It is well established that the initiating requirement of the allergic response involves the interaction of IgE with its high affinity receptor (FceRI) expressed on hematopoietic cells. Efforts to modulate this interaction through manipulation of the expression levels of either IgE or cell surface FcERI would offer the potential to intervene in the pathogenesis of the allergic response. The intracellular assembly and transport of FcERI to the plasma membrane is a complex process that has thus far been largely unexplored. Definition of critical steps in the assembly and transport pathway could open new possibilities in blocking FcERI expression with the broader aim of attenuating the allergic response. We will initially explore the assembly and transport characteristics of the human FceRI alphagamma2 receptor. In addition to association of the FceRI gamma-chain, two other critical events occur during FceRI ct-chain biosynthesis that profoundly influence the formation of a transport competent ag2 receptor: (i) glycosidase-mediated processing of a-chain core oligosaccharides in the ER and; (ii) the association of specific ER chaperones, such as calnexin, with the nascent FceRI a-chain. We will focus on the role of calnexin in promoting ag2 assembly and cell surface expression which, based on preliminary results, appears to exert a profound effect on the level of cell surface expression. These studies will then be extended to expression of transfected tetrameric FceRI abg2 receptor, as well as constitutive receptor in eosinophils and basophils. FceRI abg2 may exhibit an intrinsically higher expression phenotype than the ag2 receptor, possibly derived from improved, beta-chain-dependent intracellular assembly, stability and transport properties mediated by specific ER chaperones. Initial studies have revealed that calnexin associates with the FceRI a subunit, an association predicted to occur exclusively via the N-linked glycans found in the ct-chain ectodomain. A further goal of this proposal is compare and defined the role of each of the 7 N-linked glycosylation sites in association with calnexin and other ER chaperone and their effect on cell surface expression. A final objective in this program is the further analysis of intracellular transport characteristics of a truncated FceRI a-chain, lacking only the cytoplasmic domain sequence, which expresses at a high level on the surface of transfected cells in the absence of the y-chain. Thus we will, for the first time, determine g-chain independent a-chain-specific transport and ER quality control characteristics.
