Molecular determinants of ligand binding to the human melanocortin-4 receptor

To elucidate the molecular basis for the interaction of ligands with the human melanocortin-4 receptor (hMC4R), agonist structure-activity studies and receptor point mutagenesis were performed. Structure-activity studies of [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) identified D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full agonist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R transmembrane (TM) residues were mutated (the rationale for choosing specific receptor residues for mutation is outlined in the Results section). Mutation of TM3 residues D122 and D126 and TM6 residues F261 and H264 decreased the binding affinity of NDP-MSH 5-fold or greater, thereby identifying these receptor residues as sites potentially involved in the sought after ligand-receptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found that core melanocortin peptide residue Arg8 interacts at a molecular level with hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to alanine decreased the binding affinity of AGRP (87-132), a C-terminal derivative of the endogenous melanocortin antagonist, 8-fold, and simultaneous mutations D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4R antagonist SHU 9119. These results provide further insight into the molecular determinants of hMC4R ligand binding.     

Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity

Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. -Melanocyte stimulating hormone (-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111–113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7–9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle⁴-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH₂) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH₂). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3–5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle⁴-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH₂ peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.     

Interactions of human melanocortin 4 receptor with nonpeptide and peptide agonists

Specific interactions of human melanocortin-4 receptor (hMC4R) with its nonpeptide and peptide agonists were studied using alanine-scanning mutagenesis. The binding affinities and potencies of two synthetic, small-molecule agonists (THIQ, MB243) were strongly affected by substitutions in transmembrane alpha-helices (TM) 2, 3, 6, and 7 (residues Glu(100), Asp(122), Asp(126), Phe(261), His(264), Leu(265), and Leu(288)). In addition, a I129A mutation primarily affected the binding and potency of THIQ, while F262A, W258A, Y268A mutations impaired interactions with MB243. By contrast, binding affinity and potency of the linear peptide agonist NDP-MSH were substantially reduced only in D126A and H264A mutants. Three-dimensional models of receptor-ligand complexes with their agonists were generated by distance-geometry using the experimental, homology-based, and other structural constraints, including interhelical H-bonds and two disulfide bridges (Cys(40)-Cys(279), Cys(271)-Cys(277)) of hMC4R. In the models, all pharmacophore elements of small-molecule agonists are spatially overlapped with the corresponding key residues (His(6), d-Phe(7), Arg(8), and Trp(9)) of the linear peptide: their charged amine groups interact with acidic residues from TM2 and TM3, similar to His(6) and Arg(6) of NDP-MSH; their substituted piperidines mimic Trp(9) of the peptide and interact with TM5 and TM6, while the d-Phe aromatic rings of all three agonists contact with Leu(133), Trp(258), and Phe(261) residues.     

Molecular mechanism of the intracellular segments of the melanocortin-4 receptor for NDP-MSH signaling

Mutations of the human melanocortin-4 receptor (hMC4R) have been previously identified to be the most common cause of monogenic human obesity. Specifically, mutations of the intracellular C terminus and the third intracellular loop of hMC4R have been reported to play an important role in human obesity. However, the molecular basis of these hMC4R intracellular segments in receptor function remains unclear. In this study, we utilized deletions and mutations of specific portions of the hMC4R to determine the molecular mechanism of both the C terminus and the third intracellular loop in receptor signaling. Our results indicate that deletions of the distal 25 (the entire C terminus), 22, 18, 17, 16, and 15 amino acids of the C terminus result in the complete loss of both [Nle(4)-d-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) binding and NDP-MSH-mediated cAMP production. Deletion of the distal 14 amino acids of the C terminus significantly decreases both NDP-MSH binding affinity and potency, but deletion of the distal 13 amino acids of the C terminus does not affect NDP-MSH activity. Further analysis revealed that the proximal 12 amino acids of the C terminus are not only important for receptor signaling but also important for ligand binding. Our results also indicate that the third intracellular loop of the hMC4R is important for receptor signaling but not ligand binding. In summary, our findings suggest that the proximal region of the melanocortin-4 receptor (MC4R) C terminus is crucial not only for receptor signaling but also for ligand binding, while the third intracellular loop is important mainly for receptor signaling.     

