The Signal Peptide of the IgE Receptor α-Chain Prevents Surface Expression of an Immunoreceptor Tyrosine-based Activation Motif-free Receptor Pool*

The high affinity receptor for IgE, Fc epsilon receptor I (FcϵRI), is an activating immune receptor and key regulator of allergy. Antigen-mediated cross-linking of IgE-loaded FcϵRI α-chains induces cell activation via immunoreceptor tyrosine-based activation motifs in associated signaling subunits, such as FcϵRI γ-chains. Here we show that the human FcϵRI α-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-Kb. This single-chain isoform of FcϵRI 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 FcϵRI-α. Once the FcϵRI α-chain reached the cell surface by itself, it formed a ligand-binding receptor that stabilized upon IgE contact. Independently of the FcϵRI γ-chain, this single-chain FcϵRI was internalized after receptor cross-linking and trafficked into a LAMP-1-positive lysosomal compartment like multimeric FcϵRI. 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 FcϵRI α-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.

Fc⑀RI is part of the family of multimeric immune recognition receptors, also referred to as activating immune receptor complexes (4 -6). IgE-allergen-mediated cross-linking of the Fc⑀RI ␣-chain induces the release of inflammatory mediators via ITAMs of the associated signaling subunits, Fc⑀RI-␤ and a dimer of Fc⑀RI-␥ chains (7,8). Fc⑀RI ␣-chain transport from the ER to the cell surface is a tightly regulated trafficking process because susceptibility to IgE-mediated cell activation depends on the display of IgE-binding epitopes by Fc⑀RI-␣ (3,9,10).
The ER quality control system monitors correct folding as well as co-and post-translational modifications of proteins and protein complexes (11)(12)(13). Several regulatory mechanisms that modulate the ER exit of Fc⑀RI complexes have been described. All of the ER protein quality control steps for type I membrane proteins apply to the Fc⑀RI ␣-chain (14). Synergistic ER retrieval signals are described for Fc⑀RI-␣: two dilysine motifs, Lys 212 -Lys 216 and Lys 226 -Lys 230 , in the cytosolic tail and the charged transmembrane amino acid Asp 192 (15,16). In human cells, these ER retrieval signals are overcome by the assembly of Fc⑀RI-␣ with Fc⑀RI-␥, the common Fc receptor ␥-chain (3). In contrast, murine Fc⑀RI-␣ requires assembly with both Fc⑀RI-␥ and Fc⑀RI-␤ to reach the cell surface. Hartman et al. (17) suggested recently that the difference in ER exit requirements between human and murine Fc⑀RI-␣ is encoded entirely in the extracellular domain of the protein. Furthermore, N-linked glycosylation of the IgE-binding epitopes of Fc⑀RI-␣ has been described as a checkpoint of the ER quality control system (18). Interestingly, the formation of IgE-binding epitopes depends only on proper core glycosylation in the ER and can occur completely independent of other receptor subunits (18).
Another key control step for the formation of Fc⑀RI complexes is the requirement for cotranslational assembly of the Fc⑀RI ␣-chain with its signaling subunits (19). This is different from assembly mechanisms defined for other activating immune receptors, such as the T or B cell receptors, that do not depend on coordinated translation of their subunits for receptor complex formation (4,20). After the removal of all known transmembrane and cytosolic retention signals in Fc⑀RI-␣, a substantial amount of the protein still remains intracellular (15,18,21). These findings suggest that an as yet undefined sequence element prevents surface expression of the Fc⑀RI ␣-chain in the absence of Fc⑀RI-␥. Therefore, we revisited the regulatory mechanisms for the display of Fc⑀RI-␣ at the cell surface.
Type I membrane proteins, including Fc⑀RI-␣, contain a short cleavable N-terminal sequence called the signal peptide. The signal peptide sequence assures proper translocation into the ER; it binds the signal recognition particle and later is cleaved on the ER lumenal side by signal peptidases. Signal peptides have no bona fide consensus sequences, although they commonly contain a highly hydrophobic stretch (typically 10 -15 amino acids) long that is preceded by a basic residue and followed by a cleavage site for the signal peptidase. A growing body of evidence suggests that signal sequences are actively involved in the quality control of type I proteins (22,23). We thus hypothesized that the signal peptide of Fc⑀RI-␣ provides a control element for ER exit and surface trafficking. Such a control module could operate in two ways: it could facilitate the cotranslational assembly of the receptor subunits, and it could prevent surface expression of a single ␣-chain receptor isoform without signaling subunits. Here we show that the endogenous signal peptide of Fc⑀RI-␣ does indeed contain a regulatory element that controls ER exit and consecutively cell surface expression of this protein.

