Human epidermal melanocyte and keratinocyte melanotropin receptors: Visualization by melanotropic peptide conjugated macrospheres (polyamide beads)

The objectives of this research were to determine whether melanotropin receptors are characteristic membrane markers of human epidermal melanocytes. Methodologies were developed to visualize these receptors by light microscopy. Multiple copies (up to a thousand) of [Nle⁴,D-Phe⁷]α-MSH, a superpotent analog of α-melanocyte stimulating hormone (α-MSH), were conjugated to a macromolecular carrier, large polyamide beads (macrospheres). Incubation in the presence of the I conjugated macrospheres resulted in binding of human epidermal melanocytes to the macrospheres. Specificity of the binding of melanocytes to the melanotropin-conjugated macrospheres was demonstrated by several studies: (i) Binding of melanocytes to the conjugate was specific since it could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog; (ii) The macrospheres after removal of the bound ligand did not bind to the melanocytes; (iii) Another peptide hormone ligand (e.g., a substance-P analog) attached to the macrospheres failed to bind to the melanocytes; (iv) B16/F10 mouse melanoma cells known to express melanotropin receptors bound to the macrospheres; (v) Cells of nonmelanocyte origin (e.g., mammary cancer cells, lung cancer cells, fibroblasts) did not bind to the macrospheres. One exception was that human epidermal keratinocytes also expressed melanotropin receptors as determined by all the criteria established for epidermal melanocytes. Thus, cell specific melanotropin receptors appear to be characteristic cell surface markers of epidermal melanocytes and keratinocytes.