Structure activity studies of the melanocortin-4 receptor by in vitro mutagenesis: identification of agouti-related protein (AGRP), melanocortin agonist and synthetic peptide antagonist interaction determinants

In vitro mutagenesis of the mouse melanocortin-4 receptor (mMC4R) has been performed, based upon homology molecular modeling and previous melanocortin receptor mutagenesis studies that identified putative ligand-receptor interactions. Twenty-three mMC4 receptor mutants were generated and pharmacologically characterized using several melanocortin-based ligands [alpha-MSH, NDP-MSH, MTII, DNal (1')(7)-MTII, Nal(2')(7)-MTII, SHU9119, and SHU9005]. Selected mutant receptors possessing significant differences in the melanocortin-based peptide agonist and/or antagonist pharmacology were further evaluated using the endogenous antagonist agouti-related protein fragment hAGRP(83-132) and hAGRP(109-118) molecules. These studies of the mouse MC4R provide further experimental data suggesting that the conserved melanocortin receptor residues Glu92 (TM2), Asp114 (TM3), and Asp118 (TM3) (mouse MC4R numbering) are important for melanocortin-based peptide molecular recognition. Additionally, the Glu92 and Asp118 mMC4R residues are important for molecular recognition and binding of AGRP(83-132). We have identified the Phe176 (TM4), Tyr179 (TM4), Phe254 (TM6), and Phe259 (TM6) receptor residues as putatively interacting with the melanocortin-based ligand Phe(7) by differences between alpha-MSH and NDP-MSH agonist potencies. The Glu92, Asp118, and Phe253 mMC4R receptor residues appear to be critical for hAGRP(83-132) molecular recognition and binding while Phe176 appears to be important for functional antagonism of AGRP(83-132) and AGRP(109-118) but not molecular recognition. The Phe253 mMC4R residue appears to be important for AGRP(83-132) molecular recognition and general mMC4 receptor stimulation. The Phe254 and Phe259 mMC4R amino acids may participate in the differentiation of agonist versus antagonist activity of the melanocortin-based peptide antagonists SHU9119 and SHU9005, but not AGRP(83-132) or AGRP(109-118). The Met192 side chain when mutated to a Phe results in a constitutively active mMC4R that does not effect agonist ligand binding or potency. Melanocortin-based peptides modified at the 7 position of MTII with DPhe, DNal(1'), Nal(2'), and DNal(2') have been pharmacologically characterized at these mutant mouse MC4Rs. These data suggest a revised hypothesis for the mechanism of SHU9119 antagonism at the MC4R which may be attributed to the presence of a "bulky" naphthyl moiety at the 7 position (original hypothesis), and additionally that both the stereochemistry and naphthyl ring position (2' versus 1') are important for positioning of the ligand Arg(8) residue with the corresponding mMC4R amino acids.     

Design of novel melanotropin agonists and antagonists with high potency and selectivity for human melanocortin receptors

alpha-MSH and gamma-MSH are the natural endogenous hormones for the human melanocortin-1, 3, 4 and 5 receptors (hMC1R, hMC3R, hMC4R and hMC5R). These and more potent, stable and prolonged acting analogues such as NDP-alpha-MSH, MT-II and SHU-9119 are not very receptor selective. To develop potent and selective agonist and antagonist ligands for the melanocortin receptors we have used state-of-the-art biophysical studies, computational chemistry, and design of conformational and topographical constraints with novel templates.     

A Small Molecule Agonist THIQ as a Novel Pharmacoperone for Intracellularly Retained Melanocortin-4 Receptor Mutants

Although mutations in the melanocortin-4 receptor (MC4R) gene cause severe early-onset obesity, we still do not have effective approaches to correct the defects of these mutations. Several antagonists have been identified as pharmacoperones of the MC4R whereas no agonist of the MC4R has been reported. In the present study, we investigated the effect of a small molecule agonist of the MC4R, THIQ, on the cell surface expression and signaling of ten intracellularly retained MC4R mutants using different cell lines. We showed that THIQ increased the cell surface expression of three mutants (N62S, C84R, and C271Y) and two of them (N62S and C84R) had increased signaling in HEK293 cells. Interestingly, THIQ increased the signaling of two other mutants (P78L and P260Q) without increasing their cell surface expression in HEK293 cells. In neuronal cells, THIQ exhibited a more potent effect, correcting the cell surface expression and signaling of seven mutants (N62S, I69R, P78L, C84R, W174C, P260Q, and C271Y). Other mutants were not rescued by THIQ. We also showed that THIQ did not rescue MC4R mutants defective in ligand binding or signaling or one intracellularly retained mutant of the melanocortin-3 receptor. In summary, we demonstrated that a small molecule agonist acted as a pharmacoperone of the MC4R rescuing the cell surface expression and signaling of some intracellularly retained MC4R mutants.     

