Turnover of growth hormone receptors in rat adipocytes

Adipocytes isolated from the epididymal fat pads of normal rats specifically bound [125I]human GH [( 125I]hGH). Preincubation of cells with 20 micrograms/ml cycloheximide, an inhibitor of protein synthesis, produced a progressive loss of ability to bind [125I]hGH specifically. Loss of binding sites with time followed first order kinetics and had a half-time of about 45 min regardless of whether GH was present or absent during treatment with cycloheximide. Nonspecific binding of labeled hormone was unchanged by cycloheximide. Similar results were obtained when adipocytes were incubated with 200 micrograms/ml puromycin, another inhibitor of translation, but incubation with 5 micrograms/ml actinomycin D, an inhibitor of transcription, for 2.5 h had no effect on the binding of [125I]hGH by adipocytes. The findings are not attributable to cell death, since oxidation of [U-14C] glucose to 14CO2 and binding of [125I]insulin were unaffected in replicate cell populations exposed to the same treatments. Diminished binding could not be attributed to an effect of cycloheximide to hasten the degradation of receptor-bound hGH. Treatment of adipocytes with 0.1 mg/ml trypsin for 10 min virtually abolished their ability to bind [125I]hGH specifically, but binding capability gradually returned after removal of trypsin and was nearly restored to pretrypsin levels by 2 h. Addition of cycloheximide to the incubation medium after removal of trypsin completely prevented recovery of binding capability