Temporal variation in estrogen receptor-alpha protein turnover in the presence of estrogen

Estrogen receptor-alpha (ERalpha) is essential in the maintenance of cellular responsiveness to estrogen in the reproductive system. It is established that ligand binding induces downregulation of ERalpha protein by targeting receptor for destruction by the 26S proteasome. However, ERalpha is preserved in cells chronically exposed to estrogen and it is unknown how receptor levels are maintained in the continued presence of the signal that induces degradation. A modified pulse-chase analysis was developed using a tet-inducible ERalpha expression system to determine the rate of ERalpha protein decay following both acute and chronic estrogen treatments. Upon initial hormone treatment, ERalpha half-life is shortened from 3 to 1 h. However, ERalpha half-life increases over time, achieving a half-life of approximately 6 h in 72 h of estrogen treatment. Analysis of ERalpha half-life in the presence and absence of proteasome inhibitor, MG132, revealed that the increased stability is due in part to a decreased rate of proteolysis. In addition, we observed a time-dependent increase in phospho-S118 ERalpha and showed that the half-life of the phosphomimetic ERalpha mutant, S118E-ER, is identical to that of wild-type receptor under conditions of chronic estrogen treatment. These data provide evidence that as cells adapt to chronic stimulation, ERalpha protein is stabilized due first to a decreased rate of proteolysis, and secondarily, to the accumulation of proteasome-resistant, phosphorylated form of receptor. This temporal control of proteolysis allows for the establishment of steady-state levels of receptor and provides a protective mechanism against loss of hormone responsiveness.