Purification and Characterization of Prostaglandin E Receptor

我们曾证明牛肾上腺嗜铬细胞的前列腺素E（PGE）受体与两个信号转导系统偶联，导致通过Gi抑制cAMP积累和通过百日咳毒素不敏感方式刺激磷脂酰肌醇（PI）代谢，后者可能介导PGE_2刺激细胞释放儿茶酚胺的作用。我们今年进一步扩展了这一方法，并获得了以下结果：1）GTP结合蛋白（G蛋白）参与了NaF（G蛋白的激活剂）对PGE受体与PI代谢的偶联。2)PI水解产生两个第二信使，甘油二酯和IP 3，分别激活蛋白激酶C和增加细胞内游离Ca^2+浓度（[Ca^2+]i）。结果表明，PGE_2可通过释放胞内Ca^<2 +>和通过Ca ^<2 +>通道内流增加嗜铬细胞[Ca^<2+]i。Ca^<2+>通道的激活 ...更多信息 电生理技术证实PGE_2的作用。3)PGE_2促进的儿茶酚胺释放是由磷酸肌醇代谢引起的蛋白激酶C的激活介导的，而不是由Ca^2+动员介导的。4)蛋白激酶C的激活反过来又激活了膜上的Na^+、H^+反向转运，其次诱导[Ca^2+]i持续升高，导致儿茶酚胺的逐渐分泌。因此，我们揭示了一系列的生物学事件，从前列腺素E受体激活，从嗜铬细胞释放儿茶酚胺。我们已经完成了PGE受体及其信号转导系统的表征，超过了我们第一年的计划。另一方面，根据第二年的计划，我们合成了一种PGE_2的生物素衍生物，并尝试用WGA和亲和素两种亲和柱层析法纯化PGE_2受体-G蛋白复合物（HRG复合物）。我们分析了部分纯化的HRG复合物的SDS凝胶电泳，并检测到两个蛋白条带，一个宽的带与MR约100 K和一个尖锐的一个与40 K的凝胶上的MR。然而，由于牛肾上腺髓质中PGE受体的数量有限，且缺乏特异性强的拮抗剂，因此鉴定和纯化牛肾上腺髓质中的PGE受体非常困难。目前，我们计划通过几种方法分离PGE受体，包括受体的单克隆抗体和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