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.
