor (melanocyte α-MSH receptor) for ligand selectivity

The melanocortin-1 receptor (MC1R) is a subtype of the melanocortin receptor family and NDP-α-MSH is a non-selective agonist for MC1R. The core sequence of NDP-α-MSH, His-Phe-Arg-Trp, is important for ligand binding and biological activities at the melanocortin receptor subtypes (MCRs). A recent study indicates that Trp9 in NDP-α-MSH plays an important role in ligand selectivity. Deletion of Trp9 in NDP-α−MSH (des-Trp9-NDP-α-MSH) resulted in loss of agonist activity at MC4R, although remains agonist activity at MC1R. The molecular basis for this receptor ligand selectivity is unknown. In this study we examined what region of the MC1R is responsible for des-NDP-α-MSH selectivity. Our results indicate that (1) substitution of TM3 of MC4R with the corresponding region of MC1R switches des-Trp9-NDP-α-MSH from no activity to agonist; (2) des-Trp9-NDP-α-MSH exhibits agonistic activity at the L133M mutation of the MC4R; and (3) substitution of non-conserved amino acid residue M128 in TM3 of MC1R significantly reduced des-Trp9-NDP-α-MSH agonist activity. Our results demonstrate that amino acid residue 128 in TM3 of MC1R, or amino acid residue L133 in TM3 of the MC4R, play crucial roles in ligand des-Trp9-NDP-α-MSH selectivity at MC1R or MC4R.

Association of estradiol on expression of melanocortin receptors and their accessory proteins in the liver of chicken (Gallus gallus)

The melanocortin receptor accessory proteins (MRAP and MRAP2) are small single-pass transmembrane proteins that regulate the biological functions of the melanocortin receptor (MCR) family. MCRs comprise five receptors (MC1R–MC5R) with diverse physiological roles in mammals. Five MCR members and two MRAPs were also predicted in the chicken (Gallus gallus) genome. However, little is known about their expression, regulation and biological functions. In this study, we cloned the MRAP and MRAP2 genes. Sequencing analysis revealed that the functional domains of MRAP and MRAP2 were conserved among species, suggesting that the physiological roles of chicken MRAP and MRAP2 could be similar to their mammalian counterparts. Tissue expression analysis demonstrated that MRAP was expressed in the adrenal gland, liver, spleen, glandular stomach and lungs, while MRAP2 is predominantly expressed in the adrenal gland. All five MCRs were present in the adrenal gland, but showed different expression patterns in other tissues. The MC5R was the only MCR member that was expressed in the chicken liver. The expression levels of MRAP in chicken liver were significantly increased at sexual maturity stage, and were significantly up-regulated (P<0.05) when chickens and chicken primary hepatocytes were treated with 17β-estradiol in vivo and in vitro, respectively; however, expression levels of PPARγ were down-regulated, and no effect on MC5R was observed. Our results suggested that estrogen could stimulate the expression of MRAP in the liver of chicken through inhibiting the expression of transcription regulation factor PPARγ, and MRAP might play its biological role in a different way rather than forming an MRAP/MC2R complex in chicken liver during the egg-laying period.

Melanocortin receptor accessory proteins in adrenal disease and obesity.

Melanocortin receptor accessory proteins (MRAPs) are regulators of the melanocortin receptor family. MRAP is an essential accessory factor for the functional expression of the MC2R/ACTH receptor. The importance of MRAP in adrenal gland physiology is demonstrated by the clinical condition familial glucocorticoid deficiency type 2. The role of its paralog melanocortin-2-receptor accessory protein 2 (MRAP2), which is predominantly expressed in the hypothalamus including the paraventricular nucleus, has recently been linked to mammalian obesity. Whole body deletion and targeted brain specific deletion of the Mrap2 gene result in severe obesity in mice. Interestingly, Mrap2 complete knockout (KO) mice have increased body weight without detectable changes to food intake or energy expenditure. Rare heterozygous variants of MRAP2 have been found in humans with severe, early-onset obesity. In vitro data have shown that Mrap2 interaction with the melanocortin-4-receptor (Mc4r) affects receptor signaling. However, the mechanism by which Mrap2 regulates body weight in vivo is not fully understood and differences between the phenotypes of Mrap2 and Mc4r KO mice may point toward Mc4r independent mechanisms.

