This is a continuation application to study the molecular regulation, structure and function of the low affinity IgE receptor (Fc-epsilon-RII), using the mouse system. The overall aim is to gain further insight into the roles this molecule plays in immediate hypersensitivity and in B-cell activation and differentiation.
In aim number 1 the molecular mechanism of IL-4 induced Fc-epsilon-RII up-regulation will be determined. The promoter region of the murine Fc-epsilon-RII gene has been tentatively identified using CAT reporter plasmids. This will be further defined with additional deletion studies and the region that responds to IL-4 will be determined. The mechanism will then be further explored by analysis of DNA-binding proteins that may interact with the IL-4 responsive element. Finally, the role that mRNA stabilization plays in the IL-4- induced Fc-epsilon-RII regulation will also be determined.
In aim number 2, the Fc-epsilon-RII structure necessary for IgE binding will be determined. Current results indicate that the Fc- epsilon-RII forms a trimeric structure analogous to other """"""""coiled-coil"""""""" molecules such as tropomyosin. The region responsible for this receptor- receptor interaction has been identified; it is termed the """"""""stalk"""""""" region of the Fc-epsilon-RII. Additional deletion mutant Fc-epsilon-RII as well as chimeric Fc-epsilon-RII in which the lectin homologous region is attached to the other C-type family """"""""stalk"""""""" will be prepared and analyzed for IgE- binding and oligomer formation. A variety of new biologic activities for the sFc-epsilon-RII have been described in humans.
In aim number 3 the soluble Fc-epsilon-RII constructs prepared in aim number 2 will be analyzed for biologic activity. The sFc- epsilon-RII constructs will be tested for activity against mast cells with respect to mediator and cytokine release and B-cells with respect to costimulation, IgE production and inhibition of a proptosis. These studies will also allow the determination of whether a ligand other than IgE exists for the Fc-epsilon-RII. Two biologic activities for the intact Fc-epsilon-RII have been identified in the current grant period--enhancement of antigen presentation and modification of anti-Ig mediated B-cell activation. Using the mutant Fc-epsilon-RII molecules produced in aim number 2, the correlation between capacity for oligomer formation and biologic activity will be determined.In addition, these mutants, as well as a mutant Fc-epsilon-RII lacking a cytoplasmic tail will be examined for the capacity to modulate internalization and cytoskeletal association. Finally, as these findings indicate new roles for IgE complexes, the effect of in vivo targeting the Fc-epsilon-RII on TH phenotype will be assayed.
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