Abstract
The high affinity receptor for IgE, Fc epsilon receptor I (FcepsilonRI), is an activating immune receptor and key regulator of allergy. Antigen-mediated cross-linking of IgE-loaded FcepsilonRI alpha-chains induces cell activation via immunoreceptor tyrosine-based activation motifs in associated signaling subunits, such as FcepsilonRI gamma-chains. Here we show that the human FcepsilonRI alpha-chain can efficiently reach the cell surface by itself as an IgE-binding receptor in the absence of associated signaling subunits when the endogenous signal peptide is swapped for that of murine major histocompatibility complex class-I H2-K(b). This single-chain isoform of FcepsilonRI exited the endoplasmic reticulum (ER), trafficked to the Golgi and, subsequently, trafficked to the cell surface. Mutational analysis showed that the signal peptide regulates surface expression in concert with other described ER retention signals of FcepsilonRI-alpha. Once the FcepsilonRI alpha-chain reached the cell surface by itself, it formed a ligand-binding receptor that stabilized upon IgE contact. Independently of the FcepsilonRI gamma-chain, this single-chain FcepsilonRI was internalized after receptor cross-linking and trafficked into a LAMP-1-positive lysosomal compartment like multimeric FcepsilonRI. These data suggest that the single-chain isoform is capable of shuttling IgE-antigen complexes into antigen loading compartments, which plays an important physiologic role in the initiation of immune responses toward allergens. We propose that, in addition to cytosolic and transmembrane ER retention signals, the FcepsilonRI alpha-chain signal peptide contains a negative regulatory signal that prevents expression of an immunoreceptor tyrosine-based activation motif-free IgE receptor pool, which would fail to induce cell activation.
Highlights
Human Fc⑀RI ␣-chain with endogenous signal peptide Human Fc⑀RI ␣-chain with signal peptide of murine major histocompatibility complex class I H2-Kb Truncation mutant of endo-␣ lacking the cytosolic tail of the ␣-chain Truncation mutant of Kb-␣ lacking the cytosolic tail of the ␣-chain Transmembrane mutant of Kb-␣; the negatively charged aspartic acid in the transmembrane region was exchanged to the neutral asparagine
Transfection with an Fc⑀RI-␣ construct lacking a signal peptide resulted in the expression of a 30 –34-kDa protein corresponding to the unglycosylated protein backbone (Fig. 1B, fourth lane). These results suggested that the signal peptide is involved in the control of ER exit and the amount of Fc⑀RI ␣-chain that traffics to the Golgi in the absence of Fc⑀RI ␥-chains
Fc⑀RI-␣ was substantially higher using constructs with the H2-Kb signal peptide (Fig. 2E), it is fair to conclude that these findings argue for a strong retention of properly folded Fc⑀RI ␣-chain in the ER by its endogenous signal peptide and imply that this signal peptide is a control module to prevent surface display of a single-chain isoform of
Summary
Antibodies—Anti-human Fc⑀RI-␣ monoclonal antibodies (mAb) 19-1 and 15-1 and polyclonal rabbit anti-␣-chain serum 997 were kindly provided by Dr J.-P. Anti-Fc⑀RI-␥ polyclonal serum was purchased from Millipore, and peroxidase-conjugated anti-HA (3F10) was from Roche Applied Science. To visualize Golgi trafficking of Fc⑀RI-␣, MelJuso cells were allowed to attach to coverslips and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 20 min at room temperature prior to permeabilization in a 0.5% saponin, 3% bovine serum albumin, phosphatebuffered saline solution. The Fc⑀RI ␣-chain was detected with the polyclonal rabbit serum 997 and anti-rabbit Alexa Fluor 568 (left images, shown in red). For Fc⑀RI ␣-chain internalization, HeLa cells transiently transfected with Kb-␣ were grown on coverslips (No 1.5) stained first with purified mouse anti-Fc⑀RI-␣ antibody CRAI for 20 min at 37 °C and subsequently with an goat anti-mouse F(abЈ) fragment labeled with Alexa Fluor 568 for 30 min at 37 °C to induce receptor cross-linking. Statistical analysis was performed using the paired, two-tailed Student t test for comparison of two groups; p values of less than 0.05 were considered significant
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