Inverse agonist activity of agouti and agouti-related protein

Agouti and agouti-related protein (AgRP) are endogenous antagonists of the melanocortin receptors (MCxR). Previous data showed that recombinant full-length agouti and a synthetic fragment of AgRP, AgRP (83-132), are inverse agonists at the MC1R and MC4R, respectively. This study demonstrates the smaller analogs AgRP (87-120) and ASIP [90-132 (L89Y)], and short peptides Yc[CRFFNAFC]Y and Qc[CRFFRSAC]S are also MC4R inverse agonists. Furthermore, the relative affinity of the series of MC4R ligands for displacement of radiolabeled antagonist 125I-AgRP (86-132) versus radiolabeled agonist 125I-NDP-MSH did not correlate with ligand efficacy, which is more consistent with an induced-fit model than a simple two-state model of MC4R activation. These data shed new light on the determinants and mechanism of inverse agonism at the MC4R.     

ACTH1-17 is a more potent agonist at the human MC1 receptor than alpha-MSH.

The melanocortin receptor MC1 is expressed on melanocytes and is an important control point for melanogenesis and other responses. Alpha-MSH, which is considered to be the major ligand at the human melanocortin (MC)1 receptor (hMC1R), is produced from proopiomelanocortin (POMC) in the pituitary and in the skin by melanocytes and keratinocytes. Other POMC peptides are also produced in the skin and their concentrations exceed those of alpha-MSH by several fold. One of the most abundant is ACTH1-17. We have shown that adrenocorticotrophic hormone (ACTH)1-17 is more potent than alpha-MSH in stimulating melanogenesis in human melanocytes and unlike alpha-MSH produces a biphasic dose response curve. In this study we have examined the ability of ACTH1-17 to function as a ligand at the hMC1R. Competitive binding assays with [125I]Nle4 DPhe7 alpha-MSH as labelled ligand were carried out in HEK 293 cells transfected with the hMC1R. ACTH1-17 showed high affinity for the hMC1R with a Ki value of 0.21 +/- 0.03 nM which was slightly higher than that of 0.13 +/- 0.005 nM for alpha-MSH. ACTH1-17 was, however, more potent than alpha-MSH in increasing cAMP and IP3 production in the transfected cells. Our results demonstrate that ACTH1-17 is a potent agonist at the hMC1R. It is therefore possible that ACTH1-17, which is found in the skin in greater concentrations than alpha-MSH, has an important role in the regulation of human melanocytes and other cell types that express the hMC1R.     

Extensive structure-activity studies of lactam derivatives of MT-II and SHU-9119: their activity and selectivity at human melanocortin receptors 3, 4, and 5.

The melanocortin system is involved in the regulation of several diverse physiologic pathways. Recently we have demonstrated that replacing His6 by Pro6 in the well-known antagonist SHU-9119 resulted in a potent agonist at the hMC5R (EC50 = 0.072 nm) with full antagonist activity at the hMC3R and the hMC4R. We have designed, synthesized, and pharmacologically characterized a series of peptide analogs of MT-II and SHU-9119 at the human melanocortin receptors MC3R, MC4R and MC5R. All these peptides were modified at position 6 with a Pro instead of a His residue. In this study, we have identified new scaffolds which are antagonists at the hMC4R and hMC3R. Additionally, we have discovered a new selective agonist at the hMC4R, Ac-Nle-c[Asp-Pro-D-Phe-Arg-Trp-Lys]-Pro-Val-NH2 (6, PG-931) which will be useful in further biologic investigations of the hMC4R. PG-931 was about 100-fold more selective for the hMC4R vs. the hMC3R (IC50 = 0.58 and 55 nm, respectively). Some of these new analogs have exceptional biologic potencies at the hMC5R and will be useful in further efforts to differentiate the substructural features responsible for selectivity at the hMC3R, hMC4R, and hMC5R.     

Poor cell surface expression of human melanocortin-4 receptor mutations associated with obesity

The melanocortin-4 receptor (MC4R) plays an important role in the regulation of body weight in rodents. Mutations in the coding region of the MC4R are found more frequently in obese individuals, supporting the hypothesis that also in humans deficient melanocortin signaling may lead to obesity. Family studies that were carried out to demonstrate the relevance of single mutations for obesity were mostly inconclusive, most likely due to small sample size and complexity of the trait. In addition, the existing pharmacological data of the mutant receptors are limited in that for most mutations the effect on receptor expression level and Agouti-related protein (AgRP) pharmacology have not been studied. The aim of the present study was to gain further insight into the impact of the MC4R mutations on receptor function. Eleven missense mutations were tested for cell surface expression, affinity for alpha-melanocyte-stimulating hormone (alpha-MSH) and AgRP-(83-132), and the biological response to alpha-MSH. All mutants were poorly expressed at the cell surface, as measured by 125I-[Nle4-D-Phe7]alpha-MSH binding, and only a few mutants showed altered pharmacology for alpha-MSH and AgRP. Hemagglutinin-tagged mutant receptors were retained in the intracellular environment. These pharmacological data provide a basis to estimate the quantitative effect of MC4R mutations for the development of obesity.     

