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BASIC RESEARCH TO EXPLOIT PROPERTIES OF ENDOTHELIAL CAVEOLAE FOR DEVELOPMENT OF PREVENTIVE AND/OR TREATING MEASURES FOR VASCULAR DISEASES

利用内皮细胞小窝特性开发血管疾病预防和/或治疗措施的基础研究

基本信息

批准号：
09557001
负责人：
FUJIMOTO Toyoshi
金额：
$ 8.26万
依托单位：
NAGOYA UNIVERSITY (1999)Gunma University (1997-1998)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

Caveolae are small invaginations of the plasma membrane found in many cell types, and caveolins (caveolin-1,2,3) are a family of integral membrane proteins forming the framework of caveolae. Caveolae and caveolins have been revealed to have diverse functions, including endocytosis, signal transduction, cholesterol transport, and tumor suppression. To understand caveolae and caveolins in molecular terms, we have done several lines of research using various cell biological techniques.Caveolin-1 is heavily phosphorylated in tyrosine in v-Src-expressing cells, but consequences of the phosphorylation has not been known. We generated a polyclonal antibody specific for tyrosine-phosphorylated caveolin-1 (PY14). In v-Src-expressing cells, PY14 labeled caveolae, flat plasmalemmal areas of various sizes and shapes, and intracellular vesicles that are significantly larger than caveolae ; biochemical properties of caveolin-1, including detergent insolubility and oligomerization, were not different from the non-phosphorylated counterpart. 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.We used freeze-fracture immunoelectron microscopy to examine distribution of the two isoforms (α, β) of caveolin-1. 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 may suggest that caveolae of different depths is not be convertible each other.

小窝是细胞膜上的一种小的内陷结构，它存在于许多细胞中，而小窝蛋白（caveolin-1，2，3）是构成小窝结构的一类膜蛋白。Caveolae和Caveolins具有多种功能，包括内吞作用、信号转导、胆固醇转运和肿瘤抑制。为了从分子水平上理解小窝和小窝蛋白，我们使用各种细胞生物学技术进行了几项研究。我们制备了酪氨酸磷酸化小窝蛋白-1（PY 14）的多克隆抗体。在表达v-Src的细胞中，PY 14标记的小窝，各种大小和形状的平坦质膜区域，以及显著大于小窝的胞内囊泡;小窝蛋白-1的生化特性，包括去污剂不溶性和寡聚化，与非磷酸化的对应物没有不同。在正常大鼠体内组织中，连续毛细血管的内皮被PY 14标记，而其他血管的内皮不被标记。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caveolin-1的酪氨酸磷酸化，并引起囊泡形成。结果表明，caveolin-1的酪氨酸磷酸化可诱导囊泡形成和/或囊泡聚集。我们用冷冻断裂免疫电镜观察了caveolin-1的两种亚型（α，β）的分布。α亚型特异性抗体优先修饰深小窝，而与α和β亚型反应的抗体以相似的效率标记深小窝和浅小窝。结果表明，不同深度的小窝之间α/β值存在差异，说明不同深度的小窝之间是不可转换的。