Melanocortin Receptor Subtypes Research Articles

Endogenous and exogenous melanocortin antagonists induce anti-allodynic effects in a model of rat neuropathic pain

A number of studies suggest melanocortin (MC) system involvement in nociceptive modulation. Although the mechanism through which this occurs is still unknown, experimental evidence would suggest a primary role of MC4 receptors. To further investigate the implication of this MC receptor subtype in chronic pain, we have studied the effects of several MC antagonists on spinal nerve ligation-induced nociceptive behavior in rats. The intrathecal injection of synthetic antagonists with different selectivity to MC4 receptor and of an endogenous antagonist (Agouti related protein; AgRP) reduced mechanical allodynia in neuropathic rats, as measured by von Frey hair test. Treatments produced an anti-allodynic effect at the dose of 1.5 nmol (25–30% maximum possible effect, MPE, P < 0.05). To further investigate the possible physiological role of AgRP in pain modulation we studied its expression in both sham and neuropathic rat spinal cord and dorsal root ganglia (DRG) by quantitative real time PCR and immunohistochemistry. AgRP was present in both spinal cord and DRG, and its expression, was unchanged in neuropathic animals. In conclusion MC4 receptor antagonists with different selectivity profile, induce anti-allodynic effects in one of the most relevant neuropathic pain model. In addition the expression of AgRP in spinal cord and DRG suggests an endogenous tonic inhibitory control on MC system activity. In pathological conditions this steady control could be insufficient to cope with an over activated MC system leading to increase in nociception. These data suggest that targeting MC4 with synthetic antagonists could restore the balance and hence reduce nociception.

Phenylguanidines as selective nonpeptide melanocortin-5 receptor antagonists.

A series of phenylguanidine analogues represented by 10, 12, and 21 were synthesized and found to have high binding affinities for the human melanocortin-5 receptor. Their binding affinities for three other melanocortin receptor subtypes, MC1, MC3, and MC4, were low. Selected compounds were also tested for their functional activity and exhibited inhibition of alpha-MSH-stimulated cAMP production in cells expressing the human MC5 receptor. Compound 10 had a K(i) value of 2.1 nM in the binding assay and an IC(50) of 72 nM in the functional assay. Some analogues such as 13 from this series possessed weak agonist activity at the human MC4 receptor.

Role of hypothalamic melanocortin 3/4-receptors in mediating chronic cardiovascular, renal, and metabolic actions of leptin.

The present study examined whether blockade of melanocortin receptors subtypes 3 and 4 (MC3/4-R) inhibits chronic cardiovascular and dietary responses to leptin infusion. A cannula was placed in the lateral ventricle of male Sprague-Dawley rats for chronic intracerebroventricular (ICV) infusion via osmotic minipump, and arterial and venous catheters were implanted for measurement of mean arterial pressure (MAP) and heart rate (HR) 24 h/d and IV infusions. After a 5-day control period, rats received (1) 0.9% saline vehicle ICV for 12 days plus leptin (1 microg/kg per minute IV, n=5) during the final 7 days; (2) MC3/4-R antagonist SHU-9119 (1 nmol/h ICV) for 12 days plus leptin (1 microg/kg per minute IV, n=6) during the final 7 days; and (3) SHU-9119 (1 nmol/h ICV, n=8) for 12 days. Leptin infusion in vehicle-treated rats caused a small increase in MAP (5+/-1 mm Hg) despite reduced food intake (23+/-1 to 10+/-1 g/d) and decreased body weight (-6%+/-1%). SHU-9119 infusion completely prevented the cardiovascular and dietary actions of leptin, leading to increased food intake (23+/-1 to 49+/-4 g/d) and body weight (+30%+/-2%), markedly decreased HR (-77+/-9 bpm), and caused a decrease in MAP (-6+/-1 mm Hg). Similar results were observed when SHU-9119 was infused alone in vehicle-treated rats. Leptin decreased plasma insulin to 30% of control values, an effect that was also abolished by SHU-9119 treatment, which caused a 5-fold increase in plasma insulin concentration. Thus, MC3/4-R antagonism completely blocked the chronic cardiovascular, satiety, and metabolic effects of leptin, suggesting that the hypothalamic melanocortin system plays an important role in mediating these actions of leptin.

