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.     

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.     

Serotonergic pathways converge upon central melanocortin systems to regulate energy balance

Multiple lines of research provide compelling support for an important role for central serotonergic (5-hydroxytryptamine, 5-HT) and melanocortin pathways in the regulation of food intake and body weight. In this brief review, we outline data supporting a model in which serotonergic pathways affect energy balance, in part, by converging upon central melanocortin systems to stimulate the release of the endogenous melanocortin agonist, alpha-melanocyte stimulating hormone (alpha-MSH). Further, we review the neuroanatomical mapping of a downstream target of alpha-MSH, the melanocortin 4 receptor (MC4R), in the rodent brain. We propose that downstream activation of MC4R-expressing neurons substantially contributes to serotonin's effects on energy homeostasis.     

Potent and selective agonists of alpha-melanotropin (alphaMSH) action at human melanocortin receptor 5; linear analogs of alpha-melanotropin

Alpha-melanotropin, Ac-Ser(1)-Tyr-Ser-Met-Glu-His(6)-Phe(7)-Arg(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2)(1), is a non-selective endogenous agonist for the melanocortin receptor 5; the receptor present in various peripheral tissues and in the brain, cortex and cerebellum. Most of the synthetic analogs of alphaMSH, including a broadly used and more potent the NDP-alphaMSH peptide, Ac-Ser(1)-Tyr-Ser-Nle(4)-Glu-His(6)-D-Phe(7)-Arg(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2), are also not particularly selective for MC5R. To elucidate physiological functions of the melanocortin receptor 5 in rodents and humans, the receptor subtype selective research tools are needed. We report herein syntheses and pharmacological evaluation in vitro of several analogs of NDP-alphaMSH which are highly potent and specific agonists for the human MC5R. The new linear peptides, of structures and solubility properties similar to those of the endogenous ligand alphaMSH, are exemplified by compound 7, Ac-Ser(1)-Tyr-Ser-Met-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2) (Oic: octahydroindole-2-COOH, 4,4'-Bip: 4,4'-biphenylalanine, Pip: pipecolic acid), shortly NODBP-alphaMSH, which has an IC(50)=0.74 nM (binding assay) and EC(50)=0.41 (cAMP production assay) at hMC5R nM and greater than 3500-fold selectivity with respect to the melanocortin receptors 1b, 3 and 4. A shorter peptide derived from NODBP-alphaMSH: Ac-Nle-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8)-Trp(9) -NH(2) (17) was measured to be an agonist only 10-fold less potent at hMC5R than the full length parent peptide. In the structure of this smaller analog, the Nle-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8) segment was found to be critical for high agonist potency, while the C-terminal Trp(9) residue was shown to be required for high hMC5R selectivity versus hMC1b,3,4R.     

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.     

Conformation of the core sequence in melanocortin peptides directs selectivity for the melanocortin MC3 and MC4 receptors.

Melanocortin peptides regulate a variety of physiological processes. Five melanocortin receptors (MC-R) have been cloned and the MC3R and MC4R are the main brain MC receptors. The aim of this study was to identify structural requirements in both ligand and receptor that determine gamma-melanocyte-stimulating hormone (MSH) selectivity for the MC3R versus the MC4R. Substitution of Asp10 in [Nle4]Lys-gamma2-MSH for Gly10 from [Nle4]alpha-MSH, increased both activity and affinity for the MC4R while the MC3R remained unaffected. Analysis of chimeric MC3R/MC4Rs and mutant MC4Rs showed that Tyr268 of the MC4R mainly determined the low affinity for [Nle4]Lys-gamma2-MSH. The data demonstrate that Asp10 determines selectivity for the MC3R, however, not through direct side chain interactions, but probably by influencing how the melanocortin core sequence is presented to the receptor-binding pocket. This is supported by mutagenesis of Tyr268 to Ile in the MC4R which increased affinity and activity for [Nle4]Lys-gamma2-MSH, but decreased affinity for two peptides with constrained cyclic structure of the melanocortin core sequence, MT-II and [D-Tyr4]MT-II, that also displayed lower affinity for the MC3R. This study provides a general concept for peptide receptor selectivity, in which the major determinant for a selective receptor interaction is the conformational presentation of the core sequence in related peptides to the receptor-binding pocket.     

