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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM042388-04A3
Application #
2181332
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1990-07-01
Project End
1999-06-30
Budget Start
1995-07-01
Budget End
1996-06-30
Support Year
4
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Field, K A; Apgar, J R; Hong-Geller, E et al. (2000) Mutant RBL mast cells defective in Fc epsilon RI signaling and lipid raft biosynthesis are reconstituted by activated Rho-family GTPases. Mol Biol Cell 11:3661-73
Frigeri, L; Apgar, J R (1999) The role of actin microfilaments in the down-regulation of the degranulation response in RBL-2H3 mast cells. J Immunol 162:2243-50
Apgar, J R (1997) Increased degranulation and phospholipase A2, C, and D activity in RBL cells stimulated through FcepsilonR1 is due to spreading and not simply adhesion. J Cell Sci 110 ( Pt 6):771-80
Apgar, J R (1995) Activation of protein kinase C in rat basophilic leukemia cells stimulates increased production of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: correlation with actin polymerization. Mol Biol Cell 6:97-108
Apgar, J R (1994) Polymerization of actin in RBL-2H3 cells can be triggered through either the IgE receptor or the adenosine receptor but different signaling pathways are used. Mol Biol Cell 5:313-22
Ucker, D S; Obermiller, P S; Eckhart, W et al. (1992) Genome digestion is a dispensable consequence of physiological cell death mediated by cytotoxic T lymphocytes. Mol Cell Biol 12:3060-9
O'Rourke, A M; Mescher, M F; Apgar, J R (1992) IgE receptor-mediated arachidonic acid release by rat basophilic leukemia (RBL-2H3) cells: possible role in activating degranulation. Mol Immunol 29:1299-308
Apgar, J R (1991) Association of the crosslinked IgE receptor with the membrane skeleton is independent of the known signaling mechanisms in rat basophilic leukemia cells. Cell Regul 2:181-91