Purification and Characterization of Prostaglandin E Receptor

我们先前已经证明，牛肾上腺嗜铬细胞中的前列腺素E （PGE）受体与两个信号转导系统偶联，通过Gi抑制cAMP的积累，并通过百日咳毒素不敏感的方式刺激磷酸肌肽（PI）的代谢，后者可能介导PGE_2对细胞中儿茶酚胺释放的刺激作用。今年，我们进一步推广了这一方法，并取得了以下成果：1) gtp结合蛋白（g蛋白）参与了PGE受体通过NaF （g蛋白的激活剂）与PI代谢的偶联。2) PI水解产生两个第二信使，二酰基甘油和IP3，它们分别激活蛋白激酶C和增加细胞内游离Ca^<2+>浓度（[Ca^<2+>]i）。我们发现PGE_2可以通过细胞内储存的Ca^<2+>的释放和Ca^<2+>通过Ca^<2+>通道进入来增加[Ca^<2+>]i。电生理技术证实了PGE_2对Ca^<2+>通道的激活作用。3) pge_2增强的儿茶酚胺释放是由蛋白激酶C的激活介导的，而不是由磷酸肌肽代谢引起的Ca^<2+>的动员。4)蛋白激酶C的激活依次激活膜上的Na^+、H^+反端口，其次诱导[Ca^<2+>]i的持续升高，导致儿茶酚胺的逐渐分泌。因此，我们揭示了从PGE受体激活到从嗜铬细胞释放儿茶酚胺的一系列生物学事件。我们已经完成了PGE受体及其信号转导系统的表征，比我们第一年的计划要多。另一方面，根据第二年的计划，我们合成了PGE_2的生物素基衍生物，并试图通过WGA和亲和素两层色谱纯化PGE_2受体- g蛋白复合物（HRG复合物）。我们用SDS凝胶电泳分析了部分纯化的HRG复合物，在凝胶上检测到两条蛋白带，一条是Mr为100 K左右的宽频带，一条是Mr为40 K的锐频带。然而，由于牛肾上腺髓质中PGE受体的数量有限，且缺乏特异性和有效的拮抗剂，因此鉴定和纯化PGE受体将是相当困难的。目前，我们计划通过几种方法分离PGE受体，包括受体单克隆抗体和cDNA克隆。少

We previously demonstrated that the prostaglandin E (PGE) receptor in bovine adrenal chromaffin cells is coupled to two signal transduction systems leading to inhibition of cAMP accumulation via Gi and to stimulation of phosphoinositide (PI) metabolism via a pertussis toxin- insensitive manner, the latter of which may mediate the stimulatory effect of PGE_2 on catecholamine release from the cells. We further extended this approach this year and obtained the following results; 1) a GTP-binding protein (G-protein) is involved in the coupling of PGE receptor to PI metabolism by NaF, an activator of G-proteins. 2) PI hydrolysis produces two second messengers, diacylglycerol and IP3, which activates protein kinase C and increases intracellular free Ca^<2+> concentration ([Ca^<2+>]i), respectively. We demonstrated that PGE_2 may increase [Ca^<2+>]i in chromaffin cells by release of Ca^<2+> from intracellular stores and by Ca^<2+> entry through Ca^<2+> channels. Activation of Ca^<2+> channels … More by PGE_2 is confirmed by electrophysiological techniques. 3) PGE_2-enhanced catecholamine release is mediated by activation of protein kinase C, rather than by Ca^<2+> mobilization, resulting from phosphoinositide metabolism. 4) Activation of protein kinase C in turn activates the Na^+, H^+ antiport on the membrane, and secondly induces a sustained elevation of [Ca^<2+>]i resulting in a gradual secretion of catecholamines. Thus we have revealed a sequence of biological events from PGE receptor activation to catecholamine release from chromaffin cells. We have accomplished the characterization of the PGE receptor and its signal transduction systems more than we made the plans for the first year. On the other hand, according to the second-year plan, we synthesized a biotinyl derivative of PGE_2 and tried to purify a complex of PGE_2 receptor-G-protein (HRG complex) by two affinity column chromatographies of WGA and avidin. We analyzed a partially purified HRG complex by SDS gel electrophoresis and detected two protein bands, a broad band with a Mr of around 100 K and a sharp one with a Mr of 40 K on the gel. However, it will be quite difficult to identify and purify the PGE receptor from bovine adrenal medulla due to the limitation of its amount and lack of a specific and potent antagonist. At present we plan to isolate the PGE receptor by several approaches including monoclonal antibodies to the receptor and cDNA cloning. Less