Haug E, Naess O, Gautvik K M
Mol Cell Endocrinol. 1978 Oct;12(1):81-95. doi: 10.1016/0303-7207(78)90103-x.
Estrogens stimulate prolactin (PRL) synthesis by GH3 cells, a clonal strain of rat pituitary cells grown in culture. At 4 degrees C the binding of [3H]17 beta-estradiol to monolayer cultures of GH3 cells was specific and of limited capacity, with half-maximal and maximal binding after 1--2 h and 12 h, respectively. Scatchard analysis showed one single class of binding sites with Kd = 3.1 X 10(-10) M and n = 309 X 10(-15) mol 17 beta-estradiol/mg cell protein, calculated to give approx. 25,000 binding sites per cell. At 4 degrees C less than 10% of the specifically bound [3H]17 beta-estradiol was found in the nuclear fraction. When the incubation temperature was raised to 37 degrees C, the amount of radioactivity in the nucleus increased to 25% within 30 min with a corresponding reduction in the cytoplasm. The cytosol fractions from monolayer cultures as well as from tumors of GH3 cells contained specific 17 beta-estradiol binding proteins, having a sedimentation constant close to 8S in a salt-free buffer and 4S in the presence of 0.5 M KCl. scatchard analysis showed one single class of binding sites with Kd = 3.6 X 10(-10) M and n = 258 X 10(-15) mol 17 beta-estradiol/mg cytosol protein (GH3 tumor tissue). Thus, GH3 cells grown in culture and in the intact animal have similar binding characteristics as judged from the data for binding affinity, capacity and specificity. After the in vivo administration of [3H]17 beta-estradiol to GH3 tumor-bearing rats, radioactivity could be extracted (0.5 M KCl) from purified nuclei bound to 4.5S macromolecules. We suggest that the action of 17 beta-estradiol on GH3 cells involves an initial binding of the steroid to specific receptors in the cytoplasm, followed by transport of a fraction of the hormone-receptor complexes to the nucleus involving a temperature-sensitive step.
雌激素可刺激GH3细胞（一种在培养中生长的大鼠垂体细胞克隆株）合成催乳素（PRL）。在4℃时，[3H]17β-雌二醇与GH3细胞单层培养物的结合具有特异性且容量有限，分别在1 - 2小时和12小时后达到半数最大结合和最大结合。Scatchard分析显示存在一类单一的结合位点，Kd = 3.1×10⁻¹⁰ M，n = 309×10⁻¹⁵ mol 17β-雌二醇/毫克细胞蛋白，据此计算每个细胞约有25,000个结合位点。在4℃时，核部分中发现的特异性结合的[3H]17β-雌二醇不到10%。当孵育温度升至37℃时，30分钟内核中的放射性增加至25%，同时细胞质中的放射性相应减少。GH3细胞单层培养物以及肿瘤细胞的胞质溶胶部分含有特异性的17β-雌二醇结合蛋白，在无盐缓冲液中的沉降常数接近8S，在0.5 M KCl存在下为4S。Scatchard分析显示存在一类单一的结合位点，Kd = 3.6×10⁻¹⁰ M，n = 258×10⁻¹⁵ mol 17β-雌二醇/毫克胞质溶胶蛋白（GH3肿瘤组织）。因此，从结合亲和力、容量和特异性数据判断，培养中的GH3细胞和完整动物体内的GH3细胞具有相似的结合特性。给携带GH3肿瘤的大鼠体内注射[3H]17β-雌二醇后，放射性可从与4.5S大分子结合的纯化细胞核中提取（0.5 M KCl）。我们认为17β-雌二醇对GH3细胞的作用涉及该类固醇首先与细胞质中的特异性受体结合，随后一部分激素-受体复合物经一个温度敏感步骤转运至细胞核。
