We are working towards a complete understanding of the interaction of melanotropin (MSH) with its receptors and the way in which the proliferation and pigmentation of normal and malignant pigment cells are controlled. We want to identify the genes for the MSH receptor, for tyrosinase and for proteins affected by the phorbol ester TPA, a factor needed for the growth of normal and some malignant melanocytes in culture. We have improved on our previous procedure for preparing ?125?I-Beta-MSH and now obtain yields approximately 10-fold higher and can store the product approximately 4-fold longer than before. We developed three assays for detecting the binding of MSH to intact melanoma cells and/or homogenates. Two of the assays can also be used to detect receptors in soluble form, that is, free of other membrane components. The third assay will be useful in screening the supernatants of hybridomas for monoclonal antibodies towards the MSH receptor because it permits the completion of several hundred tests per day. We extended our studies on the expression of receptors for MSH during the cell cycle. We reported previously that receptors for MSH on Cloudman melanoma cells were active primarily during the late S-phase and in the G2-phase of the cell cycle. Because this time period represents only about 5 to 10% of the cell cycle, only 5 to 10% of the cells in non-synchronized, randomly growing populations would be expected to bind and respond to MSH. We have, therefore, partially synchronized the cultures and found that cells passing through late S and G2 synchronously, following their release from a colchicine- or thymidine-induced block, bind not 10 times but 100 times more MSH than do those in random cultures. This increased binding capacity is due to an increase in both the number and the affinity of receptors for MSH. (B)
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