Analogs of MTII, lactam derivatives of alpha-melanotropin, modified at the N-terminus, and their selectivity at human melanocortin receptors 3, 4, and 5.

In search for selective agonists at human melanocortin-4 receptor, proline-substituted analogs of MTII, a potent nonselective agonist at melanocortin receptors, were prepared by solid-phase syntheses and evaluated for their ability to bind and activate human MC-3, MC-4, and MC-5 receptors. Replacement of Nle(4) with Pro resulted in [Pro(4)]MTII with affinity to and agonist potency at hMC-4R similar to MTII, but with about 400-fold lower potency at hMC-5R and about 20-fold lower potency at hMC-3R. The substantial increase in selectivity of [Pro(4)]MTII with respect to hMC-5R prompted us to investigate additional analogs of MTII with modified N-termini. The Ac-Nle(4) segment, not encompassed in the lactam ring, was substituted with flexible, hydrophobic, or hydrophilic substituents, and also, with residues resembling proline. The similar agonist potency of these peptides to that of MTII at hMC-4R but significantly lower activity of these compounds at hMC-5R demonstrated that the N-terminal fragment of MTII has virtually no effect on the binding affinity and activation at hMC-4R, but it is essential for full potency at hMC-5R.     

Molecular basis of melanocortin-4 receptor for AGRP inverse agonism

We have investigated receptor structural components of the melanocortin-4 receptor (MC4R) responsible for ligand-dependent inverse agonism. We utilized agouti-related protein (AGRP), an inverse agonist which reduces MC4R basal cAMP production, as a tool to determine the molecular mechanism. We tested a series of chimeric receptors and utilized MC4R and MC1R as templates, in which AGRP is an inverse agonist for MC4R but not for MC1R. Our results indicate that replacements of the extracellular loops 1, 2 and 3 of MC4R with the corresponding regions of MC1R did not affect AGRP inverse agonist activity. However, replacement of the N terminus of MC4R with the same region of MC1R decreases AGRP inverse agonism. Replacement of transmembrane domains 3, 4, 5 and 6 of MC4R with the corresponding regions of MC1R did not affect AGRP inverse agonist activity but mutation of D90A in transmembrane 2 (TM2) and D298A in TM7 abolished AGRP inverse activity. Deletion of the distal MC4R C terminus fails to maintain AGRP mediated reduction in basal cAMP production although it maintains NDP-MSH mediated cAMP production. In conclusion, our results indicate that the N terminus and the distal C terminus of MC4R do appear to play important roles in AGRP inverse agonism but not NDP-MSH mediated receptor activation. Our results also indicate that the residues D90 in TM2 and D298 in TM7 of hMC4R are involved in not only NDP-MSH mediated receptor activation but also AGRP mediated inverse agonism.     

A predictive pharmacophore model of human melanocortin-4 receptor as derived from the solution structures of cyclic peptides

Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the solution structures for a series of potent agonists for the human melanocortin-4 receptor (hMC4R), based on the cyclic peptide MT-II [Ac-Nle-cyclo-(Asp-Lys) (Asp-His-(D)Phe-Arg-Trp-Lys)-NH2]. Members of this series were designed to improve selectivity for MC4R versus the other melanocortin receptors, and to reduce the flexibility of the side chains. The most selective and rigid analog [penta-cyclo(D-K)-Asp-Apc-(D)Phe-Arg-(2S,3S)-beta-methylTrp-Lys-NH2] was found to be a full agonist of hMC4R with an EC50 of 11nM against hMC4R, and to exhibit 65-fold selectivity against hMC1R. This compound represents the most constrained hMC4R peptide agonist described to date. A beta-turn structure was conserved among all of the cyclic peptides studied. The rigidity of the analogs allowed an exceptionally well-defined pharmacophore model to be derived. This model was used to perform a virtual screen using a library of 1000 drug-like compounds, to which a small set of known potent ligands had been intentionally added. The utility of the model was validated by its ability to identify the known ligands from among this large library.     

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.     