Targeting melanocortin receptors as a novel strategy to control inflammation.

Adrenocorticotropic hormone and alpha-, beta-, and gamma-melanocyte-stimulating hormones, collectively called melanocortin peptides, exert multiple effects upon the host. These effects range from modulation of fever and inflammation to control of food intake, autonomic functions, and exocrine secretions. Recognition and cloning of five melanocortin receptors (MCRs) has greatly improved understanding of peptide-target cell interactions. Preclinical investigations indicate that activation of certain MCR subtypes, primarily MC1R and MC3R, could be a novel strategy to control inflammatory disorders. As a consequence of reduced translocation of the nuclear factor kappaB to the nucleus, MCR activation causes a collective reduction of the major molecules involved in the inflammatory process. Therefore, anti-inflammatory influences are broad and are not restricted to a specific mediator. Short half-life and lack of selectivity could be an obstacle to the use of the natural melanocortins. However, design and synthesis of new MCR ligands with selective chemical properties are already in progress. This review examines how marshaling MCR could control inflammation.

Roles of the melanocortin-4 receptor in antipyretic and hyperthermic actions of centrally administered α-MSH

Activation of central melanocortin receptors (MCR) inhibits fever but can also stimulate thermogenesis, and the mechanisms involved are unknown. To determine whether the long-recognized antipyretic effect of exogenous α-MSH is mediated by the melanocortin-4 receptor (MC4R), and what thermoeffector systems are involved, we tested the effects of intracerebroventricular (icv) injection of α-MSH on lipopolysaccharide (LPS, 30 μg/kg ip)-induced fever in rats, in the presence and absence of the selective MC4R antagonist HS014. Treatment with α-MSH (1 μg, icv) suppressed LPS-induced increases in core body temperature ( T c), whereas a lower dose (300 ng) was ineffective. Nevertheless, both α-MSH doses effectively inhibited LPS-induced peripheral vasoconstriction, the principal heat-conserving thermoeffector, as determined by changes in tail skin temperature ( T sk). This implies that the net antipyretic effect of α-MSH cannot be accounted for solely by modulation of heat loss effectors, but also involves other mechanisms. Surprisingly, central MC4-R blockade by coinjected HS014 (1 μg) not only prevented, but reversed the effect of α-MSH (1 μg) on T c, thus resulting in augmented LPS-induced fever. In afebrile rats, α-MSH infusion caused a modest transient increase in T c that was blocked by coinjected HS014, but was not accompanied by altered T sk. Overall, the results support the hypothesis that the MC4R mediates the antipyretic effects of α-MSH. Paradoxically, in the presence of pharmacological MC4-R blockade during fever, exogenous α-MSH can exacerbate fever, probably by acting via other central MCR subtype(s). In normal animals, centrally injected α-MSH exerts a hyperthermic effect that is mediated by the MC4R, consistent with recent evidence that MC4R activation promotes energy expenditure in normal states through stimulation of thermogenesis.

Regulation of CRF, POMC and MC4R Gene Expression after Electrical Foot Shock Stress in the Rat Amygdala and Hypothalamus

We investigated the effects of electrical foot shock stress on the melanocortin signaling cascade and the hypothalamus-pituitary-adrenal (HPA) system by observing levels of mRNA expression of corticotropin releasing factor (CRF), pro-opiomelanocortin (POMC), and melanocortin receptor subtype 4 (MC4R) in the rat amygdala and hypothalamus. When rats were exposed to electrical shock for 0.5 hr or 1 hr, plasma ACTH and corticosterone concentrations increased, indicating stress. The rats were then sacrificed to obtain RNA preparations from the brain tissue. In the amygdala, the expression of MC4R and POMC mRNA as well as CRF mRNA was significantly increased by electrical foot shock stress. In the hypothalamus, MC4R and POMC mRNA increased, but CRF mRNA remained unchanged. The duration of increased gene expression of MC4R and POMC in the amygdala was more sustained than in the hypothalamus. These results have provided the first evidence that exposure to stress increases expression of the MC4R system in the amygdala and hypothalamus.