Third Transmembrane Domain of the Adrenocorticotropic Receptor Is Critical for Ligand Selectivity and Potency

The ACTH receptor, known as the melanocortin-2 receptor (MC2R), plays an important role in regulating and maintaining adrenocortical function. MC2R is a subtype of the melanocortin receptor (MCR) family and has unique characteristics among MCRs. Endogenous ACTH is the only endogenous agonist for MC2R, whereas the melanocortin peptides α-, β-, and γ-melanocyte-stimulating hormone and ACTH are full agonists for all other MCRs. In this study, we examined the molecular basis of MC2R responsible for ligand selectivity using ACTH analogs and MC2R mutagenesis. Our results indicate that substitution of Phe(7) with D-Phe or D-naphthylalanine (D-Nal(2')) in ACTH(1-24) caused a significant decrease in ligand binding affinity and potency. Substitution of Phe(7) with D-Nal(2') in ACTH(1-24) did not switch the ligand from agonist to antagonist at MC2R, which was observed in MC3R and MC4R. Substitution of Phe(7) with D-Phe(7) in ACTH(1-17) resulted in the loss of ligand binding and activity. Molecular analysis of MC2R indicated that only mutation of the third transmembrane domain of MC2R resulted in a decrease in D-Phe ACTH binding affinity and potency. Our results suggest that Phe(7) in ACTH plays an important role in ligand selectivity and that the third transmembrane domain of MC2R is crucial for ACTH selectivity and potency.

Molecular Characterization of Human Melanocortin-5 Receptor Ligand–Receptor Interaction

The melanocortin-5 receptor (MC5R) is a subtype receptor of the melanocortin receptor (MCR) family, which is expressed centrally, as well as in a variety of peripheral tissues. MC5R has been implicated in many different physiological fields such as lipid metabolism and exocrine function. However, the specific molecular determinants of MC5R responsible for ligand binding and receptor signaling are currently unknown. The aim of this study is to determine the molecular basis of human MC5R (hMC5R) responsible for ligand binding and receptor signaling. Twenty-four single mutations of hMC5R were created and tested. Our results indicate that (1) substituting charged amino acid residue E92 in transmembrane domain 2 (TM2), aspartic acid 115 (D115) and D119 in TM3, and histidine (H) 257 in TM6 with alanine dramatically reduced NDP-α-MSH binding affinity and receptor signaling and (2) substituting aromatic amino acids phenylalanine (F) 195 in TM5, F254 in TM6, and H276 in TM7 with alanine also significantly decreased NDP-α-MSH binding and receptor activity. Combining pharmacological results and computer modeling, our results suggest that D115 and D119 in TM3, F195 in TM5, and F254 in TM6 may form a binding pocket for NDP-α-MSH binding. Our results provide important information about the structural aspects of hMC5R responsible for ligand binding and receptor signaling.

Melanocortin receptor accessory proteins in adrenal gland physiology and beyond

The melanocortin receptor (MCR) family consists of five G-protein-coupled receptors (MC1R-MC5R) with diverse physiological roles. MC1R controls pigmentation, MC2R is a critical component of the hypothalamic-pituitary-adrenal axis, MC3R and MC4R have a vital role in energy homeostasis and MC5R is involved in exocrine function. The melanocortin receptor accessory protein (MRAP) and its paralogue MRAP2 are small single-pass transmembrane proteins that have been shown to regulate MCR expression and function. In the adrenal gland, MRAP is an essential accessory factor for the functional expression of the MC2R/ACTH receptor. The importance of MRAP in adrenal gland physiology is demonstrated by the clinical condition familial glucocorticoid deficiency, where inactivating MRAP mutations account for ∼20% of cases. MRAP is highly expressed in both the zona fasciculata and the undifferentiated zone. Expression in the undifferentiated zone suggests that MRAP could also be important in adrenal cell differentiation and/or maintenance. In contrast, the role of adrenal MRAP2, which is highly expressed in the foetal gland, is unclear. The expression of MRAPs outside the adrenal gland is suggestive of a wider physiological purpose, beyond MC2R-mediated adrenal steroidogenesis. In vitro, MRAPs have been shown to reduce surface expression and signalling of all the other MCRs (MC1,3,4,5R). MRAP2 is predominantly expressed in the hypothalamus, a site that also expresses a high level of MC3R and MC4R. This raises the intriguing possibility of a CNS role for the MRAPs.

The melanocortin receptors and their accessory proteins

The five melanocortin receptors (MCRs) named MC1R–MC5R have diverse physiological roles encompassing pigmentation, steroidogenesis, energy homeostasis and feeding behavior as well as exocrine function. Since their identification almost 20 years ago much has been learnt about these receptors. As well as interacting with their endogenous ligands the melanocortin peptides, there is now a growing list of important peptides that can modulate the way these receptors signal, acting as agonists, antagonists, and inverse agonists. The discovery of melanocortin 2 receptor accessory proteins as a novel accessory factor to the MCRs provides further insight into the regulation of these important G protein-coupled receptor.

