Estradiol receptor: phosphorylation on tyrosine in uterus and interaction with anti‐phosphotyrosine antibody.
Estradiol receptor: phosphorylation on tyrosine in uterus and interaction with anti‐phosphotyrosine antibody.
复制标题
雌二醇受体：子宫内酪氨酸的磷酸化以及与抗磷酸酪氨酸抗体的相互作用。
作者：
A. Migliaccio;A. Rotondi;F. Auricchio
用己烯雌酚-琼脂糖柱层析和肝素-琼脂糖层析法从[~(32)P]正磷酸盐孵育的大鼠子宫中纯化雌二醇受体。纯化的受体与抗纯化的雌二醇受体的单抗孵育后，经蔗糖梯度离心法分析，似乎被标记了32P。用免疫亲和层析进一步纯化了受体制剂，并进行了SDS-聚丙烯酰胺凝胶电泳法。检测到一个重32P标记的68kd蛋白和一个很弱标记的48kd蛋白，可能是68kd蛋白的蛋白水解物。对从免疫亲和柱上洗脱的受体进行的磷酸氨基酸分析表明，它的32P标记仅发生在酪氨酸上。这是关于组织中类固醇受体酪氨酸磷酸化的首次报道。这与我们之前的发现是一致的，在体外，赋予雌激素受体激素结合能力的子宫雌激素受体-激酶，仅在酪氨酸上使该受体磷酸化。小牛子宫受体与抗磷酸酪氨酸单抗共价结合(Kd=0.28 nM)，具有较高的特异性和亲和力。含有小牛子宫核磷酸酶的细胞核使受体去磷酸化，从而消除了与抗体的相互作用。这些结果表明，在小牛子宫中，雌激素受体在酪氨酸上也被磷酸化。结合琼脂糖基的抗磷酸化酪氨酸抗体已被用来部分纯化小牛子宫中的雌二醇受体。
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.