Functional and phylogenetic analyses of a melanocortin-4 receptor mutation in domestic pigs

The melanocortin-4 receptor (MC4R), a G protein-coupled seven-transmembrane receptor, which is expressed in the brain, plays an important role in the control of mammalian energy homeostasis. A missense mutation (Asp298Asn) was identified in the porcine MC4R gene, which is associated with growth and food intake traits. The Asn298 mutation occurs within a highly conserved motif, NPLIY, of all members of G protein-coupled receptors; whereas, Asp298 is conserved in all five melanocortin receptor subtypes. Functional analysis of the porcine MC4R variant was performed with an in vitro gene expression system in 293 cells. Ligand binding (NDP-αMSH) did not differ between Asp298 and Asn298 MC4R proteins. However, the Asn298 MC4R variant was unable to stimulate cAMP production in response to NDP-αMSH stimulation; whereas, the Asp298 variant could stimulate cAMP accumulation. These results demonstrate that the Asp298 is required for normal MC4R signaling to the adenylyl cyclase. Sequencing of the MC4R gene of seven diverse genera within the Suiformes that include Hippopotamidae (hippos), Tayassuidae (peccaries) and Suidae (pigs), revealed 62 nucleotide variations in MC4R. Phylogenetic relationships of MC4R variations are consistent with those previously described from morphological and physiological data among the subfamilies of the Suiformes. These findings revealed that a single missense mutation (Asp298Asn) of aspartic acid (Asp) to asparagine (Asn) in MC4R gene decreased cAMP content and MC4R signaling, but with no difference in the ligand binding was associated with growth and feed intake traits in domestic pigs.

The central melanocortin system and fever.

Fever is a phylogenetically ancient response that is mounted upon exposure of the host to pathogens or inflammatory agents. Melanocortin agonists act centrally to inhibit fever by acting at receptors, including the melanocortin-4 receptor, which is prominently expressed in key hypothalamic thermoregulatory centers. Furthermore, endogenous melanocortins act centrally as physiological modulators of fever, recruited during the febrile response to restrain its intensity. Functionally, these actions lie at the interface between the anti-inflammatory effects of melanocortins, which involve suppression of the synthesis and actions of proinflammatory cytokines, and the central control of thermoregulation. Considering the extensive neuroanatomic and functional overlaps between central pathways and peripheral effectors involved in thermoregulation and energy balance, it is not surprising that melanocortins have been found to influence the metabolic economy profoundly in pathological as well as normal states. For example, despite suppressing endotoxin-induced fever, endogenous melanocortins appear to mediate the associated anorexia, a classic component of the "illness syndrome" accompanying acute infections, and promote a negative energy balance. The thermoregulatory actions of melanocortins are in several respects functionally opposed, and are remarkably dependent on physiological state, indicating that responsiveness to melanocortins is a physiologically modulated variable. Elucidating the anti-inflammatory and thermoregulatory roles of central melanocortin receptors during inflammatory states may lead to novel pharmacotherapeutic targets based on selective targeting of melanocortin receptor subtypes, for clinical benefit in human disease states involving neuroinflammatory components and metabolic wasting.

Activation of central melanocortin-4 receptor suppresses lipopolysaccharide-induced fever in rats.

Activation of central melanocortin receptors (MCR) inhibits fever, but the identity of the MCR subtype(s) mediating this antipyretic effect is unknown. To determine whether selective central melanocortin receptor-4 (MC4R) activation produces antipyretic effects, the MC4R selective agonist MRLOB-0001 (CO-His-d-Phe-Arg-Trp-Dab-NH(2)) was administered intracerebroventricularly to rats treated with Escherichia coli lipopolysaccharide (LPS, 30 microg/kg ip). Treatment with MRLOB-0001 (150 ng icv) did not lower core body temperature (T(c)) in afebrile rats but did suppress LPS-induced increases in T(c) and associated decreases in tail skin temperature (T(sk)), an indicator of vasomotor thermoeffector function. In contrast, systemic treatment with MRLOB-0001 (150 ng iv) did not produce similar antipyretic effects. Coadministration of the selective MC4R antagonist HS014 (1 microg icv) blocked the antipyretic effects of MRLOB-0001. HS014 alone (1 microg icv) had no significant effect on LPS-induced increases in T(c) or decreases in T(sk) and in afebrile rats had no significant effects on T(c) or T(sk). We conclude that pharmacological activation of central MC4R suppresses febrile increases in T(c) and that inhibition of heat conservation pathways may contribute to this effect. These findings suggest that the central MC4R may mediate the long-recognized antipyretic effects of centrally administered melanocortins.

