Flavio Solca

Biologically active monoiodinated α-MSH derivatives for receptor binding studies using human melanoma cells

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.

B16-G4F mouse melanoma cells: An MSH receptor-deficient cell clone

The two mouse melanoma cell lines B16‐F1 and B16‐G4F retain their melanogenic capacity when cultured in vitro. Melanotropic peptides such as α‐melanocyte‐stimulating hormone (α‐MSH) induce formation and release of melanin pigment in B16‐F1 cells. In contrast, B16‐G4F cells do not respond to α‐MSH. Using receptor‐binding analysis and photoaffinity crosslinking we demonstrate that the lack of response of B16‐G4F cells to α‐MSH is due to the absence of functional MSH receptors from the cell surface. Northern blot analysis of receptor mRNA revealed that MSH receptor mRNA is not expressed in B16‐G4F cells. These cells represent a new tool for the study of signal pathways related to the control of melanogenesis in melanoma cells.

Heterogeneity of the MSH Receptor Among B16 Murine Melanoma Subclones

The heterogeneity of melanotropin receptors on B16 sublines was tested by using photoaffinity crosslinking techniques and the superpotent alpha-MSH derivative [Nle4, D-Phe7, 1-(2-nitro-4-azido-phenylsulfenyl)-Trp9]-alpha- MSH (NAPS-MSH). Specific crosslinking of this compound to B16-F1, B16-F10, B16-M2R or B16-W4 cells revealed three different subtypes of MSH receptor based on SDS-PAGE analysis. Binding of monoiodinated alpha-MSH to these different subclones is saturable and characteristic for a single class of complexes (0.9 nM less than KD less than 1.6 nM). In this article the nature of the different MSH receptor subtypes as well as their possible correlation to the melanogenic potential of a particular cell line is discussed.

Photoaffinity Labelling of Receptors for Melanocyte-Stimulating Hormone

The photoaffinity labelling technique was applied to the study of receptors for α-melanocyte-stimulating hormone (α-MSH; α-melanotropin) on mouse and human melanoma cells as well as on Xenopus and Anolis melanophores. Twenty different α-MSH analogues containing one, two or three photolabels were synthesized and tested as photoprobes. A highly potent nitrene-generating azidophenyl compound, monoiodinated [4-Nle, 7- D-Phe, 9-Trp(Naps)]-α-MSH, was most suitable for the biochemical analysis of MSH receptors on melanoma cells whereas the physiological effects following photolabelling of intact cultured cells were best studied by using carbene-generating phenyldiazirine α-MSH analogues. Covalent linkage of α-MSH to its receptor on both melanoma cells and melanophores produced a long-lasting receptor stimulation. This irreversible agonism served as a new tool for the analysis of signal transduction mechanisms. The biochemical characterization of the covalent α-MSH-receptor complex of melanoma cells revealed a 45 kDa membrane glycoprotein, probably containing a relatively high content of sialic acid, thus explaining the acidic pi (4.7) of this receptor.