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

Estrogen receptor-alpha (ER alpha) is essential in the maintenance of cellular responsiveness to estrogen in the reproductive system. It is established that ligand binding induces downregulation of ER alpha protein by targeting receptor for destruction by the 26S proteasome. However, ER alpha 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 ERa expression system to determine the rate of ERa protein decay following both acute and chronic estrogen treatments. Upon initial hormone treatment, ER alpha half-life is shortened from 3 to 1 h. However, ERa half-life increases over time, achieving a half-life of similar to 6 h in 72 h of estrogen treatment. Analysis of ERa 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 ER alpha and showed that the half-life of the phosphomimetic ER alpha 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, ER alpha 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.