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STUDY ON THE FUNCTIONAL DOMAIN STRUCTURE OF THE PLASMA MEMBRANE

质膜功能域结构的研究

基本信息

批准号：
11470003
负责人：
FUJIMOTO Toyoshi
金额：
$ 9.34万
依托单位：
NAGOYA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470003/
关键词：
PLASMA MEMBRANE CAVEOLAE RAFT CAVEOLIN-1 CAVEOLIN-2 カベオリン-1 カベオリン-2 v-Src チロシンリン酸化

项目摘要

Caveolae and rafts are microdomains of the plasma membrane. They are formed based on innate properties of membrane lipids and are thought to be related to signal transduction, cholesterol transport, and other functions. We have conducted research to study basic characteristics of the microdomains.1. To examine possible functional differences between two isoforms (α, β) of caveolin-1, we performed freeze-fracture immunoelectron microscopy. Antibodies specific to the α isoform decorated deep caveolae preferentially, whereas those reacting with both α and β isoforms labeled deep and shallow caveolae with similar efficiency. The result indicates that the α/β ratio is different among caveolae, and suggests that caveolae of different depths is not be convertible each other. Introduction of caveolin-1 to caveolae-deficient cells caused de novo formation of caveolae, but only with low efficiency. Addition of caveolin-2 significantly improved the efficiency of caveolae formation, especially tha … More t of deep caveolae. The result suggests that caveolin-2 is important for caveolae to be invaginated and take the characteristic shape.2. Caveolin-1 is one of the most heavily tyrosine-phosphorylated protein in v-Src-expressing cells. To examine direct consequences of the phosphorylation, we generated a polyclonal antibody specific for tyrosine-phosphorylated caveolin-1 (PY14). By using the antibody for immunoelectron microscopy, it was found that flat plasmalemmal areas of various sizes, and intracellular vesicles larger than caveolae are formed in v-Src-expressing cells. In normal rat tissues in vivo, the endothelium of the continuous capillary was labeled by PY14, whereas the endothelium of other vessels was not. In the cultured endothelium, tyrosine phosphatase inhibitors as well as oxidative stress induced tyrosine phosphorylation of caveolin-1, and caused vesiculation of caveolae. These results suggest that tyrosine phosphorylation of caveolin-1 induces vesiculation and/or aggregation of caveolae. Less

小窝和木筏是质膜的微域。它们是基于膜脂的固有性质而形成的，被认为与信号转导、胆固醇运输和其他功能有关。我们进行了研究，以研究微域的基本特征1。为了检测小窝蛋白-1的两种异构体(α，β)之间可能的功能差异，我们进行了冷冻断裂免疫电子显微镜检查。针对α亚型的抗体优先修饰深窝，而与α和β亚型反应的抗体以相似的效率标记深窝和浅窝。结果表明，不同深度洞穴的α/β比率不同，表明不同深度洞穴之间是不可转换的。将小窝蛋白-1导入小窝缺陷细胞可诱导小窝的新生形成，但效率较低。小窝蛋白-2的添加显著提高了小窝形成的效率，尤其是…更多的深凹陷。结果表明，小窝蛋白-2对于小窝的内陷和形成特征性形状具有重要意义。小窝蛋白-1是v-Src表达细胞中酪氨酸磷酸化程度最高的蛋白之一。为了检测磷酸化的直接后果，我们产生了一种针对酪氨酸磷酸化小窝蛋白-1(PY14)的多克隆抗体。免疫电子显微镜观察发现，v-Src表达细胞内可见大小不一的扁平质膜，胞内有比小凹更大的小泡。在活体正常大鼠组织中，连续毛细血管内皮细胞被PY14标记，而其他血管内皮细胞未被标记。在培养的内皮细胞中，酪氨酸磷酸酶抑制剂以及氧化应激诱导小窝蛋白-1的酪氨酸磷酸化，并引起小窝的囊泡形成。这些结果表明，小窝蛋白-1的酪氨酸磷酸化可诱导小窝的囊泡形成和/或聚集。较少