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 half-life of ca. 3-4 h in estrogen target cells. Sequence analysis of the human ER reveals a putative PEST sequence, sequences rich in proline (P), glutamic acid (E), serine (S) and threonine (T), in the carboxy-terminal F domain of the protein. Since PEST sequences have been implicated in the rapid turnover of some proteins, we have used site-directed mutagenesis to investigate the role of the F region containing PEST residues in the stability and bioactivity of the receptor. A truncated form of ER lacking the last 41 amino acids of the protein and encompassing the PEST sequences (amino acids 555 to 567) was made by mutagenesis of the ER cDNA. Pulse-chase experiments, involving immunoprecipitation of [35S]methionine/[35S]cysteine labeled receptors or of receptors covalently labeled with tamoxifen aziridine followed by gel electrophoresis, 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. Moreover, deletion of the last 41 amino acids (F domain) of the ER did not affect transactivation ability, ligand binding affinity, or the phosphorylation pattern of the receptor. Therefore, the role of domain F in ER function remains unclear, but it is not a determinant of the relatively rapid rate of ER turnover in cells.
Pakdel
F
F
Department of Physiology and Biophysics, University of Illinois, Urbana.
Le Goff
P
P
Katzenellenbogen
B S
BS
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003545:Cysteine / Q000378:metabolism; D017931:DNA Primers; D006207:Half-Life; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011960:Receptors, Estrogen / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D012984:Software; D013462:Sulfur Radioisotopes; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014316:Tritium
D017931:DNA Primers; D011960:Receptors, Estrogen; D013462:Sulfur Radioisotopes; D013629:Tamoxifen; D014316:Tritium; C031859:tamoxifen aziridine; D008715:Methionine; D003545:Cysteine