Cultured
melanoma cells are known targets for the pigment-inducing actions of melanotropins such as
alpha-melanocyte-stimulating hormone (
alpha-MSH). The objectives of the present studies were to determine the binding properties and functional relevance of
MSH binding sites in a mouse
melanoma cell line and to determine whether
MSH receptors are expressed in situ. The binding properties of
MSH receptors in intact cells of a highly metastatic, highly
MSH-responsive mouse
melanoma cell subline (B16-F10C23) were determined using a radiolabeled, biologically active preparation of the superpotent
alpha-MSH analogue, [Nle4,D-Phe7]-
alpha-MSH (125I-NDP-MSH). A single high-affinity class of binding site was detected (Kd for
NDP-MSH, 5.6 x 10(-11) M; Kd for
alpha-MSH, 2.6 x 10(-9) M as determined by Scatchard analysis and heterologous inhibition assays, respectively).
alpha-MSH showed nearly identical concentration-response relationships in the radioreceptor assay (inhibition of 125I-
NDP-MSH binding) and a bioassay (stimulation of intracellular
cyclic AMP accumulation). Furthermore, the respective potencies of three melanotropins,
NDP-MSH,
alpha-MSH, and
adrenocorticotropic hormone, in binding and
biological assays were highly correlated. These results indicate that the 125I-NDP-MSH binding site represents the functional
MSH receptor.
Tumors were induced by inoculation of C57BL/6 mice with B16-F10C23 cells, and the presence of 125I-NDP-MSH binding sites was determined by in situ radiolabeling of frozen tissue sections followed by autoradiography. Specific
MSH binding sites were distributed throughout the
tumor tissue, but not in associated fibrovascular elements or in neighboring nonmelanoma tissues. As in cultured B16-F10C23 cells, melanotropins inhibited 125I-NDP-MSH binding to tissue sections in a concentration-dependent manner. These results support the hypothesis that functional
MSH receptors are expressed in
melanoma in situ, suggesting that the activities of
melanoma cells in vivo may be subject to modulation by endogenous melanotropins. The methods described will be applicable for studies of the expression and regulation of
MSH receptors in human
melanoma and other target tissues.