Estradiol receptor: phosphorylation on tyrosine in uterus and interaction with anti-phosphotyrosine antibody.
Estradiol receptor from rat uteri incubated with [32P] orthophosphate has been purified by diethylstilbestrol--Sepharose followed by heparin--Sepharose chromatography. The purified receptor, analyzed by centrifugation through sucrose gradients after incubation with monoclonal antibodies against purified estradiol receptor, appears to be labeled with 32P. The receptor preparation has been further purified by immunoaffinity chromatography and submitted to SDS--poly-acrylamide gel electrophoresis. A heavily 32P-labeled 68 kd protein and a very lightly 32P-labeled 48 kd protein, probably a proteolytic product of the 68 kd protein, were detected. Phosphoamino acid analysis of the receptor eluted from the immunoaffinity column shows that its 32P-labeling occurs exclusively on tyrosine. This is the first report on phosphorylation on tyrosine of a steroid receptor in tissue. It is consistent with our previous finding that a uterus estradiol receptor-kinase, which confers hormone binding ability to the estradiol receptor, in vitro phosphorylates this receptor exclusively on tyrosine. Calf uterus receptor binds with high specificity and affinity to monoclonal anti-phosphotyrosine antibodies covalently bound to Sepharose (Kd = 0.28 nM). Dephosphorylation of the receptor by nuclei containing the calf uterus nuclear phosphatase abolishes the interaction with antibodies. These results suggest that also in calf uterus, estradiol receptor is phosphorylated on tyrosine. Anti-phosphotyrosine antibodies bound to Sepharose have been used to partially purify the estradiol receptor from calf uterus.
D000818:Animals; D000911:Antibodies, Monoclonal; D003600:Cytosol / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism; D005260:Female; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D051381:Rats; D011919:Rats, Inbred Strains; D011959:Receptors, Estradiol / Q000302:isolation & purification / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis; D014599:Uterus / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D010710:Phosphates; D011959:Receptors, Estradiol; D011960:Receptors, Estrogen; D019000:Phosphotyrosine; D014443:Tyrosine; D004958:Estradiol