Metal ion-mediated agonism and agonist enhancement in melanocortin MC1 and MC4 receptors

An endogenous metal-ion site in the melanocortin MC1 and MC4 receptors was characterized mainly in transiently transfected COS-7 cells. ZnCl(2) alone stimulated signaling through the Gs pathway with a potency of 11 and 13 microm and an efficacy of 50 and 20% of that of alpha-melanocortin stimulating hormone (alpha-MSH) in the MC1 and MC4 receptors, respectively. In the presence of peptide agonist, Zn(II) acted as an enhancer on both receptors, because it shifted the dose-response curves to the left: most pronounced was a 6-fold increase in alpha-MSH potency on the MC1 receptor. The effect of the metal ion appeared to be additive, because the maximal cAMP response for alpha-MSH in the presence of Zn(II) was 60% above the maximal response for the peptide alone. The affinity of Zn(II) could be increased through binding of the metal ion in complex with small hydrophobic chelators. The binding affinities and profiles were similar for a number of the 2,2'-bipyridine and 1,10-phenanthroline analogs in complex with Zn(II) in the MC1 and MC4 receptors. However, the potencies and efficacies of the metal-ion complexes were very different in the two receptors, and close to full agonism was obtained in the MC1 receptor. Metal ion-chelator complexes having antagonistic properties were also found. An initial attempt to map the metal-ion binding site in the MC1 receptor indicated that Cys(271) in extracellular loop 3 and possibly Asp(119) at the extracellular end of TM-III, which are both conserved among all MC receptors, are parts of the site. It is concluded that the function of the MC1 and MC4 receptors can be positively modulated by metal ions acting both as partial agonists and as potentiators for other agonists, including the endogenous peptide ligand alpha-MSH at Zn(II) concentrations that could be physiological. Furthermore, the metal ion-chelator complexes may serve as leads in the development of novel melanocortin receptor modulators.     

Modified melanocortin tetrapeptide Ac-His-dPhe-Arg-Trp-NH at the arginine side chain with ureas and thioureas.

The Ac-His-dPhe-Arg-Trp-NH2 tetrapeptide is a nonselective melanocortin agonist and replacement of Arg in the tetrapeptide with acidic, basic or neutral amino acids results in reduced potency at the melanocortin receptor (MCR) isoforms (MC1R and MC3-5R). To determine the importance of the positive charge and the guanidine moiety for melanocortin activity, a series of urea- and thiourea-substituted tetrapeptides were designed. Replacement of Arg with Lys or ornithine reduced agonist activity at the mouse mMC1 and mMC3-5 receptors, thus supporting the hypothesis that the guanidine moiety is important for receptor potency, particularly at the MC3-5 receptors. The Arg side chain-modified tetrapeptides examined in this study include substituted phenyl, naphthyl, and aliphatic urea and thiourea residues using a Lys side-chain template. These ligands elicit full-agonist pharmacology at the mouse MCRs examined in this study.     

Common requirements for melanocortin-4 receptor selectivity of structurally unrelated melanocortin agonist and endogenous antagonist, Agouti protein

The activity of melanocortin receptors (MCR) is regulated by melanocortin peptide agonists and by the endogenous antagonists, Agouti protein and AgRP (Agouti-related protein). To understand how the selectivity for these structurally unrelated agonists and antagonist is achieved, chimeric and mutants MC3R and MC4R were expressed in cell lines and pharmacologically analyzed. A region containing the third extracellular loop, EC3, of MC4R was essential for selective Agouti protein antagonism. In addition, this part of MC4R, when introduced in MC3R, conferred Agouti protein antagonism. Further mutational analysis of this region of MC4R demonstrated that Tyr(268) was required for the selective interaction with Agouti protein, because a profound loss of the ability of Agouti protein to inhibit (125)I-labeled [Nle(4),d-Phe(7)]alpha-melanocyte-stimulating hormone (MSH) binding was observed by the single mutation of Tyr(268) to Ile. This same residue conferred selectivity for the MC4R selective agonist, [d-Tyr(4)]MT-II, whereas it inhibited interaction with the MC3R-selective agonist, [Nle(4)]Lys-gamma(2)-MSH. Conversely, mutation of Ile(265) in MC3 (the corresponding residue of Tyr(268)) to Tyr displayed a gain of affinity for [d-Tyr(4)]MT-II, but not for Agouti protein, and a loss of affinity for [Nle(4)]Lys-gamma(2)-MSH as compared with wild-type MC3R. This single amino acid mutation thus confers the selectivity of MC3R toward a pharmacological profile like that observed for MC4R agonists but not for the antagonist, Agouti protein. Thus, selectivity for structurally unrelated ligands with opposite activities is achieved in a similar manner for MC4R but not for MC3R.     