QSAR and proteo-chemometric analysis of the interaction of a series of organic compounds with melanocortin receptor subtypes.

We have created quantitative structure-activity relationship (QSAR) models describing the interaction of a series of 54 organic compounds with four melanocortin (MC) receptor subtypes, MC(1), MC(3), MC(4), and MC(5). In addition to traditional QSAR analysis, we applied our recently developed proteo-chemometrics approach. Proteo-chemometrics is based on the combined analysis of series of receptors and ligands, wherein descriptions of ligands, proteins, and so-called ligand-protein cross-terms are correlated with interaction activities. The compounds were characterized by structural descriptors, including three-dimensional grid-independent descriptors (GRINDs), topological descriptors, and geometrical descriptors. Description of receptors was obtained by computing the receptors' amino acid sequence identities. Both the QSAR and proteo-chemometrics approaches resulted in models with essentially the same statistical significance: the cross-validated correlation coefficient q(2) for the proteo-chemometric model being 0.71, while for the QSAR models the q(2)s were 0.75, 0.68, 0.63, and 0.71 for the MC(1), MC(3)(-)(5) receptor, respectively. However, the proteo-chemometrics modeling provided more detailed information about receptor-ligand interactions and determinants for receptor subtype selectivity than did QSAR.

Regulation of TNF-α secretion by a specific melanocortin-1 receptor peptide agonist

The lack of specific pharmacological tools has impeded the evaluation of the role of each melanocortin receptor (MCR) subtype in the myriad physiological effects of melanocortins. 154N-5 is an octapeptide (MFRdWFKPV-NH 2) that was first identified as an MC1R antagonist in Xenopus melanophores [J. Biol. Chem. 269 (1994) 29846]. In this manuscript, we show that 154N-5 is a specific agonist for human and murine MC1R. The peptide has negligible activity at MC3R and MC4R and is 25-fold less potent and a weak agonist at MC5R. 154N-5 was tested in both a cellular and an animal model of tumor necrosis factor-α (TNF-α) secretion. The inhibitory efficacy of 154N-5 on TNF-α secretion in both models was similar to the nonselective agonist NDP-α-melanocyte stimulating hormone (NDP-αMSH), thus, we conclude that inhibition of TNF-α secretion by melanocortin peptides is mediated by MC1R. 154N-5 is a valuable new tool for the evaluation of specific contribution of MC1R agonism to physiological and pathological processes.

Differential role of melanocortin receptor subtypes in cachexia.

Animals and humans respond to starvation with a complex neuroendocrine response that ultimately leads to an increase in appetite, a sparing of lean body mass (LBM) and burning of fat, and an overall decrease in basal metabolic rate. In contrast, cachexia is a pathological state of malnutrition associated with many infections and chronic diseases, wherein appetite is diminished concomitant with an increase in metabolic rate, and a relative wasting of LBM. In previous studies, we demonstrated that anorexia and weight loss in mouse cachexia models induced by lipopolysaccharide (LPS) administration and by tumor growth are ameliorated by central melanocortin-4 (MC4) receptor (MC4-R) blockade. In contrast to the results seen with MC4 blockade, melanocortin-3 (MC3) receptor knockout (MC3-RKO) mice show illness-induced anorexia and weight loss with LPS administration and with cytokine administration, and they have similar decreases in mobility. Both MC3-RKOs and MC4-RKOs have an intact corticosterone response and fever with LPS injection. In tumor models, we show that MC4-RKO mice resist the loss of LBM brought about by tumor growth, whereas MC3-RKO animals show enhanced tissue wasting. These data underscore the importance of central melanocortin signaling in weight homeostasis and demonstrate differential effects of MC3-R and MC4-R blockade on the development of cachexia.

