Model studies of transmembrane interaction of FcEpsilonRIAlpha/FcRGamma reveal novel strategies to inhibit allergic responses

The high-affinity IgE receptor complex plays an essential part in allergic responses and involved in downstream signaling, released inflammatory mediators that cause allergic responses. The transmembrane region of the high-affinity IgE has a conserved motif [LFAVDTGL] where a polar aspartate [D194] is important for the ligand binding. This modeling study proposes novel potential binding sites between high affinity immunoglobulin E receptor Alpha subunit [FcEpsilonRIAlpha] and FcRGamma and as a consequence, we propose a new model of FcEpsilonRIAlpha and FcRGamma interaction [T194] which can mediate downstream signaling in allergic response. The docking of FcRGamma with wild-type [D194] and mutant human high affinity immunoglobulin E receptor Alpha subunit [D194T, D194I, D194L, D194A, D194V, D194E, D194S and D194R] has been performed on Autodock Vina. This modeling study is based on lab data obtained by carrying out sitedirected mutagenesis done at residue D194 of FcEpsilonRIAlpha to assess its functional importance for the mediation of intracellular signal cascade. HuFcEpsilonRIAlpha D194 residue was replaced with threonine, leucine, serine, arginine, alanine, asparagine and glutamic acid. FcRGamma docking on mutated huFcEpsilonRIAlpha [D194T] indicated a new site of interaction and emphasizes the significance of the charge and size of an amino acid at position 194 in huFcEpsilonRIAlpha subunit. Amino acids D and T at position 194 are important for cell surface localization, interactions, distribution and downstream signaling of IgE receptor subunit. These proposed models may herald in better therapeutic interventions to combat unfavorably allergic diseases

High affinity IgE-Fc receptor alpha and gamma? subunit interactions

To explore the relationships between the subunits [alpha, beta and gamma] of the high affinity IgE receptor [Fc epsilon RI] and its ability to mediate transmembrane signaling. Experimental study. Department of Molecular Biology and Biotechnology, University of Sheffield, UK, from 2008 to 2009. The approach employed was to create a chimera [human alpha gamma] using the extracellular [EC] domain of the human high affinity IgE receptor. The alpha subunit [huFc epsilon RI alpha] of IgE receptor was spliced onto the rodent gamma TM and cytoplasmic domain [CD]. This was transfected into the Rat Basophilic Leukemia cell line in order to assess the possibility of selectively activating cells transfected with this single pass construct for antigen induced mediator release. The RBLs cell lines transfected with the huFc epsilon RI alpha/gamma/gamma cDNA constructs were assessed for the cell surface expression of the huFc epsilon RI alpha subunit and the response to the antigenic stimulus by looking for degranulation and intracellular Ca[2+] mobilisation. The results obtained showed the absence of huFc epsilon RI alpha subunit expression on the surface of transfected cells as seen by flow cytometric studies, beta-hexosaminidase assays and intracellular calcium mobilisation studies. In the present study the grounds for non-expression of huFc epsilon RI alpha/gamma/gamma cDNA remains elusive but may be due to the fact that the human-rodent chimeric receptors are assembled differently than the endogenous rodent receptors as seen in study in which COS 7 cells were transfected with human/rat chimeric complexes

Protein Profiling of the secretome of Fc sigma RI Activated RBL-2H3.1 cells

Secretory proteins of IgE receptor activated mast cells and basophils play a pivotal role in the generation of immediate and long term immune responses in allergy and type I hypersensitivity. The present study aims to generate a 2-D map and profile of proteins secreted from a high secretory variant of the rat basophilic leukemia cell line, RBL-2H3.1, which in view of the difficulty associated with gaining adequate numbers of pure primary mast cell and basophiles, represents an accepted model system for the study and standardization of the methodology to characterize the secretome of these cell types. A 2-D map of secretory proteins was generated by 2-D PAGE and a shotgun mass spectrometric approach carried out for protein identification. Study resulted into identification of 299 proteins released from resting and IgE receptor activated RBL-2H3.1 cells after 90 s, 30 min and 3 h antigen challenge. Further sequence analysis identified 53% of total proteins as secretory proteins which could be attributed to classical and non-classical secretory pathways. Additionally, functional classification of classic secretory proteins verified the presence of proteins belonged to cytokines, receptors, membrane proteins, lysosomal proteins and proteins associated with specific sub-cellular localizations such as endoplasmic reticulum, mitochondria, nucleus, cytoplasm and ribosome. According to this data the presence of some secretory proteins such as cytokines [e.g. MCP-2, PF-4, CSF-1 and TGF-beta1] are all subject to Ag challenge which may point to their importance toward pathogenesis in allergic diseases. In view of both a beneficial and adverse role of mast cell mediators in health and disease, an identification of temporal changes in the secretory pattern may form the basis for future tailor made intervention strategies that may enable us to harvest the therapeutic potential inherent in mast cell exocytosis while inhibiting/attenuating negative outcomes