Assessing the role of amino acid residues in the transmembrane domains of the α- and γ-chains of the high-affinity receptor complex for immunoglobulin E in signal transduction

The dramatic rise in the incidence of allergic/asthmatic disorders in the past three decades has placed a major socio-economic burden on global health care. Exocytosis of mediators causing allergic responses follows the binding, and subsequent receptor cross-linking by cognate allergen of immunoglobulin (lg) E antibodies to Fe-receptors (FcERI), expressed predominantly on mast cells and basophils. Fe-receptors have an invariant predominantly hydrophobic ammo acid motif (LFAVDTGL) in their transmembrane (TM) domain but contain a hydrophilic aspartic acid residue (D194). The function of this potentially energetically unstable residue in the TM of the human (hu) Fci,Rla subunit was targeted by transfecting the Rat Basophilic Cell line (RBL-2H3.1) with cDNA constructs encoding the gene for native and mutant huFcERia subunits and assessing receptor expression and signalling events. RBL-2H3 transfected with cDNA constructs encoding a medium-sized polar residue hufci,Rla (D194T) demonstrated the formation of a functional rat/human chimeric receptor complex which, when activated via hulgE and antigen, supports mediator release, intracellular calcium mobilisation and tyrosine phosphorylation of ychain and Syk kinase. Transfection with mutant huFci,Rla subunit cDNA constructs encoding non-polar Ile (D1941) and V (D194V), larger sized polar Arg (D194R), smaller sized polar Ser (D194S) and non-polar Ala (D194A) abrogated surface expression of huFcERia and degranulation. An established RBL y-chain deficient cell line served as a model for assessing a- and y-chains interactions through the effect of mutations introduced into the y-chain. Compared to parental RBL-2H3 cells, the RBL y-chain deficient cells supported reduced levels of FcERI expression and mediator release. Transfection of RBL y-chain deficient cells with wild-type and mutant (T22A and T22S) y-chain cDNA constructs restored FctRI expression to levels observed in the parental cell line, but only partially restored mediator release, indicating a defective secretory response. Collectively this study identifies D194 in FctRia as a potential target for developing future anti-allergic drugs that act by inhibiting the FctRI signalling cascade.