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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Human and mouse melanoma cells have been shown to express high-affinity binding sites for a-melanocytestimulating hormone (a-MSH) (Eberle, 1988;Ghanem et al., 1988;Siegrist et al., 1989) Photo-affinity labeling revealed a molecular weight of approximately 45 kDa ( S o b et af., 1989). This subtype of the G proteincoupled melanocortin receptors was designated MC1 after its molecular cloning (Chhajlani et al., 1992; Mountjoy et ul., 1992). It was found that a-MSH upregulates its receptors in three human melanoma cell lines. The extent of up-regulation was at about two-fold above control values. In contrast, homologous downregulation was observed in six human and two mouse melanoma lines, and in two human melanoma lines no change of receptor numbers was observed. Down-regulation was particularly conspicuous in murine cells where about 80% of specific MSH binding disappeared after incubation with a-MSH. A less pronounced down-regulation was observed in some human cells where MSH reduced its receptor number by only 20 to 30% as compared to untreated cells. Up-and down-regulation of MSH receptors was confirmed by Scatchard analysis which proved that receptor affinities did not change after incubation with a-MSH. The hormone up-regulated MC1 at a half maximal effective concentration (EC,,,) of 1.6 n M in human cells. This effect was only marginal in the first 5 h of incubation but reached a maximum after 20 to 30 h. The peptide analogs ACTH,-,7 and [Nle', ~-P h e~] -a -M s H had a higher potency, whereas ACTH,_,,, desacetyl-a-MSH, and [Nle']-c(-MSH were less effective when compared to a-MSH. In mouse B16-F1 cells, a-MSH downregulated its receptors with an EC5,, of 0.23 n M . The time-course of down-regulation was maximal after 15 h and thus faster than up-regulation. In contrast to upregulation, down-regulation could be mimicked by G,-protein activation with cholera toxin (Table 1). Elevation of CAMP by treatment with forskolin or isobutylmethylxanthine (IX) alone induced only a partial down-regulation in B16 cells. Furthermore, the MSH-induced down regulation was diminished in the presence of forskolin, suggesting the existence of a CAMP-dependent mechanism counter-regulating the

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