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有效介质。我们最近分离出人和豚鼠PAF受体的cDNA克隆。利用这些克隆作为探针，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3个外显子组成，3个外显子之间由大于20kb的内含子隔开。所有外显子都位于1号染色体上。通过选择性剪接，产生白细胞型和心脏型转录本。它们受不同的启动子调控. PAF受体介导的细胞信号转导机制的研究建立了携带PAF受体的CHO细胞系。PAF可激活磷脂酶C、A_2和丝裂原相关蛋白（MAP），抑制腺苷酸环化酶。这些细胞事件似乎是由不同类型的G蛋白调节的。此外，我们发现内毒素。LPS刺激PAF受体，并在人血小板以及CHO转染子中产生Ca增加。因此，部分LPS信号，特别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PAF受体在非洲爪蟾卵母细胞中表达，建立了一个灵敏的PAF受体激动剂/拮抗剂筛选体系。此外，构建了携带过量PAF受体的COS细胞/CHO细胞。利用这些细胞的膜组分，建立了PAF受体调节剂（受体拮抗剂或受体-G蛋白解偶联剂）的高特异性筛选系统。通过对天然和人工合成的化合物的筛选，获得了几种具有免疫调节潜力的候选化合物。

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.