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Melanotropin Receptors of Murine Melanoma Characterized in Cultured Cells and Demonstrated in Experimental Tumors in Situ¹

Endocrinology Division [J. B. T., M. L. E., S. R.], Department of Medicine, Tufts University School of Medicine-New England Medical Center Hospitals, Inc., Boston, Massachusetts 02111, and Department of Laboratory Medicine and Pathology, Dight Laboratories [B. R. L.], University of Minnesota, Minneapolis, Minnesota 55455

Cultured melanoma cells are known targets for the pigment-inducing actions of melanotropins such as -melanocyte-stimulating hormone (-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 -MSH analogue, [Nle⁴,D-Phe⁷]--MSH (¹²⁵I-NDP-MSH). A single high-affinity class of binding site was detected (K_d for NDP-MSH, 5.6 x 10^-11 M; K_d for -MSH, 2.6 x 10^-9 M as determined by Scatchard analysis and heterologous inhibition assays, respectively). -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, -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.

¹ This work was supported by NIH Grants R01-MH-44694-01 (J. B. T.) and R01-DK-16884-17 (S. R.); by NIADDKD Postdoctoral Award 1-F32-07927-02 (J. B. T.); by American Cancer Society Grants RD-285 (J. B. T.) and IN-13-Z-28 (B. R. L.); and by the Leukemia Task Force, University of Minnesota (B. R. L.). Compulter facilities of the Clinical Studies Unit, Tufts-New England Medical Center, were supported by Grant RR-00054 of the Division of Research Resources, NIH.

² To whom requests for reprints should be addressed.

Received 8/10/89. Revised 11/10/89. Accepted 11/13/89.

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