ACTH is a novel regulator of bone mass

Adrenocorticotropin (ACTH) is one of several peptide hormones derived from a larger molecule, proopiomelanocortin (POMC). ACTH is a classic endocrine hormone, processed and secreted from the pituitary to stimulate cortisol production from the fasciculata cells in the adrenal gland. However, ACTH is also produced by other cells, including macrophages, at many sites in the body. ACTH binds to a specific member of the melanocortin receptor family, the MC2R. MC2R is expressed in osteoblastic cells in vivo, as shown by in situ hybridization. MC2R expression is strongest at sites of active bone deposition, and thus ACTH response probably varies with osteoblastic activity or stage of osteoblast differentiation. In vitro ACTH stimulates proliferation of osteoblasts in a dose-dependent manner. ACTH at 10 nM increases collagen I mRNA in the osteoblastic cell line SaOs2, although at lower concentrations ACTH may oppose osteoblast differentiation. ACTH is thus, at high concentrations, anabolic for the osteoblast, and it is highly likely that the hormone has concentration-dependent effects on bone metabolism in vivo.

Identification of domains responsible for specific membrane transport and ligand specificity of the ACTH receptor (MC2R)

The adrenocorticotropic hormone (ACTH) receptor has highly specific membrane expression that is limited to adrenal cells; in other cell types the polypeptide fails to be transported to the cell surface. Unlike other evolutionarily related members of the melanocortin receptor family (MC1R–MC5R) that recognize different melanocortin peptides, ACTHR (MC2R) binds only ACTH. We used a mutagenesis approach involving systematic construction of chimeric ACTHR/MC4R receptors to identify the domains determining the selectivity of ACTHR membrane transport and ACTH binding. In total 15 chimeric receptors were created by replacement of selected domains of human ACTHR with the corresponding regions of human MC4R. We developed an analytical method to accurately quantify cell-membrane localization of recombinant receptors fused with enhanced green fluorescent protein by confocal fluorescence microscopy. The chimeric receptors were also tested for their ability to bind ACTH (1–24) and the melanocyte-stimulating hormone (MSH) analog, Nle4, DPhe7-α-MSH, and to induce a cAMP response. Our results indicate that substitution of the MC4R N-terminal segment with the homologous segment of ACTHR significantly decreased membrane transport. We also identified another signal localized in the third and fourth transmembrane regions as the main determinant of ACTHR intracellular retention. In addition, we found that the fourth and fifth transmembrane domains of the ACTHR are involved in ACTH binding selectivity. We discuss the mechanisms involved in bypassing these arrest signals via an interaction with melanocortin 2 receptor accessory protein (MRAP) and the possible mechanisms that determine the high ligand-binding specificity of ACTHR.

Piecing together the pigment-type switching puzzle

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MRAP and MRAP2 are bidirectional regulators of the melanocortin receptor family

The melanocortin receptor (MCR) family consists of 5 G protein-coupled receptors (MC1R-MC5R) with diverse physiologic roles. MC2R is a critical component of the hypothalamic-pituitary-adrenal axis, whereas MC3R and MC4R have an essential role in energy homeostasis. Mutations in MC4R are the single most common cause of monogenic obesity. Investigating the way in which these receptors signal and traffic to the cell membrane is vital in understanding disease processes related to MCR dysfunction. MRAP is an MC2R accessory protein, responsible for adrenal MC2R trafficking and function. Here we identify MRAP2 as a unique homologue of MRAP, expressed in brain and the adrenal gland. We report that MRAP and MRAP2 can interact with all 5 MCRs. This interaction results in MC2R surface expression and signaling. In contrast, MRAP and MRAP2 can reduce MC1R, MC3R, MC4R, and MC5R responsiveness to [Nle4,D-Phe7]alpha-melanocyte-stimulating hormone (NDP-MSH). Collectively, our data identify MRAP and MRAP2 as unique bidirectional regulators of the MCR family.

Molecular Identification of the Human Melanocortin-2 Receptor Responsible for Ligand Binding and Signaling

The melanocortin-2 receptor (MC2R), also known as the adrenocorticotropic hormone (ACTH) receptor, plays an important role in regulating and maintaining adrenocortical function, specifically steroidogenesis. Mutations of the human MC2R (hMC2R) gene have also been identified in humans with familial glucocorticoid deficiency; however, the molecular basis responsible for hMC2R ligand binding and signaling remains unclear. In this study, both truncated ACTH peptides and site-directed mutagenesis studies were used to determine molecular mechanisms of hMC2R binding ACTH and signaling. Our results indicate that ACTH1-16 is the minimal peptide required for hMC2R binding and signaling. Mutations of common melanocortin receptor family amino acid residues E80 in transmembrane domain 2 (TM2), D107 in TM3, F178 in TM4, F235 and H238 in TM6, and F258 in TM7 significantly reduced ACTH-binding affinity and signaling. Furthermore, mutations of unique amino acids D104 and F108 in TM3 and F168 and F178 in TM4 significantly decreased ACTH binding and signaling. In conclusion, our results suggest that the residues in TM2, TM3, and TM6 of hMC2R share similar binding sites with other MCRs but the residues identified in TM4 and TM7 of hMC2R are unique and required for ACTH selectivity. Our study suggests that hMC2R may have a broad binding pocket in which both conserved and unique amino acid residues are required, which may be the reason why alpha-MSH was not able to bind hMC2R.