Screening and Development of New Immnomodulatory Drugs using PAF Receptor Gene as a Probe

血小板活化因子(PAF)是炎症、免疫紊乱和内毒素休克的重要介质。我们最近分离了人和豚鼠PAF受体的cDNA克隆。以这些克隆为探针，我们获得了以下结果。PAF受体基因的分离和结构：PAF受体基因由三个外显子组成，由&gt；20kb的内含子隔开。所有外显子都定位在1号染色体上。通过选择性剪接，产生了白细胞和心脏类型的转录本。它们受到不同的推动者的不同监管。通过克隆的PAF受体阐明信号转导。建立了携带克隆的PAF受体的稳定的转基因细胞(CHO)。在PAF的作用下，磷脂酶C、A2和丝裂原相关蛋白都被激活，而腺苷环化酶被抑制。这些细胞事件似乎受到不同类型的G蛋白的调控。此外，我们发现内毒素。脂多糖刺激PAF受体，使人血小板和CHO转染体产生钙升高。因此，部分内毒素信号，尤其是与血小板聚集相关的信号，可能是由于PAF受体的直接激活。PAF受体激动剂和拮抗剂筛选体系的建立。在非洲爪哇卵母细胞中表达PAF受体，建立了敏感的PAF受体激动剂/拮抗剂检测体系。进一步构建了携带过量PAF受体的COS细胞/CHO细胞。利用这些细胞的膜部分，建立了PAF受体调节剂(受体拮抗剂或受体-G蛋白解偶联)的高度特异性筛选体系。通过对天然化合物和合成化合物的筛选，得到了几个具有免疫调节药物潜力的候选化合物。

Platelet-activating factor (PAF) is a potent mediator of inflammation, immunological disorders, and endotoxin shock. We have recently isolated cDNA clones of human and guinea pig PAF receptors. Using these clones as probe, we have obtained following results.1. Isolation and structure of PAF receptor gene.PAF receptor gene is consisted of three exons which are separated by >20kb introns. All exons are localized on chromosome 1. By alternative splicing, the leukocyte- and heart-type transcripts are produced. They have differentially regulated by distinct promoters.2. Elucidation of signal transduction through a cloned PAF receptor.A stable transfectant cell (CHO) was established carrying a cloned PAF receptor. By the application of PAF, phospholipase C and A_2, and MAP (mitogen-associated protein) are all activated, while adenylate cyclase was inhibited. These cellular events appear to be regulated by different types of G-proteins. Furthermore, we found that endotoxin. LPS, stimulated PAF receptor and yielded a Ca increase in human platelets as well as CHO transfectants. Thus, a part of LPS signaling, especially that related to platelet-aggregation might be due to the direct activation of PAF receptor.3. Development of Screening System of PAF Receptor Agonists and Antagonists.PAF receptor was expressed in Xenopus oocytes and a sensitive assay system of PAF receptor agonists/antagonists was made. Furthermore, COS cells/CHO cells carrying an excess amount of PAF receptors were constructed. By using membrane fractions of these cells, the highly specific screening system of PAF receptor modulator (either receptor antagonists or uncoupler of receptor-G-protein) was established. Through screening of naturally-occurring and synthetic compounds, several candidate chemicals have been obtained, which have potentiality for immunomodulatory drugs.