Caveolae are a plasma membrane specialization with at least two important functions. The are the vehicle for internalizing and delivering molecules to either the cytoplasm, the ER or the opposite cell surface by a process called potocytosis. Second, caveolae are membrane domains that compartmentalize signaling activities at the cell surface. Work carried our during the grant period has identified five different signaling events that occur in caveolae: isoproterenol-stimulated activation of adenyl cyclase; regulation of potocytosis by PKCalpha; PDGF-mediated activation of MAP kinase, EGF-mediated activation of c-Raf and ceramide-mediated suppression of highly organized signaling modules. This grant outlines three projects that are designed to directly test this hypothesis. The first specific aim is to determine how two different signaling modules, one for PKCalpha nad one for MAP kinase, are functionally organized in caveolae. Our goal is to identify the protein and lipid components responsible for recruit and organizing these molecules into functional units in caveolae. Purified caveolae are enriched in several molecules known to be involved in the assembly of the actin cytoskeleton including G-actin and myosin 1beta. The second specific aim outlines experiments to determine the role of myosin 1 beta in caveolae function, whether or not caveolae contain the molecular machinery to assemble actin filaments and if they are sites that nucleate the assembly of actin filament systems in vivo. The third specific aim outlines experiments to determine the role of receptor location in signal transduction. We have found that EGF receptors in quiescent fibroblasts are concentrated in caveolae but immediately migrate to coated pits after ligand binding. Nobody knows if the receptor sends different signals from these two locations. Experiments are planned to isolate these two domains at various times during receptor migration and determine if the receptor interactions with and activates different sets of signal transducers at each location. Mutations that cause mislocalization of EGF receptors will be identified and used to explore how receptor location affects the cellular response to EGF. Finally, we will determine whether or not caveolae facilitate cross-talk between signaling pathways. Caveolae have clearly been implicated as the target in a number of disease processes including cancer, viral and protozoan parasitic infections, and certain encephalopathies. Therefore, understanding the structure and function of caveolae is important for developing rational strategies to treat these diseases.
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