Functional And Proteomics Analysis Of Lipid Rafts And Caveolae

Previously, we have found that lipid rafts/caveolae were essential for IGF-1 receptor signaling during 3T3-L1 preadipocytes differentiation induction. However, it was not identified which of the membrane lipid-ordered microdomains mediates the receptor signal. Using small double strand RNA-mediated interference (RNAi), we successfully suppressed the caveolin-1 protein expression. In cells stably transfected with vector expressing small interfering RNA (siRNA) fragment, no caveolin-1 protein or caveolae were detected. On the other hand, removal of caveolin-1 did not affect the caveolin-less lipid rafts or the localization of IGF-1 receptor in lipid rafts on plasma membrane. IGF-1 receptor signal transduction and induced cellular differentiation were normal in RNAi cells with only lipid rafts. Furthermore, these IGF-1 receptor signaling events were still sensitive to the cholesterol-binding reagents. Thus, our results suggest that lipid rafts are sufficient for IGF-1 receptor signaling and the recruitment of signal molecules by caveolin-1 is not essential for IGF-1 receptor signaling.The proteins associated with plasma membrane lipid microdomains have been indicated to be important for many of the cellular functions played by the membrane lipid microdomains. Thus, we applied qualitative and quantitative proteomic analysis to investigate the protein composition and quantitation of wild type and Cav-1 RNAi cells. The results suggested that many Lipid microdomain associated proteins including receptors, membrane channels, cell surface antigens, glycosylphosphatidylinositol (GPI)-anchored proteins, G proteins and cytoskeleton associated proteins were detected and quantitated, which do not exhibit significant qualitative and quantitative alteration. In summary, proteins associated with membrane lipid microdomains were not diminished by the suppression of caveolin-1 and ablation of caveolae. The association of proteins with membrane lipid microdomains was independent from caveolae and cveolin-1. Recently, we built a modified sucrose density gradient ultracentrifugation method, by which we could successfully isolate two forms of lipid microdomains from each other. This method will be an important tool for the component and functional analysis of lipid raft and caveolars.