Signaling through the high affinity IgE receptor, FcepsilonRI, of basophils and mast cells is initiated by receptor crosslinking and propagated by the sequential activation of two protein tyrosine kinases: Lyn, that phosphorylates tyrosines within immunoreceptor tyrosine-based activation motifs (ITAMs) in the FcepsilonRI beta and gamma subnuits; and Syk, that is recruited to the gamma subunit phospho-ITAMs and in turn activates signaling pathways leading to allergic, asthmatic and anaphylactic reactions. Since August, 1994, Dr. Janet Oliver from the University of New Mexico (UNM) and Dr. Enrique Ortega from the Universidad Nacional Autonoma de Mexico (UNAM) have conducted FIRCA-supported collaborative research on the regulation of this kinase cascade in RBL-2H3 mast cells. Their work uses unique anti-FcepsilonRI monoclonal antibodies that are signaling competent (mAb J17) or impaired (mAb H10) in comparison with multivalent antigen. Their recent results link the weak signaling activity of H10-induced dimers to their failure to complete a Lyn-mediated FcepsilonRI beta subunit phosphorylation series apparently required for Lyn dissociation, Syk recruitment and signal propagation.
Aim 1 of the renewal application tests the hypothesis that Lyn interacts with partially phosphorylated FcepsilonRI subunits and must be displaced from fully phosphorylated receptors in order to permit Syk recruitment and signal propagation. The proposed experiments consist of phosphoamino acid analyses of phospho-beta from mAb-treated RBL- 2H3 cells and biochemical and fluorescence-based measurements of Lyn s binding to synthetic phospho-betaisoforms.
Aim 2 of the renewal application tests the hypothesis that crosslinked FcepsilonRI encounter Lyn during their transient association with Lyn-enriched, detergent-insoluble membrane microdomains and must complete the phosphorylation series that releases Lyn in order to redistribute into bulk membrane and interact with Syk and downstream signaling molecules. The proposed experiments explore the membrane topography of receptors and kinases by membrane fractionation and fluorescence resonance energy transfer. Results of this continued research will provide new insights into the pathways controlling signaling through the antigen receptors of immune cells.

Agency
National Institute of Health (NIH)
Institute
Fogarty International Center (FIC)
Type
Small Research Grants (R03)
Project #
2R03TW000440-04A1
Application #
2908296
Study Section
International and Cooperative Projects 1 Study Section (ICP)
Program Officer
Michels, Kathleen M
Project Start
1999-09-30
Project End
2002-09-29
Budget Start
1999-09-30
Budget End
2000-09-29
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of New Mexico
Department
Pathology
Type
Schools of Medicine
DUNS #
829868723
City
Albuquerque
State
NM
Country
United States
Zip Code
87131
Kepley, Christopher L; Taghavi, Sharven; Mackay, Graham et al. (2004) Co-aggregation of FcgammaRII with FcepsilonRI on human mast cells inhibits antigen-induced secretion and involves SHIP-Grb2-Dok complexes. J Biol Chem 279:35139-49
Wilson, Bridget S; Pfeiffer, Janet R; Oliver, Janet M (2002) FcepsilonRI signaling observed from the inside of the mast cell membrane. Mol Immunol 38:1259-68
Wilson, Bridget S; Oliver, Janet M (2002) Effector roles of IgE antibodies: targeting allergen to the high-affinity IgE receptor for Fc epsilon RI-dependent signaling and antigen presentation. Clin Allergy Immunol 16:197-232
Kepley, C L; McFeeley, P J; Oliver, J M et al. (2001) Immunohistochemical detection of human basophils in postmortem cases of fatal asthma. Am J Respir Crit Care Med 164:1053-8
Lara, M; Ortega, E; Pecht, I et al. (2001) Overcoming the signaling defect of Lyn-sequestering, signal-curtailing FcepsilonRI dimers: aggregated dimers can dissociate from Lyn and form signaling complexes with Syk. J Immunol 167:4329-37
Oliver, J M; Kepley, C L; Ortega, E et al. (2000) Immunologically mediated signaling in basophils and mast cells: finding therapeutic targets for allergic diseases in the human FcvarepsilonR1 signaling pathway. Immunopharmacology 48:269-81
Kepley, C L; Cambier, J C; Morel, P A et al. (2000) Negative regulation of FcepsilonRI signaling by FcgammaRII costimulation in human blood basophils. J Allergy Clin Immunol 106:337-48
Ortega, E; Lara, M; Lee, I et al. (1999) Lyn dissociation from phosphorylated Fc epsilon RI subunits: a new regulatory step in the Fc epsilon RI signaling cascade revealed by studies of Fc epsilon RI dimer signaling activity. J Immunol 162:176-85