Phorbol esters induce, in the chemotactically responsive Walker 256 carcinosarcoma cells, functional responses that are similar to those induced by peptide chemotactic factors. These responses are presumed to result from ligand binding to cellular receptors, although this has not been formally demonstrated. In this study, it was shown that tritium-labeled phorbol dibutyrate ([3H]PDB) bound to the Walker cells in a time- and concentration-dependent manner. Binding was inhibited by excess unlabeled PDB and was reversible. Half-maximal binding was achieved with a 31 nm concentration of [3H]PDB and occurred within 15 min after addition of the ligand. This is in accord with biological activity (i.e., cell-to-substrate adherence). Half-maximal cell adherence was observed with 25 nm PDB. Increased adhesiveness was detected as early as 15 min after exposure of the cells to the ligand. The response peaked at 30 to 45 min after exposure and fell off rapidly thereafter. A number of phorbol esters successfully competed with [3H]PDB binding to the cells. There was a direct relationship between the ability of these agents to compete for binding and their ability to induce biological activity. Using cell-to-substrate adherence as an indicator of biological activity allowed separation of responding and nonresponding populations. The biologically responsive cells and the nonresponsive cells both bound high levels of [3H]PDB. This suggests that receptor occupancy is, by itself, not sufficient for biological activity and that, in Walker cells, one or more points of control exist subsequent to ligand binding.
This study was supported in part by Grant CA 36132 from the USPHS.