The proposed studies will advance elucidation of the features of antigen-mediated crosslinking of lgE bound to its high affinity receptor, Fc epsilonRI, on mast cells that are critical for initiating a cascade of signaling events leading to the release of key modiators in allergic responses. The studies will build on strong foundation of fluorescence and other methodologies and concepts that are derived from previous work on this well-characterized model immune cell system. We have established that a small, symmetrical bivalent ligand stimulates a cellular response depending on whether this ligand forms small cyclic complexes or linear chains with lgE bound to Fc epsilon RI , providing evidence that structural restrictions between crosslinked lgE-Fc epsilon RI can limit cell activation. This hypothesis will be tested in Specific Aim 1 with a new generation of rigid bivalent ligands made with double-stranded DNA, together with a specially prepared bi- specific lgE. The structural arrangement of crosslinked lgE-Fc epsilon Rl beta and gamma subunits by Lyn, and the binding and activation of the tyrosine kinase Syk. Binding of trivalent and multivalent antigens will also be analyzed in conjunction with their functional activities.
Specific Aim 2 will investigate the functional importance of specialized plasma membrane domains that are being characterized in this laboratory. Crosslinking of lgE-Fc epsilon Ri causes association with detergent-resistant membrane domains that contain abundant Lyn tyrosine kinase activity, and our recent results indicate that these interactions are important for initiating Fc epsilon Rl mediated signal transduction. These domain-Fc epsilon Rl interactions, their regulation by the mirofilament cytoskeleton, and the consequences of these on early and late signaling events will be investigated in biochemical preparations and intact cells. Because of the central role of ligand binding and receptor aggregation in immune cell activation and the accumulating evidence that signaling molecules localized to membrane domains are integrally involved, the proposed investigation should lead to new concepts and targets for therapeutic intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI022449-12
Application #
2671826
Study Section
Special Emphasis Panel (ZRG2-IMS (02))
Project Start
1985-08-01
Project End
2002-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
12
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Cornell University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Holowka, David; Baird, Barbara (2017) Mechanisms of epidermal growth factor receptor signaling as characterized by patterned ligand activation and mutational analysis. Biochim Biophys Acta Biomembr 1859:1430-1435
Korzeniowski, Marek K; Wisniewski, Eva; Baird, Barbara et al. (2017) Molecular anatomy of the early events in STIM1 activation - oligomerization or conformational change? J Cell Sci 130:2821-2832
Holowka, David; Baird, Barbara (2016) Roles for lipid heterogeneity in immunoreceptor signaling. Biochim Biophys Acta 1861:830-836
Wilson, Joshua D; Shelby, Sarah A; Holowka, David et al. (2016) Rab11 Regulates the Mast Cell Exocytic Response. Traffic 17:1027-41
Sun, Chao; Wakefield, Devin L; Han, Yimo et al. (2016) Graphene Oxide Nanosheets Stimulate Ruffling and Shedding of Mammalian Cell Plasma Membranes. Chem 1:273-286
Korzeniowski, Marek K; Baird, Barbara; Holowka, David (2016) STIM1 activation is regulated by a 14 amino acid sequence adjacent to the CRAC activation domain. AIMS Biophys 3:99-118
Holowka, David; Wilkes, Marcus; Stefan, Christopher et al. (2016) Roles for Ca2+ mobilization and its regulation in mast cell functions: recent progress. Biochem Soc Trans 44:505-9
Cohen, Roy; Holowka, David A; Baird, Barbara A (2015) Real-time imaging of Ca(2+) mobilization and degranulation in mast cells. Methods Mol Biol 1220:347-63
Bryant, Kirsten L; Baird, Barbara; Holowka, David (2015) A novel fluorescence-based biosynthetic trafficking method provides pharmacologic evidence that PI4-kinase III? is important for protein trafficking from the endoplasmic reticulum to the plasma membrane. BMC Cell Biol 16:5
Holowka, David; Korzeniowski, Marek K; Bryant, Kirsten L et al. (2014) Polyunsaturated fatty acids inhibit stimulated coupling between the ER Ca(2+) sensor STIM1 and the Ca(2+) channel protein Orai1 in a process that correlates with inhibition of stimulated STIM1 oligomerization. Biochim Biophys Acta 1841:1210-6

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