EXPERIMENTAL PROCEDURES
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. Kinet (Laboratory of Allergy and Immunology, Beth Israel Deaconess Medical Center, Boston, MA) and used as published (24,25). The mAb 19-1 reacts only with Fc⑀RI-␣ chain that expresses the IgE binding epitopes (ER and Golgi modified forms). IgE (Serotec) and antibody 15-1 recognize the IgE-binding epitope and were used for detection of the properly folded and N-glycosylated form of the ␣-chain. Phycoerythrin-conjugated or allophycocyanin-conjugated anti-human Fc⑀RI-␣ antibody CRA1 and the appropriate mIgG2b isotype controls were purchased from eBioscience. Anti-Fc⑀RI-␥ polyclonal serum was purchased from Millipore, and peroxidase-conjugated anti-HA (3F10) was from Roche Applied Science. Anti-eGFP serum was a kind gift from Dr. H. Ploegh (Whitehead Institute, Cambridge, MA). Mouse anti-human GM130 antibody (BD Biosciences) was used to visualize the Golgi compartment.
Fc⑀RI ␣-Chain Constructs-cDNAs encoding the Fc⑀RI-␣and ␥-chains have been previously published (19). The first 25 amino acids of the endogenous ␣-chain (endo-␣) contain the predicted signal peptide of this protein and were replaced with the first 22 amino acids of the H2-K b protein using a two-step PCR cloning strategy according to the literature (26), and this construct was termed K b -␣. Cytosolic tail truncations of endo-␣ as well as K b -␣ were generated by introduction of a stop codon following the transmembrane region at position 199 using PCR cloning. A construct that lacks the signal peptide was generated with an N-terminal primer starting at position 26 of Fc⑀RI-␣. HA tags were introduced by using a C-terminal primer that contained the tag sequences prior to the stop codon. Signal peptide point mutants at position 6 of endo-␣, endo-␣ E/K , and endo-␣ E/A were generated with the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's guidelines. All of the constructs were subcloned into pcDNA3.1 and pIRES2-eGFP expression vectors and verified by sequencing.
Cell Lines and Transient Transfections-293T, HeLa, and MelJuso cells were maintained in Dulbecco's modified Eagle's medium as previously described (19). Lipofectamine 2000 (Invitrogen) and FuGENE 6 (Roche Applied Science) were used to transiently transfect cells following the manufacturer's protocols. The cells were trypsinized and harvested 48 h post-transfection.
Immunoprecipitation and Immunoblotting-The cells were solubilized in lysis buffer (0.5% Brij 96, 20 mM Tris, pH 8.2, 20 mM NaCl, 2 mM EDTA, 0.1% NaN 3 ) containing protease inhibitors (Complete; Roche Applied Science) for 30 min on ice. Immunoprecipitation was performed with IgE specific for the hapten NP (Serotec) and NIP beads (Sigma) as previously described (27). The proteins were eluted from beads in nonreducing Laemmli sample buffer, and samples were run on 12% nonreducing SDS-PAGE gels, transferred to polyvinylidene difluoride membrane (Pierce) and probed with the anti-Fc⑀RI-␣ antibody 19-1 followed by peroxidase-conjugated goat anti-mouse IgG for detection of precipitated ␣-chain. HA-tagged proteins were detected with peroxidase coupled 3F10 (Roche Applied Science). Fc⑀RI-␥ was detected with polyclonal anti-Fc⑀RI-␥ serum followed by peroxidase-conjugated goat anti-rabbit IgG. Peroxidase activity was detected using SuperSignal chemiluminescent substrate reagents (Pierce). Endoglycosidase H (EndoH) and PNGase F (Roche Applied Science) digestions were performed according to manufacturer's instructions.
FACS Analysis and Cell Sorting-The cells were stained with either phycoerythrin-conjugated or allophycocyanin-conjugated anti-Fc⑀RI-␣ antibody CRA1 or appropriate isotype control antibodies and analyzed on a FACScan flow cytometer (Becton Dickinson) using CellQuest software for acquisition and analysis. For staining of intracellular Fc⑀RI-␣, the cells were fixed and permeabilized using Fix & Perm reagents (CALTAG Laboratories; Invitrogen) prior to staining. Cell sorting was performed on a Moflow cell sorter (DAKO).
