Dattatreyamurty B, Zhang S B, Reichert L E
Department of Biochemistry, Albany Medical College, New York 12208.
Endocrinology. 1990 Mar;126(3):1318-26. doi: 10.1210/endo-126-3-1318.
We have raised polyclonal antibodies in rabbits against the FSH receptor, purified from calf testis and isolated the IgG fraction from the immune serum (immune IgG) by protein A affinity chromatography. When the immune IgG was incubated with purified, radioiodinated FSH receptor, the resulting complex could be immunoprecipitated by goat anti-rabbit gamma globulin. The immunoprecipitate, after dissociation of receptor from antibody, separation by SDS-PAGE under reducing conditions, and autoradiography, showed the presence of a approximately 60 kDa protein previously identified as a component of the FSH receptor. Binding of 125I-hFSH to membrane-bound receptors was inhibited in a concentration-dependent manner by immune IgG (Ed50 = 90 micrograms/ml). Nonimmune serum or IgM/IgA fractions from immune serum had no effect. 125I-labeled immune IgG bound specifically to testis membranes and the binding could be inhibited in a concentration-dependent manner by ovine FSH. These results suggest that the FSH-binding site and the antibody-binding site on the receptor are proximate or identical. Immune IgG mimicked the ability of FSH to stimulate basal adenylate cyclase activity and conversion of androstenedione to estradiol in cultured immature rat Sertoli cells. Stimulatory but submaximal effects of FSH were augmented by immune IgG. Rat Sertoli cells treated with IgG fractions from immune serum showed an intense fluorescent staining of plasma membrane receptor. No fluorescent staining of receptor was seen when preimmune IgG was used or in the presence of excess ovine FSH. These observations suggest that the polyclonal receptor antibody capable of recognizing FSH receptor behaved as an FSH binding competitor, but was also active as an agonist producing the biological effect of FSH in vitro. The effectiveness of antibodies against FSH receptor in stimulating estradiol synthesis suggests that the information needed for FSH signal transduction resides in the membrane receptor rather than in the hormone molecule. Such antibodies may offer a useful probe for further study of FSH receptor structure and mechanism of hormone action.
我们用从牛睾丸中纯化得到的促卵泡激素（FSH）受体免疫家兔，制备了多克隆抗体，并通过蛋白A亲和层析从免疫血清中分离出IgG组分（免疫IgG）。当免疫IgG与纯化的、经放射性碘标记的FSH受体一起孵育时，形成的复合物可被山羊抗兔γ球蛋白免疫沉淀。免疫沉淀产物在使受体与抗体解离后，于还原条件下进行SDS-PAGE分离及放射自显影，结果显示存在一种约60 kDa的蛋白质，该蛋白质先前已被鉴定为FSH受体的一个组分。免疫IgG以浓度依赖的方式抑制125I-hFSH与膜结合受体的结合（半数有效剂量[Ed50]=90微克/毫升）。非免疫血清或免疫血清中的IgM/IgA组分无此作用。125I标记的免疫IgG特异性结合睾丸膜，且该结合可被绵羊FSH以浓度依赖的方式抑制。这些结果提示，受体上的FSH结合位点与抗体结合位点相邻或相同。免疫IgG模拟了FSH刺激培养的未成熟大鼠支持细胞中基础腺苷酸环化酶活性以及将雄烯二酮转化为雌二醇的能力。FSH的刺激作用但未达最大效应可被免疫IgG增强。用免疫血清的IgG组分处理的大鼠支持细胞显示质膜受体有强烈的荧光染色。当使用免疫前IgG或存在过量绵羊FSH时，未观察到受体的荧光染色。这些观察结果提示，能够识别FSH受体的多克隆受体抗体表现为FSH结合竞争者，但在体外也作为激动剂发挥作用，产生FSH的生物学效应。抗FSH受体抗体在刺激雌二醇合成方面的有效性提示，FSH信号转导所需的信息存在于膜受体而非激素分子中。此类抗体可能为进一步研究FSH受体结构和激素作用机制提供有用的探针。