Cyclic analogs of alpha-melanocyte-stimulating hormone (alphaMSH) with high agonist potency and selectivity at human melanocortin receptor 1b

Alpha-melanotropin (alphaMSH), Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2,(1) has been long recognized as an important physiological regulator of skin and hair pigmentation in mammals. Binding of this peptide to the melanocortin receptor 1 (MC1R) leads to activation of tyrosinase, the key enzyme of the melanin biosynthesis pathway. In this study, interactions of the human MC1bR (an isoform of the receptor 1a) with the synthetic cyclic analogs of alphaMSH were studied. These ligands were analogs of MTII, Ac-Nle4-cyclo-(Asp5-His6-D-Phe7-Arg8-Trp9-Lys10)-NH2, a potent pan-agonist at the human melanocortin receptors (hMC1,3-5R). In the structure of MTII, the His6-D-Phe7-Arg8-Trp9 segment has been recognized as "essential" for molecular recognition at the human melanocortin receptors (hMC1,3-5R). Herein, the role of the Trp9 in the ligand interactions with the hMC1b,3-5R has been reevaluated. Analogs with various amino acids in place of Trp9 were synthesized and tested in vitro in receptor affinity binding and cAMP functional assays at human melanocortin receptors 1b, 3, 4 and 5 (hMC1b,3-5R). Several of the new peptides were high potency agonists (partial) at hMC1bR (EC50 from 0.5 to 20 nM) and largely inactive at hMC3-5R. The bulky aromatic side chain in position 9, such as that in Trp, was found not to be essential to agonism (partial) of the studied peptides at hMC1bR.     

Cysteine residues are involved in structure and function of melanocortin 1 receptor: Substitution of a cysteine residue in transmembrane segment two converts an agonist to antagonist

Reduction of disulfide bonds in human melanocortin 1 receptor (hMC1R) with increasing concentrations of DTT (dithiothreitol) resulted in a decrease in the binding of [125I]-ACTH (adrenocorticotropic hormone, L-isomer) in an uniphasic manner and a decrease in [125I]-NDP-MSH ([Nle(4),D-Phe(7)]-alpha-melanocyte stimulating hormone; D-isomer) binding in a biphasic manner. Pretreatment of hMC1R with 10 mM DTT resulted in a 36-fold loss of affinity for alpha-MSH (L-isomer) without affecting the affinity of NDP-MSH (D-isomer). To characterize the role of individual cysteine residues, we employed site-directed mutagenesis to substitute cysteine by glycine at all fourteen positions in hMC1R and analysed wild-type and mutant receptors for ligand binding and cAMP signalling. Single point mutation of four cysteine residues in extracellular loops to glycine (C35G, C267G, C273G, and C275G) resulted in a complete loss of binding for [125I]-NDP-MSH. Moreover, mutants with normal ligand binding, at positions C191G (transmembrane segment 5), C215G (third intracellular loop), and C315G (C-terminal loop) failed to generate cAMP signal in response to both agonists alpha-MSH and NDP-MSH. Mutant at position C78G (with wild-type binding to alpha-MSH as well as NDP-MSH) generated a cAMP signal in response to alpha-MSH (identical to wild-type hMC1R) but interestingly could not be stimulated by NDP-MSH. Moreover, this single amino acid substitution converted NDP-MSH from being an agonist to antagonist at the C78G mutant receptor. These findings demonstrate that (i) alpha-MSH and ACTH (L-isomers) are different from D-isomer NDP-MSH in their sensitivity to DTT for receptor binding, (ii) cysteine residues in N-terminus and extracellular loop three make disulfide bridges and are needed for structural integrity of hMC1R, (iii) cysteine residues in transmembrane segments and intracellular loops are required for receptor-G-protein coupling, (iv) C78 in transmembrane segment two is required for generating a functional response by D-isomer agonist (NDP-MSH) but not by L-isomer agonist (alpha-MSH), and (v) wild-type receptor agonist NDP-MSH is an antagonist at the mutant C78G receptor.     

Kinetic evidence for tandemly arranged ligand binding sites in melanocortin 4 receptor complexes

