alpha-MSH Sequence Research Articles

New Melanocortin 1 Receptor Binding Motif Based on the C‐Terminal Sequence of α‐Melanocyte‐Stimulating Hormone

The C-terminal tripeptide of the alpha-melanocyte stimulating hormone (alpha-MSH11-13) possesses strong antiinflammatory activity without known cellular target. In order to better understand the structural requirements for function of such motif, we designed, synthesized and tested out Trp- and Tyr-containing analogues of the alpha-MSH11-13. Seven alpha-MSH11-13 analogues were synthesized and characterized for their binding to the melanocortin receptors recombinantly expressed in insect (Sf9) cells, infected with baculovirus carrying corresponding MC receptor DNA. We also tested these analogues on B16-F1 mouse melanoma cells endogenously expressing the MC1 receptor for binding and for ability to increase cAMP levels as well as on COS-7 cells transfected with the human MC receptors. The data indicate that HS401 (Ac-Tyr-Lys-Pro-Val-NH2) and HS402 (Ac-Lys-Pro-Val-Tyr-NH2) selectively bound to the MC1 receptor and stimulated cAMP generation in a concentration dependent way while the other Tyr- and Trp-containing alpha-MSH11-13 analogues neither bound to MC receptors nor stimulated cAMP. We have thus identified new MC receptor binding motif derived from the C-terminal sequence of alpha-MSH. The tetrapeptides have novel properties as the both act via MC-ergic pathways and also carry the anti-inflammatory alpha-MSH11-13 message sequence.

The melanocortin receptor subtypes in chicken have high preference to ACTH-derived peptides.

1 Melanocortin (MC) receptors are widely distributed throughout the body of chicken, like in mammals, and participate in a wide range of physiological functions. 2 To clarify the pharmacological impact of ligands acting in the MC system, we expressed the chicken MC1, MC2, MC3, MC4 and MC5 (cMC1-5) receptors in eukaryotic cells and performed comprehensive pharmacological characterization of the potency of endogenous and synthetic melanocortin peptides. 3 Remarkably, the cMC receptors displayed high affinity for ACTH-derived peptides and in general low affinity for alpha-MSH. It is evident that not only the cMC2 receptor but also the other cMC receptors interact with ACTH-derived peptide through an epitope beyond the sequence of alpha-MSH. 4 The synthetic ligand MTII was found to be a potent agonist whereas HS024 was a potent antagonist at the cMC4 receptor, indicating that these ligands are suitable for physiological studies in chicken. 5 We also show the presence of prohormone convertase 1 (PC1) and PC2 genes in chicken, and that these peptides are coexpressed with proopiomelanocortin (POMC) in various tissues.

NMR studies on turn mimetic analogs derived from melanocyte-stimulating hormones.

Oligomers with alpha-aminooxy acids are reported to form very stable turn and helix structures, and they are supposed to be useful peptidomimetics for drug design. A recent report suggested that homochiral oxa-peptides form a strong eight-member-ring structure by a hydrogen bond between adjacent aminooxy-acid residues in a CDCl3 solution. In order to design an alpha-MSH analog with a stable turn conformation, we synthesized four tetramers and one pentamer, based on alpha-MSH sequence, and determined the solution structures of the molecules by two-dimensional NMR spectroscopy and simulated annealing calculations. The solution conformations of the three peptidomimetic molecules (TLV, TDV, and TLL) in DMSO-d6 contain a stable 7-membered-ring structure that is similar to a gamma-turn in normal peptides. Newly-designed tetramer TDF and pentamer PDF have a ball-type rigid structure that is induced by strong hydrogen bonds between adjacent amide protons and carbonyl oxygens. In conclusion, the aminooxy acids, easily prepared from natural or unnatural amino acids, can be employed to prepare peptidomimetic analogues with well-defined turn structures for pharmaceutical interest.

Alpha-MSH can control the essential cofactor 6-tetrahydrobiopterin in melanogenesis.

