The down-modulation of receptors for phorbol ester tumor promoter in primary epidermal cells

The specific [20-³H]phorbol 12,13-dibutyrate ([³H]PDBu) binding to intact epidermal cells displayed the phenomenon of down-modulation, i.e., the specific binding of [³H]PDBu to its receptors on primary epidermal cells reached a maximum within 1 h and steadily declined thereafter (Proc. Natl. Acad. Sci. USA, 78, 2549, 1981). The apparent down-modulation of radiolabel resulted from a partial loss in the total number of receptors; the affinity of receptors for the ligand was essentially unchanged. A number of agents such as chloroquine, methylamine, or arginine which are known to prevent clustering, down-modulation, and/or internalization of several hormone receptors did not affect the down-modulation of phorbol ester receptors. Furthermore, cydoheximide had no effect either on down-modulation or on the binding capacity of cells. The surface binding capacity of down-modulated cells following a 90-min incubation with unlabeled ligand was almost returned to normal within 1 h. This recovery did not depend on protein synthesis, as it was insensitive to cydoheximide. However, the surface binding capacity of epidermal cells was sensitive to the serum. Cells maintained for more than 3.5 h at 37°C in the serum-free medium showed a loss of specific [³H]PDBu binding; no such loss was seen in medium containing serum. The effect of the antidepressant drug chlorpromazine, which is known to interact with calmodulin, on [³H]PDBu binding was also investigated. The cells pretreated with chlorpromazine showed a significant decline in their cell binding capacity for [³H]PDBu; this was due to a decrease in the number of available binding sites, because the affinity of binding remained almost unchanged. Our data indicate that the effect of chlorpromazine on [³H]PDBu binding is probably unrelated to its calmodulin-binding activity.