STUDIES ON PHYSIOLOGICAL FUNCTION OF PLATELET-ACTIVATING FACTOR

研究了血小板活化因子(PAF)的生理作用及其细胞信号转导途径：(1)PAF受体介导的细胞信号转导：利用CHO细胞中的PAF受体，检测PAF作用后的细胞信号转导。PAF刺激磷脂酰肌醇、丝裂原活化蛋白(MAP)激酶、磷脂酶A2，抑制腺苷环化酶。从PTX处理的结果来看，PAF受体似乎与两种类型的G蛋白偶联。Wortmannin可抑制MAP激酶的非钙激活，提示Gi和MAP激酶之间存在一个Wortmannin敏感分子，如磷脂酰肌醇3-激酶。为了确定PAF信号脱敏的分子机制，我们制备了携带野生型、C端缺失突变体和替换突变体(Ser，Thr to Ala)受体的CHO细胞。我们发现，C末端胞质尾巴不是正向信号转导所必需的，但在信号关闭过程中是必不可少的。GRK家族可能参与Ser/Thr残基的磷酸化，导致信号关闭。(2)PAF受体的基因组结构。该基因由三个相互分离的外显子组成&gt；20kb内含子。Two5‘-非编码外显子具有明显的转录起始点和启动子。这些外显子被交替地剪接到共同的剪接受体上，以产生两个不同的mRNA。转录本1存在于几乎所有的组织中，并被PAF或内毒素通过其核因子-kB位点上调。转录本2以更具组织特异性的方式表达，受雌激素上调，受转化生长因子-β下调。这些启动子的生理和病理意义目前正在研究中。

Following results were obtained on the physiological role and cellular signaling of platelet-activating factor (PAF).(1)Cellular signaling via PAF receptorBy using transfected receptor in CHO cells, cellular signaling after PAF application was determined. PAF stimulates phosphoinositide turnover, mitogen-activated protein (MAP) kinase, phospholipase A2, and inhibits adenylate cyclase. From the results of PTX-treatment, PAF receptor appears to couple with two types of G-proteins. Ca-independent activation of MAP kinase was inhibited by wortmannin, suggesting a wortmannin-sensitive molecule such as phosphatidylinositol 3-kinase is present between Gi and MAP kinase.To determine the molecular mechanisms of signal desensitization by PAF,we prepared CHO cells carrying wild-type, C-terminal deletion mutant, and substitution mutant (Ser, Thr to Ala) receptors. We found that C-terminal cytoplasmic tail is not required for forward signal transduction, but it is essential for signal-shut down. A family of GRK (G-protein receptor kinase) may be involved in the phosphorylation of Ser/Thr residues leading to signal shut down.(2)Genomic structure of PAF receptor.Human PAF receptor gene is present as a single copy in haploid on the chromosome 1q. The gene consists of three exons, separated each other > 20kb intron. Two5'-noncoding exons has distinct transcriptional start site, and promoters. These exons are alternatively spliced to a common splice acceptor to yield two different mRNAs. Transcript 1 is present in almost all tissues, and up-regulated by PAF or endotoxin through its NF-kB site. Transcript 2 is expressed in more tissue-specific way, and up-regulated by estrogen, and down-regulated by TGF-b. The physiological and pathological significance of these promoters are currently under investigation.