STUDY ON THE FUNCTIONAL DOMAIN STRUCTURE OF THE PLASMA MEMBRANE

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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