Uterine estrogen receptor in vivo: phosphorylation of nuclear specific forms on serine residues.
We have characterized further the heterogeneous nuclear-specific doublet forms of the mouse uterine estrogen receptor (ER). Estrogen treatment produced the multiple nuclear ER forms of 65 and 66.5 kDa, which were isolated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soluble ER preparations exhibited only a single 65-kDa form. Isolation of the individual nuclear ER forms and reanalysis demonstrated that formation of the multiple bands was not due to artifacts of nuclear sample preparation or the presence of contaminating proteins. Analysis of individual uterine cell types (epithelial and stromal/myometrium) indicated that both ER forms were present in both cell fractions. Fractionation of nuclear components with low salt showed that both ER forms were found in the salt-resistant fraction. Extraction of nuclei with high salt (0.6 M KCl) solubilized both ER forms. Phosphorylation was studied as a protein modification to account for the multiple forms. Incorporation of 32P into uterine protein both in vivo and in intact tissue incubation indicated 32P labeling of uterine nuclear ER after hormone treatment. Both nuclear ER forms are labeled, although the 66.5-kDa form appears to be more heavily labeled. Phosphoamino acid analysis of the immunopurified 32P-labeled ER from intact uterine tissue indicated phosphoserine as the only phospholabeled residue. These data suggest that phosphorylation is associated with the physiological functioning of the ER in response to hormone and produces the heterogeneous ER forms in the nucleus.
Washburn
T
T
Receptor Biology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Hocutt
A
A
Brautigan
D L
DL
Korach
K S
KS
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D007163:Immunosorbent Techniques; D051379:Mice; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D014599:Uterus / Q000648:ultrastructure*
D010761:Phosphorus Radioisotopes; D011960:Receptors, Estrogen; D010768:Phosphoserine; D004958:Estradiol