PLATELET ACTIVATING FACTOR SIGNALING IN LIVER

血小板活化因子(PAF)是急性心肌梗死的一种独特的脂质介质。 过敏是一种炎症反应，会引起强烈的 An对肝糖原分解有极强的促进作用 间接机制涉及肝血管收缩。我们的 PAF体内调节肝糖原分解和血流量的研究 在抗原诱导的过敏反应过程中，PAF在 为肝外组织提供能量底物并调节 急性病理生理状态下的肝脏微循环。 这项拟议的研究将推动我们对PAF在肝脏中的作用的研究 病理到分子水平，并提供详细信息 需要推进这一领域。使用基于聚合酶链式反应的策略，我们成功地 克隆的大鼠肝组织纤溶酶原激活物受体的编码基因，仅缺少极端的5‘端 PAF R基因编码区。肝组织纤溶酶原激活物受体的序列分析 表明它是G蛋白偶联受体家族的成员 与最近克隆的白细胞PAF-R在细胞内的不同 与其他几种G蛋白的信号作用有关的结构域- 偶联受体。我们还扩增了PAF R基因的一个截短形式的cDNA， 编码缺少C末端胞质结构域的蛋白质，提供 首次证明存在PAF R的另一种简写形式。 原位杂交显示PAF-R mRNA在大鼠脑内显著表达 我们的研究表明肝窦细胞是原发的 PAF在肝脏中的作用部位。全长PAF受体cDNA的可用性 将使我们能够进行详细的研究，以解决与 纤溶酶原激活剂受体的异质性及其基因表达调控机制 阐明了PAF受体结构域的结构和功能 R为PAF在肝脏中作用的分子基础提供新的见解。 在建议的研究中，我们会1)扩增余下的5‘端 短的和长的PAF受体cDNA或者筛选大鼠肝脏的cDNAs 文库，以获得编码这两种形式的 PAF R我们将利用这些PAF R cDNA的瞬时表达来 确定配体结合、信号传递和脱敏特性 这两个PAF受体，包括鉴定激活的G蛋白 感受器。这个项目的一个主要焦点将是评估 黄曲霉毒素受体胞内结构域的结构和功能 在利用共表达的研究中，信号传递增加了脱敏作用， 这些受体结构域的截断和定点突变。最后， 我们将对表达PAF R mRNA的肝细胞进行鉴定，确定 是否存在长短形式的表达差异 PAF受体mRNA的表达及肝脏对PAF反应是否受到调节 在转录水平上由PAP和内毒素引起。这些研究将 为PAF在肝脏中作用的分子基础提供新的见解。 因为还没有研究评估PAF R功能的分子基础 受体的水平，很可能与 了解PAF在其他组织中作用的分子基础将 从这项工作中脱颖而出。

Platelet-activating factor (PAF) is a unique lipid mediator of acute allergic an inflammatory reactions that elicits a powerful and exceptionally potent stimulatory effect on hepatic glycogenolysis by an indirect mechanism involving constriction of the hepatic vasculature. Our findings that PAF regulates hepatic glycogenolysis and blood flow in vivo and during antigen-induced anaphylaxis suggests a crucial role for PAF in supplying extrahepatic tissues with energy substrates and regulating the hepatic microcirculation during acute pathophysiologic situations. The proposed research will carry our studies of the role of PAF in liver pathology to the molecular level and provide the detailed information needed to advance this area. Using a PCR-based strategy, we successfully cloned cDNAs encoding the rat liver PAF R, lacking only the extreme 5' coding region of the PAF R cDNA. Sequence analysis of the hepatic PAF R demonstrates that it is a member of the G protein-coupled receptor family and differs from the recently cloned leukocyte PAF R in intracellular domains implicated in the signaling actions of several other G protein- coupled receptors. We also amplified a truncated form of the PAF R cDNA, encoding a protein lacking a C terminal cytoplasmic domain, providing the first evidence for the existence of an alternate short form of the PAF R. In situ hybridization revealed prominent expression of the PAF R mRNA in hepatic sinusoidal cells which our studies have implicated as the primary site of PAF action in liver. The availability of full length PAF R cDNAs will enable us to pursue detailed studies to address questions relating to PAF R heterogeneity, mechanisms for regulation of PAF R gene expression and elucidating the structure and function of receptor domains of the PAF R to provide new insights into the molecular basis of PAF action in liver. In the proposed studies, we will 1) amplify the remaining 5' end of the short and long PAF R cDNAs or alternatively, screen a rat liver cDNA library, to obtain the full length cDNAs encoding these two forms of the PAF R. We will 2) utilize transient expression of the these PAF R cDNAs to determine the ligand binding, signaling and desensitization properties of these two PAF Rs, including identification of the G proteins activated by the receptors. A major focus of this project will be to 3) evaluate the structure and function of intracellular domains of the receptor in signaling add desensitization in studies utilizing coexpression, truncation and site-directed mutations of these receptor domains. Finally, we will 4) identify the hepatic cells expressing the PAF R mRNA, determine whether there is differential expression of the short and long forms of the PAF R mRNA and assess whether hepatic responses to PAF are regulated at the transcriptional level by PAP and endotoxin. These studies will provide new insights into the molecular basis of PAF action in liver. Because no studies have evaluated the molecular basis of PAF R function at the level of the receptor, it is likely that concepts relevant to understanding the molecular basis of PAF action in other tissues will emerge from this work.