The melanocortin 4 receptor (MC(4)R) binding of the peptide analogue of melanocyte stimulating hormone, [(125)I]NDP-MSH, and the low molecular weight radionucleid 1-(D-1,2,3,4-tetrahydroisoquinoline-3-carboxy-D-4-(125)iodophenylalanyl)-4-cyclohexyl-4-[(1,2,4-triazol-1-yl)methyl]piperidine trifluoroacetate ([(125)I]THIQ) were compared. Kinetic analysis indicated heterogeneity in the binding of both radioligands, the binding apparently proceeding to two tandemly arranged interconnected mutually dependent binding sites. Steric considerations and BRET analysis of Rluc and GFP tagged receptors proposed that these sites are located on different subunits of receptor dimers, which form receptor complexes. According to the minimal model proposed, ligand binding proceeds consecutively to the two binding sites of the dimer. After binding of the first ligand conformational transformations of the complex occur, which is followed by binding of the second ligand. When both receptor units have bound [(125)I]NDP-MSH, the radioligand can be released only from one unit. The [(125)I]NDP-MSH bound to the remaining unit stays practically irreversibly bound due to a very slow retransformation rate of the transformed complex. The considerably faster binding of [(125)I]THIQ did not allow accurate kinetic differentiation of the two binding sites. However, addition of NDP-MSH as well as a fragment of the human agouti protein, hAGRP(83-132) to the preformed [(125)I]THIQ-MC(4)R complex drastically retarded the release of [(125)I]THIQ from the complex, blocking conformational transformations in the complex by binding into the second binding site. The consecutive binding of ligands to the MC(4)R dimers has substantial impact on the apparent ligand potencies, when determined in competition with the two different radioligands applied herein; the apparent potencies of the same ligand differing up to three orders of magnitude when assayed in competition with [(125)I]NDP-MSH or [(125)I]THIQ.     

Melanocortin-4 receptor-mediated inhibition of apoptosis in immortalized hypothalamic neurons via mitogen-activated protein kinase

The melanocortin-4 receptor (MC4R) is a seven transmembrane member of the melanocortin receptor family. The GT1-1 cell line exhibits endogenous expression of MC4R. In this study, GT1-1 cells were used to study MC4R signaling pathways and to examine the effects of melanocortin receptor agonist NDP-MSH on apoptosis. MC4R mRNA expression was demonstrated by RT-PCR. Functional melanocortin receptor expression was implied by specific binding of NDP-MSH and cAMP production. NDP-MSH-stimulated GnRH release in a dose-dependent manner. Serum deprivation-induced apoptosis in GT1-1 cells, and the NDP-MSH inhibited this effect. The melanocortin receptor antagonist SHU9119 blocked the antiapoptotic actions of NDP-MSH, and the MAP kinase inhibitor PD98059 significantly attenuated the antiapoptotic effect. NDP-MSH-stimulated ERK1/2 phosphorylation in a dose-dependent manner. ERK1/2 phosphorylation could be abolished by SHU9119. In GT1-1 cells, melanocortin receptor activation causes ERK1/2 phosphorylation. In these cells, MC4R activation is also associated with antiapoptotic effects.     

Substituted NDP-MSH peptides paired with mutant melanocortin-4 receptors demonstrate the role of transmembrane 6 in receptor activation

The melanocortin-4 receptor (MC4R) is involved in regulating energy homeostasis and is a potential therapeutic target for obesity and cachexia. Molecular interactions between peptide ligands and MC4R have been studied in detail. Less is known regarding the role of these interactions in the mechanism of MC4R activation. The aim of this study was to investigate the molecular mechanism of human MC4R activation by [Nle4, d-Phe7]alpha-melanocyte-stimulating hormone (NDP-MSH), by first defining the role of the His6-d-Phe7-Arg8-Trp9 residues in receptor activation (Emax for stimulation of cAMP accumulation) using modified peptides, then understanding how their interaction with the receptor modulates activation using site-directed mutagenesis and a molecular model of NDP-MSH bound to the active state of the receptor. Alanine substitution indicated that the d-Phe7, Arg8, and Trp9 side chains contribute binding energy but are not essential for the receptor activation event. Conversely, His6 to Ala6 substitution reduced receptor activation but did not affect affinity. Chlorine substitutions on the d-Phe7 side chain also inhibited receptor activation. F261(6.51)A and F284(7.35)A receptor mutations acted as gain-of-function mutations, restoring efficacy to the His6 and d-Phe7 substituted peptides that had lost efficacy at the wild-type receptor. Based on a model of NDP-MSH and MC4R interaction, the antagonist behavior of these peptides is consistent with the prevention of transmembrane 6 (TM6) rotation. This data supports the hypothesis that increasing the size of d-Phe7 directly interferes with TM6 rotation, preventing receptor activation. We further propose that removing the interaction with the His6 side chain reorients the peptide within the binding pocket, indirectly impeding TM6 rotation by strengthening peptide interaction with F261(6.51) and F284(7.35). These findings refine the molecular basis for the mechanism of ligand-stimulated hMC4R activation and will be useful for the development of hMC4R agonists and antagonists.     

