The studies described in this application will utilize FcepsilonRI as a model system for antigen receptor mediated cell activation. The attractiveness of FcepsilonRI as a model system is due in particular to three features: 1) the complete multi-subunit receptor can be studied using currently available molecular techniques; 2) its rapid engagement and disengagement capability permits the analysis of both positive and negative regulations of kinase-dependent signaling pathways; 3) homogenous FcepsilonRI+ cell populations can be easily generated from the rapidly expanding repertoire of mice with targeted inactivations of signal transduction proteins. Utilizing these features specific to FcepsilonRI, several intriguing questions about antigen receptor signaling can be answered: 1) Is a multi-subunit receptor more than the sum of its parts? Two signaling chains of FcepsilonRI (the beta and gamma subunits) participate cooperatively to amplify FcepsilonRI- mediated signals. The experiments proposed will analyze how interactions among the beta and gamma subunits and two tyrosine kinases produce a signal amplifier. 2) How is antigen receptor signaling controlled? A specific negative regulatory molecule - hematopoietic cell phosphatase, and its interactions with FcepsilonRI alone and in conjunction with an inhibitory antigen receptor will be analyzed. 3) How are NRTKs which don't interact directly with an antigen receptor controlled by antigen receptor clustering? A model of btk kinase activation by the src family kinase lyn will be tested and the data used to understand how FcepsilonRI clustering initiates the events leading to btk activation and interaction with downstream effectors. 4) How do antigen receptors mobilize calcium? FcepsilonRI is shown to activate a sphingosine kinase (SK) pathway for calcium mobilization. Experiments are proposed to analyze how FcepsilonRI is linked to SK activation. The answers to these questions will provide a molecular dissection of FcepsilonRI function which will be relevant to the understanding of other multi-subunit antigen receptors.