Molecular Determinants of Human Melanocortin-4 Receptor Responsible for Antagonist SHU9119 Selective Activity

The hypothalamic melanocortin-4 receptor (MC4R), a seven transmembrane G-protein-coupled receptor, plays an important role in the regulation of body weight. The synthetic melanocortin analog SHU9119 has been widely used to characterize the physiological role of MC4R in feeding behavior and energy homeostasis. Previous studies indicated that SHU9119 is an agonist at the melanocortin-1 receptor (MC1R) but an antagonist at the MC4R. However, the molecular basis of the interaction between hMC4R and SHU9119 has not been clearly defined. To gain insight into the molecular determinants of hMC4R in the selectivity of SHU9119 chimeras and mutants hMC1R and hMC4R were expressed in cell lines and pharmacologically analyzed. A region of receptor containing the third transmembrane of hMC4R was found to be required for selective SHU9119 antagonism. Further mutagenesis studies of this region of hMC4R demonstrated that the amino acid residue leucine 133 in the third transmembrane was critical for the selective antagonist activity of SHU9119. The single substitution of leucine 133 to methionine did not affect SHU9119 binding to hMC4R. However, this substitution did convert SHU9119 from an antagonist to an agonist. Conversely, exchange of Met(128) in hMC1R to Leu, the homologous residue 133 of hMC4R, displayed a reduction in SHU9119 binding affinity and potency. This report provides the details of the molecular recognition of SHU9119 antagonism at hMC4R and shows that amino acid Leu(133) of hMC4R plays a key role in melanocortin receptor subtype specificity.

Functional properties of an agouti signaling protein variant and characteristics of its cognate radioligand.

Agouti signaling protein (ASIP), the human (h) homolog of agouti, is an endogenous melanocortin peptide antagonist. To date, characterization of this protein has been performed with recombinant protein only and without the availability of an ASIP/agouti radioligand. In this report we describe the functional characteristics of a chemically synthesized truncated ASIP variant, ASIP-[90-132 (L89Y)], and the binding characteristics of its cognate radioligand, (125)I-ASIP-[90-132 (L89Y)]. Similar to full-length recombinant ASIP/agouti, ASIP-[90-132 (L89Y)] was a potent inhibitor of alpha-melanocyte-stimulating hormone cAMP generation at the cloned human melanocortin receptor (hMCR) subtypes hMC1R and hMC4R. It also displayed a lesser degree of inhibition at the hMC3R and hMC5R. However, ASIP-[90-132 (L89Y)] was found to be less potent than full-length recombinant ASIP and, surprisingly, only exhibited weak inhibitory activity at the hMC2R. In competition binding assays with the radioligand (125)I-ASIP-[90-132 (L89Y)], ASIP-[90-132 (L89Y)] displayed a hierarchy of binding affinity that roughly paralleled its rank order of inhibitory potency at the various MCR subtypes, i.e., hMC1R approximately hMC4R > hMC3R approximately hMC5R > hMC2R. Structure-activity studies revealed that ASIP-[90-132 (L89Y)] possessed greater pharmacological potency than either the further truncated ASIP variants ASIP-(116-132) or cyclo(CRFFRSAC). Interestingly, the latter molecules were both weak agonists at the hMC1R. These studies further support the concept that ASIP/agouti inhibits melanocortin action by directly binding to target MCRs and provide additional insight into the structural requirements for maximal inhibitory potency.

Minimization of MC1R selectivity by modification of the core structure of α-MSH-ND

