IgE sensitized mast cells and basophils can be triggered by antigen to degranulate and release inflammatory agents, such as histamine, which cause the allergic reaction seen in atopic individuals. This response is generated when multivalent antigen binds to and crosslinks the IgE which is bound to the IgE receptor on the surface of these cells. Crosslinking of the receptors leads to an interaction with the membrane skeleton- cytoskeleton and immobility of the complex in the plane of the membrane. In addition to degranulation, receptor crosslinking induces changes in cell shape, cell spreading, increased F-actin content, aggregation of the receptors into patches, and internalization of the crosslinked complexes. Although the exact sequence of events is not known, many of these responses involve the membrane skeleton and the cytoskeleton. The purpose of the proposed research is to investigate the role of the membrane skeleton- cytoskeleton in the stimulation and degranulation of RBL cells. The initial work will focus on identifying the components of the crosslinking reagents and an actin binding assay will be used to determine which of the components is interacting with microfilaments. Once the actin binding proteins are identified, experiments will be done to determine the signalling mechanism used to recruit microfilaments to the plasma membrane. An excellent candidate is phosphorylation since it is known that PMA can mimic many of the cytoskeletal changes seen in stimulated cells. Furthermore, it is also known that agorin, the major component of the membrane skeleton, is a phosphoprotein which binds actin.
The final aim of the project is to determine if membrane skeleton-cytoskeletal involvement is necessary for the degranulation signal or whether it is part of a separate system for modulating the response by clearing aggregated receptors from the cell surface. This will be investigated by using transfected cells containing mutant IgE receptors and by comparing the role of the membrane skeleton-cytoskeleton in the immobilization, capping, and internalization of the IgE receptor versus RT1.A, the class 1 major histocompatibility antigen, which is independent of the IgE receptor and is not involved in the degranulation process.
