Fishman P H, Bradley R M, Rebois R V, Brady R O
J Biol Chem. 1984 Jun 25;259(12):7983-9.
A clonal line of murine Leydig tumor cells (MLTC-1) bound both human chorionic gonadotropin (hCG) and cholera toxin (CT) with high affinity and accumulated cyclic AMP in response to either effector. The major cellular ganglioside was GM3 with small amounts of GM2, GM1, and GD1a. The gangliosides became labeled when the cells were grown in medium containing [3H] galactose or were exposed to galactose oxidase or NaIO4 followed by NaB3H4. CT specifically protected GM1 from surface labeling whereas hCG did not protect any gangliosides from being labeled. When the cells were exposed to sialidase, surface GD1a was eliminated, and GM1 increased with a corresponding increase in CT binding. When sialidase-treated cells were first incubated with the B component of CT, binding and action of CT was blocked. The cells, however, retained their ability to bind and respond to hCG. Addition of purified gangliosides to the medium effectively inhibited the binding and action of CT but not hCG. The cells incorporated the exogenous gangliosides and exhibited increased binding of and responsiveness to CT but not hCG. Both hCG- and CT-receptor complexes were extracted from the cells with nonionic detergent and analyzed by sucrose gradient centrifugation. The hCG-receptor complex had an apparent molecular weight of 190,000 whereas the CT-receptor complex sedimented only slightly faster than CT itself. MLTC-1 gangliosides were separated on thin layer chromatograms which were overlayed with either iodinated CT or hCG. The toxin bound to a ganglioside corresponding to GM1 whereas the hormone did not bind to any of the gangliosides. When the cells were incubated overnight with hCG, they lost their hCG receptors but exhibited an increase in CT binding and gangliosides. Our results indicate that GM1 is the specific receptor for CT whereas gangliosides are not involved in the binding and action of hCG.
小鼠睾丸间质细胞瘤细胞系（MLTC - 1）能以高亲和力结合人绒毛膜促性腺激素（hCG）和霍乱毒素（CT），并在受到任何一种效应物刺激时积累环磷酸腺苷。主要的细胞神经节苷脂是GM3，还有少量的GM2、GM1和GD1a。当细胞在含有[3H]半乳糖的培养基中生长，或暴露于半乳糖氧化酶或高碘酸钠后再用硼氢化钠（NaB3H4）处理时，神经节苷脂会被标记。CT能特异性地保护GM1不被表面标记，而hCG不能保护任何神经节苷脂不被标记。当细胞暴露于唾液酸酶时，表面的GD1a被去除，GM1增加，同时CT结合相应增加。当先用唾液酸酶处理的细胞与CT的B亚基孵育时，CT的结合和作用被阻断。然而，这些细胞保留了它们结合和响应hCG的能力。向培养基中添加纯化的神经节苷脂可有效抑制CT的结合和作用，但对hCG无影响。细胞摄取了外源性神经节苷脂，并表现出对CT的结合增加和反应性增强，但对hCG无此现象。用非离子去污剂从细胞中提取hCG和CT受体复合物，并通过蔗糖梯度离心进行分析。hCG受体复合物的表观分子量为190,000，而CT受体复合物的沉降速度仅比CT本身略快。MLTC - 1神经节苷脂在薄层层析图上分离，然后用碘化CT或hCG覆盖。毒素与对应于GM1的神经节苷脂结合，而激素不与任何神经节苷脂结合。当细胞与hCG孵育过夜时，它们失去了hCG受体，但CT结合和神经节苷脂增加。我们的结果表明，GM1是CT的特异性受体，而神经节苷脂不参与hCG的结合和作用。
