AN ASSESSMENT OF THE ROLE OF DOMAIN-F AND PEST SEQUENCES IN ESTROGEN-RECEPTOR HALF-LIFE AND BIOACTIVITY

The estrogen receptor (ER) is a rapidly turning over protein, with a h alf-life of ca. 3-4 h in estrogen target cells. Sequence analysis of t he human ER reveals a putative PEST sequence, sequences rich in prolin e (P), glutamic acid (E), serine (S) and threonine (T), in the carboxy -terminal F domain of the protein. Since PEST sequences have been impl icated in the rapid turnover of some proteins, we have used site-direc ted mutagenesis to investigate the role of the F region containing PES T residues in the stability and bioactivity of the receptor. A truncat ed form of ER lacking the last 41 amino acids of the protein and encom passing the PEST sequences (amino acids 555 to 567) was made by mutage nesis of the ER cDNA. Pulse-chase experiments, involving immunoprecipi tation of [S-35]methionine/[S-35]cysteine labeled receptors or of rece ptors covalently labeled with tamoxifen aziridine followed by gel elec trophoresis, were used to determine the half-life of the wild-type and truncated ERs. These experiments showed that the turnover rate of the receptors expressed in Chinese hamster ovary and monkey kidney (COS-1 ) cells was 3 to 5 h and that elimination of the PEST residues did not have a significant effect on the degradation rate of the protein. Mor eover, deletion of the last 41 amino acids (F domain) of the ER did no t affect transactivation ability, ligand binding affinity, or the phos phorylation pattern of the receptor. Therefore, the role of domain F i n ER function remains unclear, but it is not a determinant of the rela tively rapid rate of ER turnover in cells.