Yoshikuni M, Nagahama Y
Department of Developmental Biology, National Institute for Basic Biology, Okazaki, Japan.
Dev Biol. 1994 Dec;166(2):615-22. doi: 10.1006/dbio.1994.1341.
To investigate the mechanism by which the hormone 17 alpha,20 beta-dihydroxy-4-pregnen-3-one(17 alpha,20 beta-DP) acts on a receptor on the external surface of rainbow trout oocytes to induce maturation, the interaction between 17 alpha,20 beta-DP receptors and G-proteins was examined. Pertussis toxin (PT) catalyzed the ADP ribosylation of a 40-kDa protein in crude membranes from rainbow trout postvitellogenic oocytes, and cholera toxin (CT) labeled several proteins, including a major protein with an apparent molecular weight of 43 kDa. The 40-kDa protein was recognized by an antibody against the alpha subunit of inhibitory G-proteins (Gi), whereas the 43-kDa protein was recognized by an antibody against the alpha subunit of stimulatory G-proteins (Gs). Treating the membrane fraction with 17 alpha,20 beta-DP decreased the PT-catalyzed ADP ribosylation of the 40-kDa protein. In contrast, there was no significant change in the CT-catalyzed ribosylation of the 43-kDa protein after exposure to 17 alpha,20 beta-DP. The specific binding of 17 alpha,20 beta-DP to membrane fractions was decreased by PT. 17 alpha,20 beta-DP binding was also inhibited by nonhydrolyzable GTP analogs such as guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanylylimidodiphosphate (GppNHp), but not by either ATP or guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). Scatchard analysis revealed that GppNHp induced a 3.8-fold increase in the dissociation constant without a significant change in the number of binding sites, suggesting that the GppNHp-induced decrease in 17 alpha,20 beta-DP binding is due to the decrease in binding affinity between 17 alpha,20 beta-DP and its receptors. We conclude that the PT-sensitive Gi is involved in the signal transduction pathway of 17 alpha,20 beta-DP in rainbow trout oocytes.
为了研究激素17α,20β-二羟基-4-孕烯-3-酮（17α,20β-DP）作用于虹鳟鱼卵母细胞外表面受体以诱导成熟的机制，研究了17α,20β-DP受体与G蛋白之间的相互作用。百日咳毒素（PT）催化虹鳟鱼卵黄生成后期卵母细胞粗膜中一种40 kDa蛋白的ADP核糖基化，霍乱毒素（CT）标记了几种蛋白，包括一种表观分子量为43 kDa的主要蛋白。40 kDa蛋白可被抗抑制性G蛋白（Gi）α亚基的抗体识别，而43 kDa蛋白可被抗刺激性G蛋白（Gs）α亚基的抗体识别。用17α,20β-DP处理膜组分可降低PT催化的40 kDa蛋白的ADP核糖基化。相反，暴露于17α,20β-DP后，CT催化的43 kDa蛋白的核糖基化没有显著变化。PT降低了17α,20β-DP与膜组分的特异性结合。17α,20β-DP的结合也受到不可水解的GTP类似物如鸟苷5'-O-（3-硫代三磷酸）（GTPγS）和鸟苷亚氨二磷酸（GppNHp）的抑制，但不受ATP或鸟苷5'-O-（2-硫代二磷酸）（GDPβS）的抑制。Scatchard分析表明，GppNHp使解离常数增加了3.8倍，而结合位点数没有显著变化，这表明GppNHp诱导的17α,20β-DP结合减少是由于17α,20β-DP与其受体之间结合亲和力的降低。我们得出结论，PT敏感的Gi参与了虹鳟鱼卵母细胞中17α,20β-DP的信号转导途径。