Background: Melanocortin, through its distinct receptor subtypes, has many different effects. Receptor-selective ligands are required to reduce the undesirable effects of melanocortin. To investigate which conformation is preferable to a given melanocortin receptor subtype, a structural and functional analysis of the ligand–receptor interactions was made by studying the biological activity, the nuclear magnetic resonance structures, and the patterns of the ligand–receptor interaction for each receptor subtype by homology modeling analysis. Results: Among the several analogues examined, [Gln 6]α-melanocyte-stimulating hormone (MSH)-ND was found to have 10 000 times less biological activity than α-MSH-ND for the MC1R, whereas, the potencies of both oligopeptides were comparable in both the melanocortin-3 receptor (MC3R) and MC4R. [Gln 6]α-MSH-ND exhibited a type I′ β-turn that was similar to the type I β-turn structure of α-MSH-ND. However, a remarkable structural difference was observed with respect to the side chain orientations of the sixth and seventh residues of [Gln 6]α-MSH-ND, which were found to be mirror images of α-MSH-ND. By homology modeling analysis, the His 6 of α-MSH-ND was found to interact with the TM2 regions of all three receptors (Glu 94 of MC1R, Glu 94 of MC3R, and Glu 100 of MC4R), but [Gln 6]α-MSH-ND did not. The phenyl ring of the D-Phe 7 residue of [Gln 6]α-MSH-ND revealed an interaction with the TM3 regions of both the MC3R and MC4R (Ser 122 of MC3R or Ser 127 of MC4R). However, in the MC1R, these serine residues corresponded to Val 122, which contains two methyl groups that induce steric hindrance with D-Phe 7 of [Gln 6]α-MSH-ND. This is a possible explanation for the biological activity of [Gln 6]α-MSH-ND for the MC1R being significantly lower than that for either the MC3R or MC4R. Conclusions: Minimization of the MC1R selectivity whilst preserving its comparable potency for both the MC3R and MC4R could be achieved by modifying the D-Phe 7 orientation of α-MSH-ND, while maintaining the ‘type I β-turn’-like structure.

Identification of avian alpha-melanocyte-stimulating hormone in the eye: temporal and spatial regulation of expression in the developing chicken.

The presence and possible physiological roles of alpha-melanocyte-stimulating hormone (alpha-MSH) in the peripheral tissues of birds have not been established. By a combination of RT-PCR, immunocytochemistry and in situ hybridization, we have examined alpha-MSH expression in the eye of the chicken during development. In the 1-day-old chick, alpha-MSH was expressed in the retinal pigment epithelial (RPE) cells, and also at a lower level in the cone cells. The melanocortin receptor subtypes, CMC1, CMC4 and CMC5, were expressed in the layers of the choroid and the neural retina, but not in the RPE cells. It is probable that the RPE cells secrete alpha-MSH to exert paracrine effects on the choroid and neural retina. During embryonic development, alpha-MSH immunoreactivity in the RPE cells was initially detected at embryonic day 10, and increased in intensity as development proceeded. No cone cells were stained with anti-alpha-MSH antiserum in any of the embryonic stages tested. The immunoreactivities for two prohormone convertases, PC1 and PC2, were co-localized to the RPE cells with a pattern of staining similar to that of alpha-MSH. Despite containing alpha-MSH immunoreactivity, the RPE cells in 1-day-old chicks expressed no immunoreactivity for the endoproteases. Furthermore, in a 3-day-old chick, pro-opiomelanocortin mRNA was detectable by in situ hybridization only in the photoreceptor layer and not in the RPE cells. These results suggest that the RPE cells and the cone cells are intraocular sources of alpha-MSH in the embryonic and postnatal life of the chicken respectively. Embryonic expression of alpha-MSH in the RPE cells implies a possible role for the peptide in ocular development.

The Melanocortin 5 Receptor is Expressed in Human Sebaceous Glands and Rat Preputial Cells

Melanocortins regulate pigmentation, adrenal hormone secretion, immune functions, lipid metabolism, and feeding behaviors in rodents. These peptides include adrenocorticotrophic hormone, melanocyte stimulating hormone, beta-lipotrophin, and the endorphins. Lipid metabolism in sebaceous glands and preputial glands of rodents is regulated by alpha-melanocyte stimulating hormone, the major agonist for melanocortin receptors. Five melanocortin receptor subtypes have been identified that differ in their tissue localization and affinities for melanocortin ligands. Targeted disruption of the melanocortin 5 receptor in transgenic mice results in widespread dysfunction of exocrine glands, including a marked decrease in sebum production. A role for melanocortins in the modulation of human sebum production has not been established. The goal of this study is to determine which melanocortin receptors are expressed in human sebaceous glands. Messenger RNA was isolated from human sebaceous glands and the reverse transcriptase polymerase chain reaction was performed using primers specific for each of the melanocortin receptor subtypes. Transcripts were detected for the melanocortin 5 receptor. A polyclonal chicken antihuman antibody to the melanocortin 5 receptor localized to sebaceous glands, eccrine glands, hair follicles, and epidermis in human skin, rat skin, cultured human sebocytes, and rat preputial cells. Presence of the melanocortin 5 receptor protein in human sebaceous glands and rat preputial glands was further verified by Western blotting. These data support further investigation of the role of melanocortins in the regulation of human sebum production and support the use of the rat preputial system as an experimental model in sebaceous gland physiology.

