RESUMO
The high-affinity IgE receptor (FcÉRI) is formed by the IgE-binding α subunit, ß subunit and γ subunits homodimer. All three subunits are required for proper expression of the receptor on the plasma membrane of mast cells and basophils. However, the exact molecular mechanism of inter-subunit interactions required for correct expression and function of the FcÉRI complex remains to be identified. A recent study suggested that polar aspartate at position 194 within the transmembrane domain of the α subunit could interact by hydrogen bonding with polar threonine at position 22 in the transmembrane domains of the γ subunits. To verify this, we used previously isolated rat basophilic leukemia (RBL)-2H3 variant cells deficient in the expression of the FcÉRI-γ subunit (FcR-γ), and transfected them with DNA vectors coding for FcR-γ of the wild-type or mutants in which T22 was substituted for nonpolar alanine (T22A mutant) or polar serine (T22S mutant). Analysis of the transfectants showed that both T22A and T22S mutants were capable to restore surface expression of the FcÉRI similar to wild-type FcR-γ. Furthermore, cells transfected with wild-type, T22A or T22S FcR-γ showed comparably enhanced FcÉRI-mediated degranulation. Our data indicate that substitution of FcR-γ T22 with non-polar amino acid does not interfere with surface expression of the FcÉRI and its signaling capacity.
Assuntos
Basófilos/imunologia , Receptores de IgE/química , Receptores de IgE/metabolismo , Alanina/química , Substituição de Aminoácidos , Animais , Degranulação Celular/imunologia , Linhagem Celular Tumoral , Ligação de Hidrogênio , Leucemia Basofílica Aguda/genética , Leucemia Basofílica Aguda/imunologia , Leucemia Basofílica Aguda/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas , Ratos , Receptores de IgE/deficiência , Receptores de IgE/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Transdução de Sinais , Treonina/química , TransfecçãoRESUMO
The high-affinity receptor complex for IgE plays a pivotal role in allergic responses since cross-linking of the high-affinity IgE receptor (FcÉRI) 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 FcÉRI-mediated receptor signaling was assessed by site-directed mutagenesis. Rat basophilic leukemia cells (RBL-2H3) transfected with the human IgE binding subunit (FcÉRIα) 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 huFcÉRIα 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.
Assuntos
Ácido Aspártico/química , Receptores de IgE/química , Transdução de Sinais/imunologia , Animais , Ácido Aspártico/imunologia , Western Blotting , Linhagem Celular , Separação Celular , Citometria de Fluxo , Humanos , Imunoprecipitação , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Ratos , Receptores de IgE/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Allergen-mediated cross-linking of the high-affinity receptor for IgE on mast cells triggers the release of diverse preformed and de novo synthesized immunoregulatory mediators that further the allergic response. A proteomic screen applied to the detection of proteins secreted by the model rat mast cell line, RBL-2H3 (rat basophilic leukaemia, subline 2H3.1), led to the identification of the cholesterol-binding glycoprotein, NPC2/RE1 (Niemann-Pick Type C2/epididymal secretory protein 1). Glycosylated NPC2 is secreted early in response to an IgE-mediated stimulus and co-localizes with the lysosomal membrane marker, CD63. NPC2 belongs to the ML (MD-2-related lipid-recognition) protein family (155 members), which includes the Toll-like receptor co-factors, MD-1 and MD-2, and perhaps most interestingly, seven major house dust mite allergens of unknown function (including Der p 2 and Der f 2). Possible role(s) for the protein in the allergic response and future applications of this approach are discussed.
Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas/metabolismo , Mastócitos/metabolismo , Proteômica , Receptores de IgE/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/análise , Antígenos CD/metabolismo , Antígenos de Dermatophagoides/imunologia , Antígenos de Dermatophagoides/metabolismo , Linhagem Celular , Reagentes de Ligações Cruzadas/química , Proteínas Secretadas pelo Epidídimo/metabolismo , Glicosilação , Peptídeos e Proteínas de Sinalização Intracelular , Mastócitos/imunologia , Dados de Sequência Molecular , Glicoproteínas da Membrana de Plaquetas/análise , Glicoproteínas da Membrana de Plaquetas/metabolismo , Ratos , Tetraspanina 30RESUMO
A comparison of IgE recognition by cognate receptors expressed on the C2 canine mastocytoma cell line with analogous events in a rat basophilic leukemia cell line transfected with the alpha-chain subunit of the canine high-affinity IgE receptor using flow cytometry show that canine IgE recognizes the alpha-chain of its cognate receptor on both cell lines. Our study confirms the expression of functional IgE receptors in both cell lines, but receptor-mediated signaling in the C2 line only supports the early stages of downstream signaling as shown by the phosphorylation of the gamma-chain and the failure to effect the phosphorylation of Syk. In contrast RBL-2H3 cells respond to sensitization with IgE and challenge with cognate antigen with tyrosine phosphorylation of the gamma-subunits of the receptor complex followed by downstream phosphorylation of Syk and Ca(2+) mobilization, culminating in beta-hexosaminidase release. We propose that the identification of the precise signaling defect in C2 cells will yield useful information regarding the pathway leading to mast cell exocytosis and facilitate the restoration of the complete signaling cascade through complementation of the missing/defective signal transducer since signaling events downstream of Ca(2+) mobilization are intact as demonstrated by beta-hexosaminidase release following non-immunologic stimulation with the calcium ionophore, A23187.