Receptors for .beta.-melanocyte-stimulating hormone exhibit positive cooperativity in synchronized melanoma cells

Abstract

Cloudman S91 mouse melanoma cells respond in culture to .beta.-melanocyte-stimulating hormone (B-MSH) with changes in morphology, growth rates, and melanin production. The effects of MSH appear to be mediated through a stimulation of the cyclic AMP system. It was reported earlier that at least some of the responses to MSH (increased cyclic AMP production and tyrosinase activity) occur in the G2 phase of the cell cycle [Wong, G., Pawelek, J., Sansone, M., and Morowitz, J. (1974) Nature (London) 248, 351-354] and that the apparent reason for this cell cycle restriction is that receptors for MSH are most active in the G2 phase [Varga, J. M., DiPasquale, A., Pawelek, J., McGuire, J., and Lerner, A. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 1590-1593]. In this report, we found that by two separate methods of obtaining populations of cells in the G2 phase of their cycle.sbd.centrifugal elutriation or synchronization with thymidine.sbd.we observed increased binding of MSH by cells in the G2 and possibly late S phases of their cycle. However, cultures of cells passing through their cycle in synchrony were quite different from nonsynchronized (random) cultures. Both synchronized and random cultures expressed receptors for MSH in the G2 and possibly late S phases of their cycle, but synchronized cultures bound severalfold more MSH per cell than random cultures. This increased binding of MSH by synchronized cells was accompanied by an increase in tyrosinase activity and pigment production. Analyses by Scatchard and Hill methods revealed a potential basis for the increased binding capacity and responsiveness to MSH by synchronized cells: receptors from synchronized cells exhibited positive cooperativity, while receptors from random populations of cells exhibited no cooperativity. Understanding the mechanisms regulating cooperative interactions in the MSH receptor system could be of fundamental value in understanding the regulation of proliferation and pigmentation by MSH.