Signal transduction by the high affinity receptor for immunoglobulin E, FcepsilonRI, in the mast cell line RBL-2H3

Antigenic crosslinking of the FcϵRl on mast cells results in the expression of inflammatory function. A complex mixture of cytokines, chemokines and allergic mediators is released into the context of an inflammatory site. The interplay between these effector molecules is poorly understood, as are the signalling pathways which control their generation. The experiments described in this thesis derived from two proposed ideas. First, that the FcϵR1 may regulate adapter molecule cassettes analogous to those which link other antigen receptors to Ras family GTPases and second, that FcϵR1 regulation of Ras family GTPases may control aspects of mast cell function. The FcϵR1 regulates components of protein complexes formed around the adapter molecule Grb2. A 33kDa tyrosine phosphoprotein is postulated to be the major adapter between the Grb2 SH2 domain and the FcϵR1 ITAMs. The SH3 domains of Grb2 adapt to multiple candidate effector proteins. These include the SLP-76 protein, a newly recognised adapter molecule and the Sos protein, a guanine nucleotide exchange factor for Ras. Protein complexes nucleated around the Grb2 molecule may therefore link the FcϵR1 to the regulation of Ras family GTPases. The Ras GTPase regulates distinct nuclear targets of FcϵR1 signals by different effector signalling pathways. FcϵR1 regulation of transcription factors is required for induction of multiple genes observed after antigenic crosslinking of the FcϵR1 on mast cells. Ras signals are necessary and sufficient for FcϵR1 regulation of Elk-1, a transcription factor important in immediate early gene induction. Ras signals to Elk-1 via the Raf-l/MEK/ERK pathway; dominant inhibition of this effector cascade ablates FcϵR1 stimulation of Elk-1 transactivation. Ras is also involved in FcϵR1 regulation of the Nuclear Factor of Activated T cells (NFAT) transcription factor. NFAT regulates the promoters of multiple cytokines, growth factors and other immune response genes in a co-operative transcriptional complex with a dimer of AP-1 proteins. FcϵR1 induction of Ras is necessary but not sufficient for activation of an NFAT/AP-1 reporter derived from the murine IL-4 promoter and does not involve the Raf-l/MEK/ERK cascade. Dominant inhibition by Ras of NFAT/AP-1 transcriptional activity can be rescued by constitutive activation of Rac-1. These data suggest that regulation of a Rac-1 signalling pathway may be the effector mechanism through which Ras regulates NFAT/AP-1 transcriptional activity. Rac-1 activity is absolutely required for FcϵR1 regulation of NFAT. Rac-1 regulates the subcellular localisation of NFAT protein in RBL2H3. Dominant inhibition of Rac-1, but not Ras, prevents FcϵR1 regulated dephosphorylation and nuclear import of NFAT, providing a mechanism for Rac-1 involvement in regulation of NFAT transcriptional activity. This suggests that both Ras dependent and independent Rac-1 signalling pathways contribute to FcϵR1 induction of the NFAT/AP-1 transcriptional complex. These are 1) A Ras/Rac-1 pathway postulated to regulate the AP-1 dimer component of the NFAT/AP-1 transcriptional complex and 2) A Ras independent requirement for Rac-1 in FcϵR1 regulation of NFAT phosphorylation status and hence subcellular localisatio