Receptors for α-melanocyte-stimulating hormone (α-MSH) on human malignant melanoma cell lines were investigated with a specific binding assay and characterized with structural analogues of α-MSH and adrenocorticotropic hormone and by photoaffinity cross-linking of the hormone-receptor complex. Specific binding of high-performance liquid chromatography-purified, monoiodinated α-MSH in the presence of 1 mm 1,10-phenanthroline as protease inhibitor was highest after a 2-h incubation at 37°C. The nonspecific binding was <20% and dissociation of the ligand-receptor complex was relatively slow. Ten out of 12 human cell lines showed specific binding sites for α-MSH with Kd values ranging from 0.195 to 2.87 nm and the sites/cell being ∼400 to ∼1600. Virtually identical results were obtained in an assay where the cells remained attached to the culture dishes during the entire experiment. The study of hormone analogues with the D10 cell line showed that oxidized α-MSH had an ∼40-fold lower affinity than α-MSH whereas [Nle4,d-Phe7]-α-MSH displayed a threefold and the adrenocorticotropic hormone fragments (1–17) and (1–24) a 20- and 8-fold higher affinity. Cross-linking of the α-MSH-receptor complex of three cell lines using monoiodinated [Nle4,d-Phe7,Trp(2-nitro-4-azidophenylsulfenyl)9]-α-MSH as photoaffinity label revealed a major Mr 45,000 protein band on sodium dodecyl sulfate-polyacrylamide gels, analogous to the MSH receptor of mouse B16 melanoma cells.
This work was supported by the Swiss National Science Foundation (Grant 3.287-0.85) and the Swiss and Basel Cancer Leagues (Grant For. 314.85).