Synthesis and activity of the melanocortin Xaa-d-Phe-Arg-Trp-NH tetrapeptides with amide bond modifications.

The melanocortin receptor (MCR) pathway has been identified as participating in several physiologically important pathways including pigmentation, energy homeostasis, inflammation, obesity, hypertension, and sexual function. All the endogenous MCR agonists contain a core His-Phe-Arg-Trp sequence identified as important for receptor molecular recognition and stimulation. Several structure-activity studies using the Ac-His-d-Phe-Arg-Trp-NH2 tetrapeptide template have been performed in the context of modifying N-terminal 'capping' groups and amino acid constituents. Herein, we report the synthesis and pharmacologic characterization of modified Xaa-d-Phe-Arg-Trp-NH2 (Xaa = His or Phe) melanocortin tetrapeptides (N-site selective methylation, permethylation, or amide bond reduction) at the mouse MC1, MC3, MC4 and MC5 receptors. The modified peptides generated in this study resulted in equipotent or reduced MCR potency when compared with control ligands. The reduced amide bond analog of the Phe-d-Phe-Arg-Trp-NH2 peptide converted its agonist activity into an antagonistic at the central mMC3 and mMC4 receptors involved in the regulation of energy homeostasis, while retaining full agonist activity at the peripheral MC1 and MC5 receptors.     

Interactions between the melanocortin system and the hypothalamo-pituitary-thyroid axis

Recent studies of transgenic mice and humans have provided compelling evidence for the importance of the hypothalamic melanocortin system in the regulation of energy balance. Energy homeostasis is a balance between food intake (energy input) and energy expenditure. The melanocortin system regulates feeding via effects of the endogenous agonist, alpha-melanocyte stimulating hormone (alpha-MSH) and the endogenous antagonist agouti-related protein (AGRP) on melanocortin 3 and 4 receptors (MC3-Rs and MC4-Rs). It has been demonstrated that the melanocortin system interacts with the hypothalamo-pituitary-thyroid (HPT) axis. Thyroid hormones influence metabolism and hence energy expenditure. Therefore, an interaction between the HPT axis and the melanocortin system would allow control of both sides of the energy balance equation, by the regulation of both energy input and energy expenditure. Here we will discuss the evidence demonstrating interactions between the melanocortin system and the HPT axis.     

Design of cyclic peptides with agonist activity at melanocortin receptor-4

A series of cyclic pentapeptides, c(His-D-Phe-Arg-Trp-Z) (Z=omega-amino acid), were prepared and biologically evaluated. The effects of increasing alkyl chain length of omega-amino acid on the functional activities and the receptor binding affinities for human melanocortin receptors (hMC-Rs) were studied. Compound 2 was an agonist for hMC-4R with an EC50 value of 15.4 nM, which was 4.7 times more potent than that of alpha-MSH. Compound 2 also showed a 4.3-fold higher hMC-4R selectivity over hMC-1R, thus providing us with information concerning size and chemical structure of the lactam ring for the development of the agonist with hMC-4R selectivity.     