Agouti-related protein has an inhibitory paracrine role in the rat adrenal gland

alpha-Melanocyte-stimulating-hormone (alpha-MSH) is an agonist at the melanocortin 3 receptor (MC3-R) and melanocortin 4 receptor (MC4-R). alpha-MSH stimulates corticosterone release from rat adrenal glomerulosa cells in vitro. Agouti-related protein (AgRP) an endogenous antagonist at the MC3-R and MC4-R, is expressed in the adrenal gland. We investigated the expression of the MC3-R and MC4-R and the role of AgRP in the adrenal gland. MC3-R and MC4-R expression was detected in rat adrenal gland using RT-PCR. The effect of AgRP on alpha-MSH-induced corticosterone release was investigated using dispersed rat adrenal glomerulosa cells. AgRP administered alone did not affect corticosterone release, but co-administration of AgRP and alpha-MSH attenuated alpha-MSH-induced corticosterone release. To investigate glucocorticoid feedback, adrenal AgRP expression was compared in rats treated with dexamethasone to controls. AgRP mRNA was increased in rats treated with dexamethasone treatment compared to controls. Our findings demonstrate that adrenal AgRP mRNA is regulated by glucocorticoids. AgRP acting via the MC3-R or MC4-R may have an inhibitory paracrine role, blocking alpha-MSH-induced corticosterone secretion.     

Chimeras of the agouti-related protein: insights into agonist and antagonist selectivity of melanocortin receptors

The specific melanocortin receptors, MC3R and MC4R, are directly linked to metabolism and body weight control. These receptors are activated by the peptide hormone alpha-MSH and antagonized by the agouti-related protein (AGRP). Whereas alpha-MSH acts broadly on most members of the MCR family (with the exception of MC2R), AGRP is highly specific for only MC3R and MC4R. AGRP is a complex ligand of approximately 100 amino acids. Within AGRP, MCR recognition and antagonism is localized to a 34 residue, cysteine-rich domain that adopts an inhibitor cystine knot (ICK) fold. An oxidatively folded peptide corresponding to this domain, referred to as mini-AGRP, exhibits full antagonist function and selectivity for MC3R and MC4R. Here we investigate a series of chimera proteins based on the mini-AGRP scaffold. Amino acid sequences derived from peptide agonists are grafted into the mini-AGRP active loop, implicated in receptor recognition, with the goal of producing ICK based agonists specific for MC3R and MC4R. Several constructs indeed exhibited potent agonist activity; however, with all chimeras, receptor selectivity is significantly altered. Pharmacologic data indicate that the chimeras do not interact with MC receptors through native AGRP like contacts. A model to explain the data suggest that there is only partial overlap of the agonist versus antagonist binding surfaces within MC receptors. Moreover, accessibility to the binding pocket is highly receptor specific with MC3R being the least tolerant of ligand alterations.     

Contribution of the conserved amino acids of the melanocortin-4 receptor in [corrected] [Nle4,D-Phe7]-alpha-melanocyte-stimulating [corrected] hormone binding and signaling

Melanocortin 4 receptor (MC4R) plays an important role in the regulation of food intake and body weight. To determine the molecular basis of human MC4R (hMC4R) responsible for alpha-melanocortin-stimulating hormone (alpha-MSH) binding, in this study, we utilized both receptor domain exchange and site-directed mutagenesis studies to investigate the molecular determinants of hMC4R responsible for alpha-MSH binding and signaling. alpha-MSH is a potent agonist at hMC4R but not at hMC2R. Cassette substitutions of the second, third, fourth, fifth, and sixth transmembrane regions (TM) of the hMC4R with the homologous regions of hMC2R were performed and alpha-MSH binding and signaling were examined. Our results indicate that each chimeric receptor was expressed at the cell surface and the expression levels remain similar to that of the wild-type receptor. The cassette substitutions of the second, fourth, fifth, and sixth TMs of the hMC4R with homologous regions of the hMC2R did not significantly alter alpha-MSH binding affinity and potency except substitution of the TM3 of the hMC4R, suggesting that the conserved residues in TMs of the hMC4R are crucial for alpha-MSH binding and signaling. Further mutagenesis studies indicate that conserved residues Glu(100) in TM2, Asp(122), Asp(126) in TM3 and Trp(258), Phe(261), His(264) in TM6 are involved in alpha-MSH binding and signaling. In conclusion, our results suggest that the conserved residues in the TM2, TM3, and TM6 of the hMC4R are responsible for alpha-MSH binding and signaling.     

Sustained orexigenic effect of Agouti related protein may be not mediated by the melanocortin 4 receptor

Intracerebroventricular (ICV) injection of Agouti related protein (AgRP), an endogenous melanocortin 3 and 4 receptor (MC3/4-R) antagonist, produces a prolonged increase in food intake. To clarify the roles of the MC3-R and MC4-R in AgRP-induced hyperphagia, the feeding effect of AgRP (83-132) was compared with that of the selective MC4-R antagonist, JKC-363 (cyclic [Mpr11, D-Nal14, Cys18, Asp22-NH2]-beta-MSH11-22). Single ICV administration of AgRP (83-132) increased food intake for 48 h whilst ICV JKC-363 increased food intake for 8h. An increase in body weight at 24 and 48 h was observed following AgRP (83-132) but not JKC-363 treatment. These data suggest that the sustained orexigenic action of AgRP (83-132) may not be through MC4-R antagonism.     