In the human epidermis both keratinocytes and melanocytes express POMC m-RNA. Immunohistochemical studies of both cell types demonstrate significantly higher levels of alpha-MSH in melanocytes than in keratinocytes. Both cell types also hold the full capacity for de novo synthesis/recycling of the essential cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4). 6BH4 is critical for the hydroxylation of the aromatic amino acids L-phenylalanine, L-tyrosine, and L-tryptophan, for nitric oxide production and in various immune modulatory processes. Recently it was shown that tyrosinase activity is regulated by 6BH4 through a specific allosteric inhibition. The tyrosinase/6BH4 inhibition can be activated by 1:1 complex formation between 6BH4 and alpha-MSH, but an excess of alpha-MSH over 6BH4 can inhibit tyrosinase due to complex formation by tyr2 in the alpha-MSH sequence. In both melanocytes and keratinocytes 6BH4 controls the L-tyrosine supply via phenylalanine hydroxylase (PAH). Recently we were able to show that the cellular uptake of L-phenylalanine and its intracellular turnover to L-tyrosine is crucial for melanogenesis. alpha-MSH can promote the production of L-tyrosine via PAH due to activation of the PAH tetramer to the more active dimer by removing 6BH4 from the regulatory binding domain on the enzyme. In conclusion, alpha-MSH can control (1) intracellular L-tyrosine formation from L-phenylalanine in both melanocytes and keratinocytes, and (2) tyrosinase activity, directly, in melanocytes.

The melanocortin 1, 3, 4 or 5 receptors do not have a binding epitope for ACTH beyond the sequence of alpha-MSH.

ACTH(1-39), and several shorter N- and/or C-terminally truncated fragments of ACTH, with and without N-terminal acetylation and/or C-terminal amidation, were tested for binding on a single eukaryotic cell line transiently and independently expressing the melanocortin MC1, MC3, MC4 and MC5 receptors. The results show that none of these MC receptors has specific binding epitopes for the ACTH peptides beyond the amino acid sequence of alpha-MSH, when tested for their ability to compete with 125I-labelled [Nle4,D-Phe7]alpha-MSH and ACTH. The MC3 receptor favours the natural desacetylated N-terminal end of the ACTH peptides, and it has generally more than 10-fold higher affinity for the ACTH peptides than the MC4 receptor. Considering earlier anatomical localisation data, together with the present data, we suggest that the MC3 receptor is the most likely candidate of the MC receptors to mediate the short-loop negative feedback release of corticotrophin-releasing factor (CRF) caused by ACTH/MSH peptides.

Alpha-melanocyte-stimulating hormone (MSH) inhibits insulin secretion in HIT-T 15 cells.

alpha-Melanocyte-stimulating hormone (MSH) antagonizes the binding of interleukin-1 beta (IL-1) to its type I receptors on T cells. IL-1 modulates insulin secretion in the insulin-secreting tumor cell line, HIT-T 15 cells. In the present studies, we examined a hypothesis that alpha-MSH may modulate insulin secretion in HIT-T 15 cells. The addition of alpha-MSH over the concentrations of 10(-10) M inhibited insulin secretion for 4 h. In addition, the carboxyl-terminal tripeptide of alpha-MSH [alpha-MSH(11-13)], which is supposed to be an important sequence of alpha-MSH on its antipyretic effects, significantly inhibited insulin secretion at the concentration of 10(-9) M. In contrast, acetyl-[Nle4,D-Phe7] alpha-MSH(4-10)-amide (10(-11) to 10(-9) M) failed to inhibit insulin secretion. The maximal effect of alpha-MSH on insulin secretion was obtained at the concentration of 10(-9) M. The addition of alpha-MSH from 10(-11) to 10(-9) M significantly inhibited 7.5 mM glucose-stimulated insulin secretion at each period of 0-5, 5-15, and 15-30 min. Radioreceptor assay suggested that HIT-T 15 cells possess the specific binding sites for 125I-labeled alpha-MSH. The present studies indicated that alpha-MSH inhibits insulin secretion through the specific binding site in HIT-T 15 cells.

Discovery and structure-function analysis of alpha-melanocyte-stimulating hormone antagonists.

Structure-function relationships of alpha-melanocyte-stimulating hormone (alpha-MSH, alpha-melanotropin) were investigated and novel alpha-MSH receptor antagonists were identified. Based on the alpha-MSH-[5-13] peptide sequence, a multi-use peptide library consisting of 31,360 structurally different candidates was generated, and approximately 40% of the peptides were individually screened for their ability to block receptor function. This led to the identification of antagonists with a range of potencies and revealed structural requirements necessary for receptor inactivation. The most potent antagonist Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val-NH2 has an IC50 value of 11 +/- 7 nM. Analysis revealed that D-Trp5 and Phe6 were crucial to its antagonistic properties which could be potentiated by D-Phe3. This study demonstrates that residues in positions 5-6, 7-9, and 10 of the alpha-MSH sequence constitute crucial determinants for potent antagonist activity.

