Assessing the role of Asp 194 in the transmembrane domains of the α-chain of the high-affinity receptor complex for immunoglobulin E in signal transduction.
The high-affinity receptor complex for IgE plays a pivotal role in allergic responses since cross-linking of the high-affinity IgE receptor (FcɛRI) on target cells initiates a signaling cascade facilitating release of inflammatory mediators causing allergic responses. The transmembrane regions of the ligand binding domains of the high-affinity IgE and low-affinity IgG receptors share an invariant motif (LFAVDTGL) containing a polar aspartate within a predominantly non-polar setting. The functional importance of this aspartate residue (D194) in FcɛRI-mediated receptor signaling was assessed by site-directed mutagenesis. Rat basophilic leukemia cells (RBL-2H3) transfected with the human IgE binding subunit (FcɛRIα) incorporating polar substitutions like asparagine (D194N) or threonine (D194T) resulted in the formation of a functional rat/human chimeric receptor complex. When activated via huIgE and antigen, cells transfected with these variant receptor subunits supported mediator release, intracellular calcium mobilisation and tyrosine phosphorylation of γ-chain and Syk kinase while a non-polar substitution (D194L) gave rise to cell surface expression of the mutated receptor subunit but failed to initiate downstream signaling. No cell surface expression of huFcɛRIα gene constructs was observed when D194 was replaced with the non-polar Ile (D194I) residue of similar size, the larger positively charged Arg (D194R) or lysine (D194K) residues, or the negatively charged glutamate (D194E) and smaller polar Ser (D194S) non-polar Ala (D194A) and V (D194V). These observations highlight importance of the size and charge of amino acid residue at position 194 in determining IgE receptor subunit interactions, cell surface localization, and initiation of downstream signaling events.
Rashid
Amir
A
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom. dramiramc@yahoo.com
Iodice
Marco W
MW
Carroll
Kathleen M
KM
Housden
Jonathan E M
JE
Hunter
Michael
M
Sabban
Sari
S
Artymiuk
Peter J
PJ
Helm
Birgit A
BA
D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry* / Q000276:immunology; D015153:Blotting, Western; D002460:Cell Line; D002469:Cell Separation; D005434:Flow Cytometry; D006801:Humans; D047468:Immunoprecipitation; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D051381:Rats; D017455:Receptors, IgE / Q000737:chemistry* / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000276:immunology*; D014162:Transfection
D017455:Receptors, IgE; D001224:Aspartic Acid