Immunofluorescence Microscopy-Immunofluorescence experiments were performed essentially as previously described (28) with minor modifications. To visualize Golgi trafficking of Fc⑀RI-␣, MelJuso cells were allowed to attach to coverslips and then 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. Polyclonal rabbit serum 997 was used to detect ␣-chains. An antibody against GM130 was used to visualize the Golgi compartment. Goat anti-mouse F(abЈ) 2 fragment labeled with Alexa Fluor 488 (Molecular Probes) and goat anti-rabbit Alexa F(abЈ) 2 fragment labeled with Fluor 568 (Molecular Probes) were used as the fluorescent probes. The images were collected on a Zeiss Axiophot upright microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY) with a Spot scientific grade cooled CCD camera (Diagnostic Instruments, Inc., Sterling Heights, MI).
For Fc⑀RI ␣-chain internalization, HeLa cells transiently transfected with K b -␣ 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) 2 fragment labeled with Alexa Fluor 568 for 30 min at 37°C to induce receptor cross-linking. The cells were fixed with 4% paraformaldehyde for 20 min and mounted using Prolong Antifade reagent (Invitrogen). Both antibodies were diluted in Hanks' balanced salt solution supplemented with 10 mM HEPES (Invitrogen) and 5% NuSerum (Invitrogen). CRA I was diluted at 1:100, and anti-mouse Alexa Fluor 568 was diluted at 1:400. Plasma membranes of fixed cells were stained with Alexa Fluor 647-conjugated wheat germ agglutinin (diluted at 1:1000) for 10 min. All of the incubation steps were carried out in a humidified chamber. Confocal images were acquired on a Nikon TE2000 inverted microscope coupled to a Yokogawa spinning disk confocal unit (PerkinElmer Life Sciences) and an Orca AG scientific grade cooled CCD camera (Hamamatsu Photonics K.K.). Slidebook software (Intelligent Imaging Innovations Inc.) was used for image capture, processing, and analysis. The LAMP-1-eGFP vector, kindly provided by Dr. Tomas Kirchhausen (Immune Disease Institute, Children's Hospital Boston), was transfected into HeLa cells, and stably transduced cells were selected with G418 (1 mg/ml).
Statistical Analysis-All of the graph data represent the means Ϯ S.E. of the indicated number of independent experiments (at least three). 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.

Efficiency of the ER Exit of the Fc⑀RI ␣-Chain Depends on Its
Signal Peptide-We wanted to test the hypothesis that the signal peptide of the Fc⑀RI ␣-chain contains a module to regulate ER exit of this protein in its properly folded IgE-binding form. Therefore, we compared the intracellular trafficking of Fc⑀RI-␣ with its endogenous signal peptide (referred hereafter as endo-␣) with a chimeric Fc⑀RI ␣-chain that had its signal peptide swapped for that of H2-K b (referred hereafter as K b -␣). A comparison of both signal peptides is shown in Fig. 1A. The H2-K b signal peptide is highly efficient at driving expression of the murine major histocompatibility complex class I H2-K b molecule and is thus commonly used to optimize expression of type I membrane proteins in vivo and in vitro (19,20,29,30). A FIGURE 1. Properly folded Fc⑀RI-␣ reaches the Golgi in the absence of Fc⑀RI-␥. A, signal peptide (SP) sequences of Fc⑀RI-␣ (NCBI RefSeq NP_001992.1) and mouse major histocompatibility complex class I H2-K b (NCBI RefSeq NP001001892.2) are depicted. The stretch of hydrophobic amino acids representing the predicted transmembrane region of the signal peptide is underlined. The construct containing the wild type Fc⑀RI-␣ chain with its endogenous signal peptide is referred to as endo-␣, and the chimeric construct where the endogenous signal peptide was swapped for the signal peptide of H2-K b is referred to as K b -␣. B, Fc⑀RI ␣-chain shows a protein pattern characteristic for ER-and Golgi-modified forms in the absence of ␥-chain. 293T cells were transfected with indicated HA-tagged ␣-chain constructs or cotransfected with ␥-chain as control (first lane 1). Smaller quantities of ␣-chain from endo-␣ transfected cells reached the Golgi compared with K b -␣ transfected cells (second and third lanes). A construct without signal peptide (no SP) did not enter the ER (fourth lane). Immunoblot analysis was performed under nonreducing conditions with the anti-HA antibody 3F10 (upper blot). The blot was stripped and reprobed with a polyclonal anti-␥-chain reagent (lower blot). C, glycosylation study performed on the Fc⑀RI ␣-chain after immunoprecipitation with IgE. Fc⑀RI ␣-chain (first, fourth, and seventh lanes) is compared for its susceptibility to EndoH (second, fifth, and eighth lanes) or PNGase F (third, sixth, and ninth lanes) digestion. D, visualization of Fc⑀RI-␥ independent trafficking to the Golgi. Immunofluorescence staining of MelJuso cells transfected with K b -␣. The Fc⑀RI ␣-chain was detected with the polyclonal rabbit serum 997 and anti-rabbit Alexa Fluor 568 (left images, shown in red). The Golgi was visualized with anti-GM130 followed by antimouse Alexa Fluor 488 (middle image, shown in green). The merged images are depicted in the right picture. E, molecular characteristics of ␣-chain from MelJuso cells transfected with K b -␣. Immunoprecipitation was performed with IgE; IgG was used to control for specificity of IgE binding. F, RT-PCR analysis confirmed lack of ␥-chain expression in 239T, HeLa, and MelJuso cells. cDNA from human tonsil tissue was used as positive control (CTRL).
