Impact of reactive oxygen species on surface localization of membrane proteins

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Cells interact with one other and with their surroundings through proteins that reside at the cell surface. Proteins located at the surface of the cell play a role in forming connections that hold cells together, responding to the chemicals and hormones our bodies produce, responding to drugs and in many other processes. These proteins do not remain fixed in one location, but in many cases, they move around both within the cell surface membrane allowing complex interactions with other proteins, as well as to and from the surface membrane allowing control over their availability. Some of this movement is controlled by the formation of caveolae. Caveolae are invaginations in the cell membrane that can act as “signaling domains” or can bud off to remove proteins from the surface. Caveolin-1 is a caveolae-associated scaffolding protein that can be activated by reactive oxygen species (ROS) such as hydrogen peroxide. One of the main sources of reactive oxygen species in cells is a family of enzymes called NADPH oxidases, or NOX enzymes. We aim to define the relationship between NADPH oxidase derived ROS, caveolin-1 phosphorylation and activation, movement of cell surface proteins. ******This research will focus on three surface proteins: the interlekin-1 receptor (Il1R1) which undergoes caveolin-1 dependent movement, the TGF-beta receptor (TGFBR) which can undergo caveolin-1 dependent or caveolin-1 independent movement, and the epidermal growth factor receptor (EGFR) which primarily undergoes caveolin-1 independent movement. We will investigate whether NOX enzymes regulate caveolin-1 dependent movement, and whether the type of movement is influenced by cell surface NOX isoforms such as NOX1 and NOX2 versus the intracellular NOX isoform NOX4.******The lung fibroblast cell-line IMR-90 and the skin fibroblast cell line BJ were selected because they express these surface proteins and respond to TGF-beta stimulation by upregulating the NOX4 enzyme. In addition, we will genetically manipulate NOX1, NOX2, NOX4, and caveolin-1 using miRNA and over-expression vectors to compare the roles of subcellular NOX localization on caveolin-1 phosphorylation and the movement surface proteins. Membrane protein localization will be followed by confocal immunofluorescence; NADPH oxidase activity will be blocked by inhibitors DPI and celastrol and monitored by Amplex Red and NBT assays; phosphorylation of caveolin-1 will be monitored by Western blotting; and signaling events will be monitored by western blotting and quantitative RT-PCR. ******This work will provide important information about a cellular process of fundamental importance in biology of cell signaling.