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.     

Functional characterization of the modified melanocortin peptides responsible for ligand selectivity at the human melanocortin receptors

The melanocortin system plays an important role in energy homeostasis as well as skin pigmentation, steroidogenesis and exocrine gland function. In this study, we examined eight Ac-His-Phe-Arg-Trp-NH(2) tetrapeptides that were modified at the Phe position and pharmacologically characterized their activities at the human MCR wild-types and their mutants. Our results indicate that at the hMC1R, all D stereochemical modified residues at the Phe position of peptides increase cAMP production in a dose-dependent manner. At the hMC3R, the DPhe peptide dose dependently increases cAMP production but all other three tetrapeptides were not. At the hMC4R, both the DPhe and DNal(1') peptides induce cAMP production. However, both DTyr and DNal(2') were not able to induce cAMP production. Further studies indicated that at the hMC1R M128L mutant receptor, the all D-configured tetrapeptides reduce their potencies as compared to that of hMC1R wild-type. However, at the hMC3R and hMC4R L165M and L133M mutant receptors, the DNal(2') and DTyr tetrapeptides possess agonist activity. These findings indicate that DPhe in tetrapeptide plays an important role in ligand selectivity and specific residue TM3 of the melanocortin receptors is crucial for ligand selectivity.     

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.     

Long-term orexigenic effects of AgRP-(83---132) involve mechanisms other than melanocortin receptor blockade

Overexpression of agouti-related peptide (AgRP), an endogenous melanocortin (MC) 3 and 4 receptor antagonist (MC3/4-R), causes obesity. Exogenous AgRP-(83---132) increases food intake, but its duration and mode of action are unknown. We report herein that doses as low as 10 pmol can have a potent effect on food intake of rats over a 24-h period after intracerebroventricular injection. Additionally, a single third ventricular dose as low as 100 pmol in rats produces a robust increase in food intake that persists for an entire week. AgRP-(83---132) completely blocks the anorectic effect of MTII (MC3/4-R agonist), given simultaneously, consistent with a competitive antagonist action. However, when given 24 h prior to MTII, AgRP-(83---132) is ineffective at reversing the anorectic effects of the agonist. These results support a critical role of MC tone in limiting food intake and indicate that the orexigenic effects of AgRP-(83---132) are initially mediated by competitive antagonism at MC receptors but are sustained by alternate mechanisms.     

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.     

Synthesis and biological evaluation on hMC3, hMC4 and hMC5 receptors of gamma-MSH analogs substituted with L-alanine.

To elucidate the molecular basis of the interaction of the native dodecapeptide gamma-MSH with the melanocortin receptors, we performed a structure-activity study in which we systematically replaced l-Ala in each position of this peptide. Here we report the binding affinity and agonist potency on human MC3R, MC4R and MC5R. Intracellular cAMP concentration was measured on CHO cells, and binding assays were carried out using membranes prepared from these cell lines which stably express hMC3R, hMC4R and hMC5R. Our results indicate that the last four amino acids in the C-terminal region of gamma-MSH are not important determinants of biological activity and selectivity at human melanocortin receptors. Interesting results were obtained when l-Ala was substituted for His6, Phe7, Arg8 and Trp9. For these peptides, the affinity and activity at all three human receptors (MC3R, MC4R and MC5R) decreased significantly, demonstrating that the His-Phe-Arg-Trp sequence in gamma-MSH is important for activity at these three melanocortin receptors. Similar results were obtained when Met3 was replaced with l-Ala, suggesting the importance of this position in the interaction with all three receptors. This study highlights the role played by the His-Phe-Arg-Trp sequence in receptor binding and in agonist activity of gamma-MSH.     

黑皮质素受体-4调节通路的研究进展

黑皮质素系统来自阿片-促黑素细胞皮质素原,在中枢摄食行为和能量平衡代谢中起到重要作用,此系统生理功能的发挥主要通过与下丘脑神经元细胞上特定膜受体（黑皮质素受体）结合完成。黑皮质素受体（MCR）有五种亚型（MC1R-MC5R）,其中参与体重调节的受体主要是黑皮质素受体3（MC3R）和黑皮质素受体4（MC4R）。MC4R属于G蛋白耦联受体,具有七次跨膜结构。作为一种膜受体,MC4R发挥体重调节作用,一方面受外界激动剂或拮抗剂的调节;另一方面,此受体活化后会影响到细胞内的信号调节通路。研究MC4R的功能首先要了解受体的结构,本文对G蛋白耦联受体的结构进行了较详细的叙述,MC4R经信号调节通路,激活腺苷酸环化酶,增加cAMP的浓度,最终通过影响细胞内基因的转录和翻译,来调节体重和能量的消耗。     

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 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.     

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).