Parathyroid hormone-related protein and lung injury after pulmonary thromboendarterectomy

Agouti-related protein (AGRP) is one of two naturally occurring antagonists of G-Protein coupled receptors (GPCRs) identified to date, and has been physiologically implicated in regulating food intake, body weight, and energy homeostasis. AGRP has been identified in vitro, as competitively antagonizing the brain melanocortin-4 (MC4R) and melanocortin-3 (MC3R) receptors, and when over expressed in transgenic mice, results in an obese phenotype. Emerging data propose that AGRP has additional targets in the hypothalamus and/or physiologically functions via a mechanism in addition to competitive antagonism of alpha-MSH at the brain melanocortin receptors. We report data herein supporting an alternative mechanism for AGRP involvement in feeding behavior. A constitutively active MC4R has been generated which possess EC(50) values for melanocortin agonists (alpha-MSH, NDP-MSH, and MTII) and a pA2 value for the synthetic peptide antagonist SHU9119 identical to the wildtype receptor, but increases basal activity to 50% maximal response. AGRP possesses inverse agonist activity at this constitutively active MC4R. These data support the hypothesis for an additional physiological mechanism for AGRP action in feeding behavior and energy homeostasis.     

Molecular characterization of human melanocortin-3 receptor ligand-receptor interaction

Melanocortin-3 receptor (MC3R), primarily expressed in the hypothalamus, plays an important role in the regulation of energy homeostasis. MC3R-deficient (MC3R(-)(/)(-)) mice demonstrate increased fat mass, higher feeding efficiency, hyperleptinaemia, and mild hyperinsulinism. At least one specific mutation of MC3R has been identified to be associated with human obesity. Functional analysis of this altered MC3R (I183N) has indicated that the mutation completely abolishes agonist-mediated receptor activation. However, the specific molecular determinants of MC3R responsible for ligand binding and receptor signaling are currently unknown. The present study is to determine the structural aspects of MC3R responsible for ligand binding and receptor signaling. On the basis of our theoretical model for MC1R, using mutagenesis, we have examined 19 transmembrane domain amino acids selected for these potential roles in ligand binding and receptor signaling. Our results indicate that (i) substitutions of charged amino acid residues E131 in transmembrane domain 2 (TM2), D154 and D158 in TM3, and H298 in TM6 with alanine dramatically reduced NDP-MSH binding affinity and receptor signaling, (ii) substitutions of aromatic amino acids F295 and F296 in TM6 with alanine also significantly decreased NDP-MSH binding and receptor activity, (iii) substitutions of D121in TM2 and D332 in TM7 with alanine resulted in the complete loss of ligand binding, ligand induced receptor activation, and cell surface protein expression, and (iv) interestingly, substitution of L165 in TM3 with methionine or alanine switched antagonist SHU9119 into a receptor agonist. In conclusion: Our results suggest that TM3 and TM6 are important for NDP-MSH binding, while D121 in TM2 and D332 in TM7 are crucial for receptor activity and signaling. Importantly, L165 in TM3 is critical for agonist or antagonist selectivity. These results provide important information about the molecular determinants of hMC3R responsible for ligand binding and receptor signaling.     

Analogs of sub-nanomolar hMC1R agonist LK-184 [Ph(CH2)3CO-His-D-Phe-Arg-Trp-NH2]. An additional binding site within the human melanocortin receptor 1?

Twenty nine analogs of a superpotent MC1R agonist LK-184 (1) were tested at human melanocortin receptors (hMC1, hMC3, and hMC4Rs). All derivatives with the spacer between the N-terminus and the aromatic ring longer or shorter than C(3) were much less potent at hMC1R than 1. Only LK-312 PhCO(CH(2))(3)CO-His-d-Phe-Arg-Trp-NH(2) (3), partially mimicking the pi-system of 1, had an EC(50) of 0.05 nM at hMC1R, which confirms the localization of the pi-binding zone of the receptor. Truncation of 1 to Ph(CH(2))(3)CO-His-d-Phe-Arg-NH(2) gave a full MC1 agonist, LK-394 (30), with an EC(50) of 5 nM and a weak partial agonism at MC3/4Rs. This suggests the existence of an additional binding site within hMC1R next to that for the core sequence His-d-Phe-Arg-Trp-NH(2).