MEMBRANE DOMAINS IN SECRETORY GRANUES OF RAT PAROTID ACINAR CELLS

膜生物学的最新进展表明，富含糖脂和胆固醇的膜微域是质膜的侧向结构成分。这些微域被称为脂筏，被认为是信号转导和膜运输的平台。木筏被认为是由胆固醇隔开的糖脂的长的和大部分饱和的酰基链紧密堆积而成的。脂筏的成分根据其在洗涤剂Triton X-100中的不溶性被生化分离为DRM(抗洗涤剂膜)或DIGS(洗涤剂不可溶糖脂富集区)。由于DRM的高脂含量，经梯度离心法可以从低密度组分中分离出DRM。DRMS集中了糖基磷脂酰肌醇锚定蛋白(GPI锚定蛋白)、包括流感血凝素在内的一些跨膜蛋白，以及细胞内信号蛋白如二酰化的Src-PTKs Lck、Lyn和Fyn或异三聚体GTP结合蛋白和磷脂酰肌醇二磷酸。因此，木筏很可能充当细胞外蛋白质和细胞内分子的支架。在免疫系统中，脂筏对高亲和力Fc受体介导的酪氨酸磷酸化的需求已被报道。然而，脂筏对FcγR介导的酪氨酸磷酸化的需求尚未被研究。在本研究中，我们研究了脂筏在维甲酸分化的HL60细胞中FcγR信号转导中的作用。我们的结果表明，脂筏是FcγRIIA聚集性诱导src-PTKs激活以启动酪氨酸磷酸化途径的关键机制。我们还尝试在大鼠腮腺腺泡细胞的分泌颗粒中鉴定脂筏。

Recent advances in membrane biology suggest the glycolipids- and cholesterol-rich membrane microdomains as lateral structural components of the plasma membranes. These microdomains are called as lipid rafts and have been proposed to function as platforms for both signal transduction and membrane trafficking. Rafts are considered to be formed by tight packing of long and mostly saturated acyl chains of glycolipids interspaced by cholesterol. The components of lipid rafts are biochemically separated as DRMs (detergent-resistant membranes) or DIGs (detergent-insoluble glycolipids-enriched domains) based on their insolubility in the detergent Triton X-100 in the cold. Because of their high lipid content, DRMs can be isolated in the low density fraction after gradient centrifugation. DRMs concentrate glycosyl-phosphatidylinositol-anchored proteins (GPI-anchored proteins), some transmembrane proteins including influenza hemagglutinin, and also intracellular signaling proteins such as dually acylated Src-PTKs Lck, Lyn and Fyn or heterotrimeric GTP-binding proteins and phosphatidylinositol bisphosphate. Rafts are therefore likely to act as scaffolding for both extracellular proteins and intracellular molecules. In the immune system, the requirement of lipid rafts for tyrosine phosphorylation mediated via high affinity Fc receptor for IgE (FceRI) and TCR has been reported. However, the requirement of lipid rafts for FcγR-mediated tyrosine phosphorylation has not been investigated.In the present study, we investigated the role of lipid rafts in FcγR-signaling in retinoic acid-differentiated HL60 cells (RA-HL60 cells). Our results suggest that lipid rafts are crucial machinery in which clustering of FCγRIIa induces the activation of Src-PTKs to initiate the tyrosine phosphorylation pathway. We have also tried to identify lipid raft in the secretory granules of rat parotid acinar cells.