Identification of the binding pocket for the TRH peptide in the melanocortin 1 receptor

We found recently that thyrotropin-relasing hormone (TRH) acts as a selective agonist on the melanocortin MC1 receptor. While the TRH was capable of fully activating the MC1 receptor, it did not interact with any of the other MSH peptide binding G-protein coupled melanocortin receptor subtypes MC3–5. The MC1 receptor is a promising target for the development of anti-inflammatory and immuno-modulatory drugs, and it was of wide interest to explore the binding site of the TRH in this receptor. Here we have investigated the binding of TRH to MC1/MC3 chimeric receptors and used a partial least squares (PLS) modelling approach for the data evaluation. Statistically valid PLS models (R2=0.80; Q2=0.66) were obtained explaining the contribution of the amino acid sequence parts of the receptor chimeras for the binding of TRH. By using the variable importances in the projection (VIPs) deduced from the PLS-model, it was revealed that the trnsmembrane (TM) regions TM1 and TM2/TM3 contribute the most to the TRH binding. The TM6/TM7 also had a significant influence on the binding. Moreover, it was revealed that an interaction between TM1 and TM6/TM7 of the receptor contributed to the binding of TRH. The data are in full agreement with a 3D model of a TRH peptide and MC1 receptor complex and validates the location of the TRH ligand binding pocket between the TM1, TM2 and TM7 of the receptor.

Melanocortin‐3 Receptor mRNA Expression in Pro‐Opiomelanocortin Neurones of the Rat Arcuate Nucleus

The melanocortins alpha- and gamma-melanocyte-stimulating hormones (alpha- and gamma-MSH) derive from the pro-opiomelanocortin (POMC) precursor. Melanocortins exert a wide range of biological activities in the brain through activation of at least three distinct melanocortin receptor (MC-R) subtypes. In order to determine whether POMC neurones can modulate their own activity, we looked for the possible expression of the MC3-R gene in POMC-positive cell bodies in the rat hypothalamus. In situ hybridization experiments revealed that the density of MC3-R mRNA is particularly high in the arcuate nucleus which contains the main population of POMC neurones in the brain. The occurrence of MC3-R mRNA in POMC-positive cell bodies was demonstrated using a double-labelling in situ hybridization technique. The proportion of POMC neurones expressing MC3-R mRNA was significantly higher in the most rostral (43.5%) than in the most posterior part of the arcuate nucleus (8.2%). These results indicate that melanocortins likely exert a direct regulatory feedback on POMC neurones through activation of MC3-R receptors. Our data also suggest that MC3-R may be involved in the neuroendocrine responses induced by centrally administered melanocortins.

Structure activity studies of the melanocortin antagonist SHU9119 modified at the 6, 7, 8, and 9 positions☆

The melanocortin system is involved in the regulation of several diverse physiological pathways, including energy homeostasis. Several synthetic peptide analogs have been designed, synthesized, and pharmacologically characterized at the mouse melanocortin receptor subtypes MC1R, MC3R, MC4R, and MC5R. These peptides incorporate modifications of the melanocortin core amino acids His-Phe-Arg-Trp by using the cyclic lactam templates of the lead structures MTII and SHU9119. Analogs containing DNal(2′) at position 7 resulted in partial agonist and antagonistic activities at the mMC3R while possessing full antagonistic activities at the mMC4R. Recently, the melanocortin-5 receptor (MC5R) has been demonstrated to have a role in the regulation of exocrine gland function. This study has characterized the following analogs of SHU9119 that possess antagonist activity at the MC5R: Ac-Nle-c[Asp-(1-Me)His 6- DNal(2′) 7-Arg-Trp-Lys]-NH 2, pA 2 = 7.1; Ac-Nle-c[Asp-(1-Me)His 6- DNal(2′) 7-Arg-Nal(2′) 9-Lys]-NH 2, pA 2 = 7.2; and Ac-Nle-c[Asp-Trp 6- DNal(2′) 7-Arg-Nal(2′) 9-Lys]-NH 2, pA 2 = 6.6.