Melanotropin structure-activity studies on melanocytes of the teleost fish, Synbranchus marmoratus.

The minimal sequence of alpha-MSH required for full agonism on fish (Synbranchus marmoratus) melanocytes was determined to be Ac-alpha-MSH5-10-NH2 since Ac-alpha-MSH6-10-NH2 and Ac-alpha-MSH6-9-NH2 were inactive. The N-terminal tripeptide sequence, Ser-Tyr-Ser, lacked any contribution to potency since the 4-13 (Ac-[Nle4]-alpha-MSH4-13-NH2) sequence was equipotent to alpha-MSH. The important potentiating amino acids were found to be Met at position 4 of the amino terminus and Val at position 13 of the carboxy terminus of the hormone, since Ac-alpha-MSH4-10-NH2 was about 100 times more potent than the Ac-alpha-MSH5-10-NH2 sequence, and Ac-[Nle4]-alpha-MSH4-13-NH2 was about 10 times more active than Ac-[Nle4]-alpha-MSH4-12-NH2. The minimal sequence for equipotency to alpha-MSH was demonstrated to be Ac-[Nle4]-alpha-MSH4-13-NH2. [Nle4, D-Phe7]-alpha-MSH was about 10 times more active than alpha-MSH. Unexpectingly, several conformationally restricted cyclic melanotropins were either partial agonists ([Cys4, Cys10]-alpha-MSH) or totally inactive (Ac[Cys4, Cys10]-alpha-MSH4-10-NH2) on fish melanocytes. These results point out some rather remarkable differences between S. marmoratus and tetrapod melanophores relative to structural requirements for MSH receptor recognition and signal transduction.

Antipyretic properties of centrally administered alpha-MSH fragments in the rabbit.

alpha-Melanocyte stimulating hormone (alpha-MSH 1-13) has marked antipyretic effects when administered centrally or peripherally in small doses. A C-terminal fragment, alpha-MSH (11-13), contains an antipyretic message sequence of alpha-MSH; however, the lesser potency of this fragment relative to that of the entire molecule suggests that other amino acids of the alpha-MSH sequence are essential for the full antipyretic effect. Graded doses of alpha-MSH (11-13) (Ac LysProVal NH2), alpha-MSH (10-13) (Ac GlyLysProVal NH2), and alpha-MSH (8-13) (Ac ArgTrpGlyLysProVal NH2), were injected into the cerebral ventricles of rabbits made febrile by IV administration of crude interleukin-1. All three fragments reduced fever in a dose-related manner. The (8-13) sequence was much more effective than the other two fragments, and the (10-13) portion was less effective than the (11-13) tripeptide. None of the fragments was as potent as alpha-MSH (1-13). The results confirm that an antipyretic message resides within alpha-MSH (11-13) and sequential addition of amino acids to alpha-MSH (11-13) can both enhance and reduce the potency of the fragment.

Evidence for alpha-melanocyte-stimulating hormone (alpha-MSH) receptors on human malignant melanoma cells.

The presence of alpha-MSH receptors on human melanoma has so far been suggested in the literature but not proved. We describe a reproducible and specific binding assay of alpha-MSH on human melanoma cells, using a high-specific-activity 125I-labelled hormone (1.5 to 2 mCi/micrograms) with consistent receptor binding (usually exceeding 2 pg/10(6) cells) and stable for 3 weeks. Asynchronized cells in suspension were incubated for 15 min at 37 degrees C with the tracer and various concentrations of unlabelled hormones. Synthetic alpha-MSH was compared to beta-MSH, ACTH1-24, ACTH4-10, beta-LPH, CLIP, CRF, MIF I, A8VP and beta-endorphin. Out of a panel of 8 human melanoma cell lines, 3 showed specific and reproducible alpha-MSH binding curves. No significant binding to human fibroblast and human carcinoma cells was seen. alpha-MSH, beta-MSH and, to a lesser extent ACTH4-10 (a part of the alpha-MSH sequence) were the only peptides able to displace labelled alpha-MSH from its binding sites, indicating the high specificity of the MSH receptor. Affinity constants (Ka) ranged from 10(8) to 10(9) l/mole and the estimated receptor number was 1,000 to 2,000 per cell. We conclude that some human melanoma cell lines expressed specific MSH receptors with stable affinity but which are low in number.

