alpha-Melanocyte-stimulating hormone (alpha-MSH, alpha-melanotropin) and agouti control the switch between eumelanin and pheomelanin synthesis in mammalian melanocytes. Here we investigated interactions between alpha-MSH, agouti protein, cAMP elevating agents and phorbol ester on mouse B16 melanoma cells. Agouti (Kd 3.7 nmol/l) and alpha-MSH (Kd 2.3 nmol/l) had similar affinities to the MC1 melanocortin receptor. Both alpha-MSH and agouti induced MC1 receptor down-regulation. Agouti antagonized melanogenesis induced by alpha-MSH, forskolin, cholera toxin (CT), and pertussis toxin (PT). It also reduced the constitutive melanin formation of long-term cultures. Cell proliferation was inhibited by agouti (43% at 100 nM). This effect was reversed by alpha-MSH, forskolin, or CT. B16-G4F cells, a cell variant that lacks the MC1 receptor, did not respond to agouti. From these results we conclude that agouti shows the characteristics of an inverse agonist acting through the MC1 receptor.
MAGE-1 gene encodes a human melanoma antigen, recognized by syngeneic cytotoxic T lymphocytes (CTL). MAGE-1 transcripts are also detectable in breast cancers, in non-small-cell lung carcinomas and in central nervous system tumors. In order to identify, in cellular preparations, the protein encompassing the antigenic peptide, we generated a panel of monoclonal antibodies (MAbs) against the MAGE-1 gene product by using, as immunogen, a full-length recombinant preparation (rMAGE-1), obtained through expression cloning of the relevant gene in E. coli. Four reagents were obtained recognizing both rMAGE-1 and the 46-kDa native protein in cell lines expressing MAGE-1 mRNA. No positivity could be detected in MAGE-1-mRNA-negative melanoma lines. No surface labelling of MAGE-1-positive cell lines could be observed. In contrast, on permeabilization of MZ2 melanoma cells, all 4 MAbs induced efficient staining, as detected by cytofluorography. Fluorescence microscopy shows that MAGE-1 gene product is a cytoplasmic protein clustered in paranuclear organelle-like structures. Thus, MAGE-1 protein location closely resembles that of P91A and P198 murine-tumor antigens.
A radioreceptor assay for alpha-MSH is described which is based on cultured mouse B16 melanoma cells and bioactive monoiodinated [Nle4]-alpha-MSH tracer. The assay was used (1) to study the binding characteristics of alpha-MSH to B16 cells, (2) to determine the relative binding activity of MSH peptides, and (3) to measure MSH in tissue extracts. The association of alpha-MSH to B16 cells reached a stable plateau after 3 h at 15 degrees C. At 25 degrees or 37 degrees C, the binding was transient and at 0-1 degree C, the association was very slow. The hormone-receptor complex was relatively stable between 0 degrees and 15 degrees C whereas a 50% dissociation was reached after 90 min at 25 degrees C and after 35 min at 37 degrees C. The mean KD for alpha-MSH of four saturation experiments was 1.3 nM and the number of receptors 9570 per cell. 1,10-Phenanthroline had a stabilizing effect in the binding assay when used at a 0.3 mM concentration. From the MSH peptides tested in the binding assay, some showed similar potencies in three bioassays (tyrosinase, melanin and Anolis skin), whereas others displayed considerably lower bioassay values than expected from the binding data. This shows that binding and bioactivity can be dissociated in some of the MSH peptides. The biological activity of MSH from the neurointermediate lobe of the rat pituitary as measured by its binding to B16 cells corresponds fairly well with RIA results; in the anterior lobe, alpha-MSH values are overestimated because of the large amount of ACTH present.
Three different monoiodinated radioligands of alpha-MSH (alpha-melanocyte-stimulating hormone) were compared in a binding assay with human D10 melanoma cells: [Tyr(125I)2]-alpha-MSH, [Tyr(125I)2,NIe4]-alpha-MSH, and [Tyr(125I)2,NIe4,D-Phe7]-alpha-MSH. They were prepared either by the classical chloramine T method or by the Enzymobead method. A simple and rapid purification scheme was developed consisting of a primary separation on reversed-phase C18 silica cartridges immediately after the iodination, followed by HPLC purification before each binding experiment. Biological testing of the three radioligands showed that they all retained high melanotropic activity in the B16 melanin assay and the Anolis melanophore assay. However, in human D10 melanoma cells, [Tyr(125I)2,NIe4]-alpha-MSH led to a high degree of non-specific binding to the cells which could not be displaced by excess alpha-MSH and only partially by [NIe4]-alpha-MSH. The [Tyr(125I)2,NIe4,D-Phe7]-alpha-MSH tracer gave similar results but with a much lower proportion of non-specific binding. On the other hand, [Tyr(125I)2]-alpha-MSH proved to be an excellent radioligand whose non-specific binding to the D10 cells was not higher than 20% of the total binding.
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