ABSTRACT: 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 Tyr268 was required for the selective interaction with Agouti protein, because a profound loss of the ability of Agouti protein
to inhibit 125I-labeled [Nle4,d-Phe7]α-melanocyte-stimulating hormone (MSH) binding was observed by the single mutation of Tyr268 to Ile. This same residue conferred selectivity for the MC4R selective agonist, [d-Tyr4]MT-II, whereas it inhibited interaction with the MC3R-selective agonist, [Nle4]Lys-γ2-MSH. Conversely, mutation of Ile265 in MC3 (the corresponding residue of Tyr268) to Tyr displayed a gain of affinity for [d-Tyr4]MT-II, but not for Agouti protein, and a loss of affinity for [Nle4]Lys-γ2-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.
Journal of Biological Chemistry 01/2001; 276(2):931-936. · 4.77 Impact Factor
ABSTRACT: 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 γ-melanocyte-stimulating hormone (MSH) selectivity for the MC3R versus the MC4R. Substitution of Asp10 in [Nle4]Lys-γ2-MSH for Gly10 from [Nle4]α-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-γ2-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-γ2-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.
Journal of Biological Chemistry 06/1999; 274(24):16853-16860. · 4.77 Impact Factor
ABSTRACT: Cyclic lactam analogs of α-melanocyte stimulating hormone (α-MSH) have been shown to be potent agonists in the frog skin bioassay
[Al-Obeidi, F. et al., J. Med. Chem., 32 (1989) 2555], demonstrating melanocortin-1 (MC1) receptor activity. We synthesized
cyclic α-MSH(1–13) and α-MSH(4–10) lactam analogs. The peptides were synthesized using Fmoc chemistry. We improved the cyclization
procedure: side chains of Asp5 and Lys10 from the deprotected peptide were coupled in DMF to form a cyclic lactam, using an excess of PyBOP reagent and DIEA as a
base. The cyclization reaction was completed within 1 h and was almost quantitative. We also synthesized an α-MSH analog cyclized
via a disulphide bridge. The peptides were tested for their selectivity for the rat MC4 receptor. Cyclization and substitutions
at position 7 dramatically influenced the selectivity for the rMC4 receptor.
International Journal of Peptide Research and Therapeutics 04/1998; 5(2):205-208. · 0.99 Impact Factor
ABSTRACT: Since the melanocortin MC3 and melanocortin MC4 receptors are the main melanocortin receptor subtypes expressed in rat brain, we characterized the activity and affinity of nine melanocortin receptor ligands using these receptors in vitro, as well as their activity in a well-defined melanocortin-induced behavior in the rat: grooming behavior. We report here that [d-Tyr4]melanotan-II and RMI-2001 (Ac-cyclo-[Cys4, Gly5, d-Phe7, Cys10]α-MSH-NH2) have significantly higher affinity and potency on the rat melanocortin MC4 receptor as compared to the rat melanocortin MC3 receptor. Nle-γ-MSH (melanocyte-stimulating hormone) was the only ligand with higher affinity and potency on the rat melanocortin MC3 receptor. The potency order of melanocortin MC4 receptor agonists, but not that of melanocortin MC3 receptor agonists, fitted with the potency of these ligands to stimulate grooming behavior, when administered intracerebroventricularly. SHU9119 (Ac-cyclo-[Nle4, Asp5, d-Nal(2)7, Lys10]α-MSH-(4–10)-NH2) and RMI-2005 (Ac-cyclo-[Cys4, Gly5, d-Nal(2)7, Nal(2)9, Cys10]α-MSH-(4–10)-NH2) were able to inhibit α-MSH-induced melanocortin receptor activity in vitro, as well as α-MSH-induced grooming behavior. Melanotan-II, [Nle4-d-Phe7]α-MSH and RMI-2001 were also effective in inducing grooming behavior when administered intravenously. In the absence of purely selective melanocortin MC3/4 receptor ligands, we demonstrated that careful comparison of ligand potencies in vitro with ligand potencies in vivo, could identify which melanocortin receptor subtype mediated α-MSH-induced grooming behavior. Furthermore, blockade of novelty-induced grooming behavior by SHU9119 demonstrated that this physiological stress response is mediated via activation of the melanocortin system.
European Journal of Pharmacology.
ABSTRACT: Antagonists for the melanocortin receptor family were identified by analysis of the effects of four melanocortin analogues on α-MSH(α-melanocyte-stimulating hormone)-induced cAMP accumulation in 293 human embryonal kidney (HEK) cells that expressed either the rat melanocortin MC3 receptor, the human melanocortin MC4 receptor or the ovine melanocortin MC5 receptor. Two peptides, [D-Arg8]ACTH(adrenocorticotrope hormone)-(4–10) and [Pro8,10, Gly9]ACTH-(4–10), antagonized the action of α-MSH on the melanocortin MC4 and MC5 receptors, but not the melanocortin MC3 receptor. [Ala6]ACTH-(4–10) inhibited the α-MSH activation of the melanocortin MC3 and MC5, but only weakly antagonized the activation of the melanocortin MC4 receptor. [Phe-I7]ACTH-(4–10) antagonized the melanocortin MC3, MC4 and MC5 receptors equally well. These antagonists were also tested to block a behavioral response induced by α-MSH. α-MSH-induced excessive grooming behavior in rats was inhibited by [Phe-I7]ACTH-(4–10), [D-Arg8]ACTH-(4–10) and [Pro8,10,Gly9]ACTH-(4–10), but not by [Ala6]ACTH-(4–10). This suggests that α-MSH-induced excessive grooming behavior is mediated by melanocortin MC4 receptors.
European Journal of Pharmacology: Molecular Pharmacology.