Atopic individuals produce IgE antibodies which bind to the high affinity IgE receptor on the surface of mast cells and basophils. These cells are triggered to degranulate when they surface of mast cells and basophils. These cells are triggered to degranulate when they encounter multivalent antigen which binds to the IgE, thereby crosslinking the receptors. Receptor crosslinking, which is the critical event in the activation of these cells, leads to an interaction with the membrane skeleton thus causing immobilization and detergent insolubility of the receptor complex. Activation of a signalling cascade leads to degranulation and the release of inflammatory agents such as histamine. Some of these signals also lead to the polymerization of actin and the recruitment of microfilaments to the plasma membrane. Actin filaments are believed to plan a role in both cell adhesion which increases degranulation as well as the directed movement of crosslinked receptors into large, inactive aggregates on the cell surface. Thus, actin filaments may be involved in several different functions within the activated cell. Therefore, the overall purpose of the proposed research is to investigate the role of the membrane skeleton and cytoskeleton in the regulation of degranulation.
The first aim will be to study microfilament driven down-regulation. This will be accomplished by using tn assay which has been developed to measure active versus inactive receptors. Experiments will also be done to determine which protein(s) in the crosslinked receptor complex is interacting with actin filaments. In the second aim, the role of microfilament assembly in adhesion and spreading on extracellular matrix proteins will be investigated. This will include studies on how adhesion may influence degranulation and eicosanoid production through alterations in the signaling pathways.
The final aim i s to investigate the signalling mechanisms responsible for the polymerization of actin when IgE sensitized cells are activated by antigen. Protein kinase C has been shown to be crucial for this response. The upstream signaling pathway responsible for activating protein kinase C with regard to actin filament assembly will be investigated. Protein Kinase C is believed to activate the kinases responsible for the production of phosphatidylinositol mono- and bis-phosphate which may then interact with actin binding proteins such as gelsolin to release actin and allow it to polymerize. Mush of this work will then be compared to the activation of the F-actin response triggered by cellular adhesion.
