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.
The melanocortin peptides α-, β- and γ-melanocyte-stimulating hormone (γ-MSH) and adrenocorticotropic hormone (ACTH) are a group of neuropeptides derived from the pro-opiomelanocortin prohormone that contain the common amino acid sequence, His-Phe-Arg-Trp. The physiological actions of these peptides are mediated through seven transmembrane domain proteins, which are coupled to adenyl cyclase through specific G proteins, and signal through intracellular cyclic AMP (cAMP) [4], [36]. In the last decade, extensive structure–function studies on α-MSH have lead to the creation of many new, more potent and enzyme-resistant analogs of the melanocortin peptides. NDP-MSH ([Nle4, DPhe7]-α-MSH) and Melanotan II (MTII, the lactam Ac-Nle4 cycle [Asp-His6-DPhe7-Arg8-Trp9-Lys10]-amide). Both analogues were identified as potent, non-selective agonists at the human MC1, MC3, MC4 and MC5 receptors [32], [33]. Substitution of a bulky hydrophobic amino acid at the Phe position (SHU9119) converts the MTII peptide from an agonist into an antagonist at the MC3 and MC4 receptors [19], [34]. The sequences of these peptides are shown in Fig. 1. Three-dimensional homology molecular modeling of the NDP-MSH putative interactions with the MC1R and MC4R have been published and a series of amino acids in transmembranes (TM) TM2,-3,5 and 7 were suggested to form a binding pocket for the -DPhe-Arg-Trp ligand residues [12], [29], [39]. Mutagenesis studies indicated that mutation of TM3 residues Aspartic acid and TM6 residues Phenylalanine and Histidine decreased the binding affinity and potency of the NDP-MSH ligand at MC1R and MC4R. Therefore, these receptor residues sites are postulated to be involved in the ligand-receptor interactions [38], [39]. Chimeric receptor and receptor mutagenesis studies of the human MC3R and hMC4R identified the L165 in hMC3R and L133 in hMC4R residues in the putative TM3 domain as important for converting the SHU9119 from an antagonist into an agonist at both the hMC3R and hMC4R [37]. The sequences of TM3 of MCRs are shown in Fig. 2. However, the molecular basis of SHU9119 antagonism at MC3R and MC4R remains undefined. In this study, we utilized the tetrapeptide template Ac-His-Xaa-Arg-Trp-NH 2, where Xaa is substituted with aromatic amino acids in either the L or D stereochemical configuration to examine the melanocortin ligand side chain properties important for melanocortin receptor selectivity, potency and differentiation of agonist versus antagonist activities at the human MC1R, MC3R and MC4R.
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NDP-MSH, MTII and SHU9119 were purchased from Peninsula Laboratories, Inc. (Belmont, CA). Tetrapeptide synthesis was performed using standard Fmoc methodology on an automated synthesizer (Advanced ChemTech 440MOS, Louisville, KY) as previously reported [14], [20]. Approximately, 250 mg of rink-amide MHBA resin (0.1 mmol) was added to a manual reaction vessel (Peptides International). The resin was allowed to swell for 2 h in DMF followed by Na deprotection of the Fmoc group using 20% piperidine in
We first examined the effect of MTII and SHU9119 on MCR function. Our results indicate that MTII and SHU9119 dose dependently displaced 125 I-NDP-MSH binding at the hMC1R, hMC3R and hMC4R wild-type (WT) (Fig. 3A and C). MTII induces cAMP production in a dose-dependent manner at hMC1R, hMC3R and hMC4R wild-type (Fig. 3B). However, SHU9119 was not able to stimulate cAMP production at hMC3R and hMC4R (Fig. 3D; Table 1), which are consistent with the previous finding [37]. To examine the role of Phe
A major breakthrough in understanding melanocortin action came with the cloning and characterization of five members or subtypes of the melanocortin receptor family. In the last decade, extensive agonist structure activity studies and receptor mutagenesis studies have been performed [1], [3], [7], [23], [24], [29], [30], [38], [39]. For melanocortin peptides, extensive studies indicate that His-DPhe-Arg-Trp is crucial for ligand binding and receptor activation but tetrapeptide could not
This work has been supported by NIH grants R03HD047312-01A (Y.K.Y.), CNRC pilot/feasibility grant (NIH P30 DK56336 (David B. Allison), and RO1DK57080 (C.H.L.) and an American Diabetes Association Research Award (C.H.L.).
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