ACTH and adrenal aerobic glycolysis. II: Effects of aminoterminal peptide fragments on lactic acid and steroid production by mouse adrenocortical cells.

The effects of shortening the ACTH molecule from either end of the peptide chain on adrenal glycolysis and steroidogenesis were examined in mouse adrenal cell suspensions. Shortening the (1-24) sequence to (1-17), (1-16) and (1-14), thereby interfering with the basic tetrapeptide (15-18) assigned to the address message, progressively reduced both glycolytic and steroidogenic potencies by four, six and ten orders of magnitude respectively, without impairing the capacity for maximal excitation. The glycolytic potency of the (1-18) sequence, which was amidated at the C-terminal, equalled that of ACTH (1-24), but the steroidogenic potency was reduced by an order of magnitude. The (1-13) sequence of alpha-MSH, which contains substitutions at both terminals, had glycolytic and steroidogenic potencies intermediate between those of ACTH(1-16) and ACTH(1-17). Deletion of Ser1,Tyr2 from ACTH(1-18)-NH2 reduced both potencies by an order of magnitude. ACTH(11-24) and (7-38) were inactive or inhibitory. The capacity for excitation was further examined by comparing responses to peptide fragments (1-4), (1-10), (1-13), (4-10), (4-11), (5-10), (5-14), (7-13) and (11-24) at a concentration of 1 mmol/l. All fragments, excepting (1-4), (5-10) and (11-24) were active. The activities of fragments (5-14) and (7-13), as opposed to (5-10), suggest that the requirements for methionine in position 4 may be replaced by the (11-13) tripeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide sequence of cloned cDNA for pro-opiomelanocortin in the amphibian Xenopus laevis.

The function of alpha-melanocyte-stimulating hormone (alpha-MSH) is not known in mammals. It is well-established in the amphibian Xenopus laevis in which alpha-MSH mediates the process of adaptation to a dark background. The amino acid sequence of this hormone is, however, not known in amphibians. In order to determine the primary structure of the precursor protein for alpha-MSH, which in mammals has been called pro-opiomelancortin (POMC), we constructed a cDNA library from Xenopus pituitary mRNA. A pool of synthetic oligodeoxyribonucleotide tetradecamers corresponding to part of the mammalian alpha-MSH sequence was used to screen the library. The nucleotide sequence of a 1050-base pair hybridization-positive cDNA clone was determined and the deduced amino acid sequence of Xenopus POMC revealed the sequences of Xenopus gamma-MSH, alpha-MSH, corticotropin-like intermediate lobe peptide, beta-MSH, and beta-endorphin. Interestingly, the N-terminal amino acid of Xenopus alpha-MSH, which is N alpha-acetylated in the biologically active form of the hormone, is different from that of mammalian alpha-MSH. The distribution of the bioactive domains within Xenopus POMC is remarkably similar to that in other known POMC molecules and as in mammals the domains in the amphibian prohormone are flanked on both sides by pairs of basic amino acids.

Melanotropic peptides: Presence in brain of normal and hypophysectomized rats, and subcellularly localized in synaptosomes

A major and several minor trichloroacetic acid (TCA) soluble, bioassayable melanotropic peptides, as well as bioreactive and immunoreactive alpha-MSH have been found in the hypothalamus, olfactory bulb and cerebral cortex of normal and hypophysectomized rats. By employing subcellular fractionation procedures it was demonstrated that alpha-MSH and the major TCA soluble melanotropic peptide (MMPB) were localized in synaptosomes and were released by hypoosmotic shock. The analysis of MMPB by electrophoretic and chromatographic procedures reveales that it is not ACTH4-10, ACTH1-10, ACTH1-24, NAcACTH1-10 or alpha-MSH. MMPB was found to cross-react with an antiserum specific for the Lys-Pro-Val NH2 sequence in alpha-MSH, indicating that this C-terminal sequence of alpha-MSH may be present in its structure. MMPB was also shown to differ electrophoretically from the two major TCA soluble melanotropic peptides found in the neurointermediate lobe of the pituitary. In view of their synaptosomal localizations MMPB and alpha-MSH may play a role in synaptic function in the nervous system.