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

Abstract

The specific [20-3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding to intact [mouse] 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. 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 Arg which are known to prevent clustering, down-modulaton, and/or internalization of several hormone receptors did not affect the down-modulation of phorbol ester receptors. Cycloheximide had no effect either on down-modulation or on the binding capacity of cells. The surface binding capacity of down-modulated cells following a 9-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 cycloheximide. The surface binding capacity of epidermal cells was sensitive to the serum. Cells maintained for > 3.5 h at 37.degree. 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. The effect of chlorpromazine on [3H]PDBu binding is probably unrelated to its calmodulin-binding activity.