Richards J S, Sehgal N, Tash J S
J Biol Chem. 1983 Apr 25;258(8):5227-32.
The responsiveness of granulosa cells to the gonadotropins and cAMP increases as ovarian follicles mature. To determine if this change in response might be related to either the content or cAMP-dependent phosphorylation of specific proteins, we labeled proteins in 30,000 X g supernatant fractions (cytosol) with [gamma-32P] ATP in the presence or absence of cAMP. Using two-dimensional gel electrophoresis, we observed that granulosa cells of preantral follicles exhibited low amounts of cAMP-dependent phosphorylation of two proteins with apparent molecular weights of 54,000-56,000 and 43,000. Using [32P]8-N3cAMP and photoaffinity labeling procedures, the Mr = 54,000-56,000 protein was identified as RII, the regulatory subunit of type II protein kinase. Polychromatic silver staining, as well as the photoaffinity labeling, revealed that RII exists in three forms, each of which was also labeled by [gamma-32P] ATP. Based on the relative isoelectric points and specific silver staining of highly purified actin and phosphorylated actin, the Mr = 43,000 protein has been provisionally identified as actin. Five proteins (Mr = 37,500, 27,500, 22,500, 19,000, and 15,000), in addition to RII and actin, were phosphorylated in cytosol of granulosa cells from preovulatory follicles. By adding increasing concentrations of exogenous catalytic subunit to the cytosols, we demonstrated that the content, as well as the phosphorylation of these proteins, was increased selectively in granulosa cells of antral follicles. By using hypophysectomized rats, we demonstrated that these five proteins are induced by follitropin (FSH). Because they were not present in cytosols of thecal cells or corpora lutea, they appear to be specific markers for granulosa cells. The content and phosphorylation of RII was also dramatically increased in cytosols of granulosa cells from antral follicles, whereas that of actin remained unchanged. These observations indicate that granulosa cell differentiation involves regulation by FSH of specific proteins which are substrates for cAMP-dependent protein kinase. Thus, FSH and cAMP appear to regulate the intracellular content and phosphorylation of a cAMP response system in granulosa cells. The extent to which RII and the five specific phosphoproteins themselves regulate granulosa cell responsiveness remains to be determined.
随着卵巢卵泡成熟，颗粒细胞对促性腺激素和环磷酸腺苷（cAMP）的反应性增强。为了确定这种反应变化是否可能与特定蛋白质的含量或cAMP依赖性磷酸化有关，我们在有或无cAMP存在的情况下，用[γ-32P]ATP对30,000×g上清液组分（胞质溶胶）中的蛋白质进行标记。通过二维凝胶电泳，我们观察到窦前卵泡的颗粒细胞中，两种表观分子量分别为54,000 - 56,000和43,000的蛋白质的cAMP依赖性磷酸化水平较低。使用[32P]8-N3cAMP和光亲和标记程序，将分子量为54,000 - 56,000的蛋白质鉴定为II型蛋白激酶的调节亚基RII。多色银染以及光亲和标记显示，RII以三种形式存在，每种形式也都被[γ-32P]ATP标记。根据高度纯化的肌动蛋白和磷酸化肌动蛋白的相对等电点和特异性银染，分子量为43,000的蛋白质已被初步鉴定为肌动蛋白。除RII和肌动蛋白外，排卵前卵泡颗粒细胞胞质溶胶中的五种蛋白质（分子量分别为37,500、27,500、22,500、19,000和15,000）发生了磷酸化。通过向胞质溶胶中添加浓度递增的外源性催化亚基，我们证明这些蛋白质的含量以及磷酸化水平在窦状卵泡颗粒细胞中选择性增加。通过使用垂体切除的大鼠，我们证明这五种蛋白质是由促卵泡激素（FSH）诱导产生的。由于它们不存在于膜细胞或黄体的胞质溶胶中，它们似乎是颗粒细胞的特异性标志物。RII的含量和磷酸化水平在窦状卵泡颗粒细胞的胞质溶胶中也显著增加，而肌动蛋白的则保持不变。这些观察结果表明，颗粒细胞分化涉及FSH对作为cAMP依赖性蛋白激酶底物的特定蛋白质的调节。因此，FSH和cAMP似乎调节颗粒细胞中cAMP反应系统的细胞内含量和磷酸化。RII和这五种特异性磷酸化蛋白本身对颗粒细胞反应性的调节程度仍有待确定。