Analogs of alpha-melanocyte stimulating hormone with high agonist potency and selectivity at human melanocortin receptor 1b: the role of Trp(9) in molecular recognition

alpha-Melanocyte stimulating hormone (alphaMSH), Ac-Ser(1)-Tyr(2)-Ser(3)-Met(4)-Glu(5)-His(6)-Phe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH(2), is an endogenous agonist for the melanocortin receptor 1 (MC1R), the receptor found in the skin, several types of immune cells, and other peripheral sites. Three-dimensional models of complexes of this receptor with alphaMSH and its synthetic analog NDP-alphaMSH, Ac-Ser(1)-Tyr(2)-Ser(3)-Nle(4)-Glu(5)-His(6)-D-Phe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH(2), have been previously proposed. In those models, the 6-9 segment of the ligand was considered essential for the ligand-receptor interactions. In this study, we probed the role of Trp(9) of NDP-alphaMSH in interactions with hMC1bR. Analogs of NDP-alphaMSH with various amino acids in place of Trp(9) 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 new compounds displayed high agonist potency at hMC1bR (EC(50) = 0.5-5 nM) and receptor subtype selectivity greater than 2000-fold versus hMC3-5R. The Trp(9) residue of NDP-alphaMSH was determined to be not essential for molecular recognition at hMC1bR.     

Potent and selective peptide agonists of alpha-melanotropin action at human melanocortin receptor 4: their synthesis and biological evaluation in vitro

alpha-Melanotropin (alphaMSH) and several of its derivatives are potent but not selective agonists at melanocortin receptors 3, 4, and 5 present in the brain (MC3-5R). To differentiate between the physiological role of hMC-4R (believed to be involved in regulation of energy balance) from those of melanocortin receptors 3 and 5, potent and receptor-specific agonists are needed. Therefore, the cyclic derivatives of alphaMSH of a general structure, cyclo(X-His-d-Phe-Arg-Trp-Y)-NH(2), where X is succinic acid or an omega-amino-carboxylic acid, and Y is an alpha,omega-di-amino-carboxylic acid or an omega-carboxy-alpha-amino acid, were prepared and tested in binding assays and in cAMP assays on CHO cells expressing hMC3-5R. Several of the 21-membered or larger lactams turned out to be potent and hMC-4R-selective agonists. For instance, cyclo(CO-CH(2)-CH(2)-CO-His-d-Phe-Arg-Trp-Dab)-NH(2) (Dab: 2,4-di-amino-butyric acid) was a potent agonist at hMC-4R (EC(50) = 4 nM) with 55-fold selectivity over hMC-3R and greater than 1000-fold selectivity over hMC-5R. Another potent and selective compound was cyclo(NH-CH(2)-CH(2)-CO-His-d-Phe-Arg-Trp-Glu)-NH(2): EC(50) about 1 nM at hMC-4R, with 90-fold selectivity over hMC-3R and greater than 2000-fold selectivity over hMC-5R.     

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.     

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.     

Rate limiting factors in melanocortin 1 receptor signalling through the cAMP pathway

The melanotropic actions of alpha-melanocyte-stimulating hormone (alpha-MSH) and other melanocortins are mediated by activation of the melanocortin 1 receptor (MC1R). This G protein-coupled receptor is positively coupled to Gs and triggers the cyclic adenosine mono-phosphate (cAMP) pathway. Mutations of the MC1R gene are associated with skin type and pigmentation phenotypes, and with increased risk of skin cancers. Genetic studies have demonstrated an heterozygote carrier effect for these associations, suggesting the importance of variant allele dosage. This could be accounted for, at least partially, if the number of MC1R molecules, rather than the Gs protein or the effector enzyme, adenylyl cyclase, is limiting for the activation of the signalling pathway. However, the nature of the limiting factor(s) in MC1R signalling has not been investigated. We addressed this question by comparing the cAMP output of clones of human melanoma cell lines enriched in MC1R by stable transfection. We also analysed heterologous cell systems widely used for functional studies of MC1R. We show that cAMP production in clones of Chinese hamster ovary cells stably expressing the MC1R is a linear function of receptor number up to high, supraphysiological levels of approximately 50,000 alpha-MSH binding sites per cell. Enrichment of human melanoma cell lines with MC1R also results in increased cAMP levels, with a small leftward shift of the agonist dose-response curves. Therefore, at physiological expression levels second-messenger generation is dependent on receptor density. Within melanoma cells and also likely in normal melanocytes, MC1R appears the limiting factor controlling the output of the cAMP signalling pathway.