summary of all constructs used in this study is given in Table 1. Because Fc⑀RI-␣ becomes highly glycosylated on its way from the ER to the cell surface, modifications of N-glycans on the Fc⑀RI ␣-chain can be used to monitor proper ER to Golgi trafficking of the protein. 293T cells were transiently transfected with Fc⑀RI endo-␣ or K b -␣ constructs, and the molecular properties of the translation products were compared (Fig. 1, A and  B). All of the constructs were tagged with HA at the C terminus to allow for detection of the Fc⑀RI-␣-chain irrespective of its folding stage. We used the molecular weight characteristics of endo-␣ in the presence of Fc⑀RI-␥ as the published standard for comparison (Fig. 1B, first lane) (18,25,31). Immunoblotting experiments showed that the protein pattern between 40 and 60 kDa obtained with single Fc⑀RI␣-chains (Fig. 1B, second and third lanes) is comparable with the pattern in ␣and ␥-chain cotransfected cells (Fig. 1B, first lane). In the absence of Fc⑀RI-␥, endo-␣ seems to be expressed predominantly near ϳ46 kDa; based on the literature this is most likely the ER glycosylated form of the protein (ER form; Fig. 1B, second lane) (19,24). We detected some unglycosylated protein backbone at ϳ34 kDa, probably because of incomplete insertion into the ER. Interestingly, some higher molecular weight forms, most likely Golgi-modified protein (Golgi form; Fig. 1B, second lane), were also detected (19,24,31). This was not expected from the literature and indicates that a small amount of endo-␣ by itself can exit the ER (Fig. 1B, second lane). Exchange of the endogenous signal peptide with the H2-K b signal peptide (K b -␣) dramatically enhanced the amounts of potentially Golgi-modified protein despite the absence of Fc⑀RI-␥ (Fig. 1B, third lane). 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.
We next confirmed that the observed molecular weight changes of the single-chain Fc⑀RI-␣ resulted from post-translational glycosylation because of receptor trafficking from ER to Golgi and not from polyubiquitination during targeting for proteasomal degradation. Thus, we immunoprecipitated Fc⑀RI ␣-chain with IgE to select for properly folded protein and examined the extent of N-glycosylation by immunoblotting. Endo-␣ in the presence of Fc⑀RI-␥ was used as a control (Fig. 1C, left  panel). The ER form of Fc⑀RI-␣ was sensitive to EndoH digestion as evidenced by a drop in the molecular weight of the 46-kDa ER form to the protein backbone (34 kDa; Fig. 1C, compare first and second lanes). In the presence of Fc⑀RI-␥, a large amount of Fc⑀RI-␣ remained EndoH-resistant because of glycosylation patterns acquired in the Golgi (Fig. 1C, second lane).
Deglycosylation of Fc⑀RI-␣ precipitated from endo-␣ or K b -␣ transfectants with EndoH and PNGase F yielded a comparable protein pattern to that of endo-␣ in the presence of Fc⑀RI-␥ (Fig. 1C, compare left immunoblot with middle and right immunoblots). In line with our observations in whole cell lysates (Fig. 1B), the amount of EndoH-insensitive protein varied depending on the nature of the signal peptide. The Golgi form precipitated from endo-␣ and K b -␣ transfectants remained sensitive to PNGase F (Fig. 1C, third, sixth, and ninth lanes). Therefore, we concluded that the single-chain isoform of Fc⑀RI was properly glycosylated in the Golgi. Sensitivity to PNGase F digestion also excluded that single Fc⑀RI ␣-chain was simply aggregated or ubiquitinated protein and targeted for degradation.
We next visualized the ER exit of the single Fc⑀RI ␣-chain. We transiently transfected MelJuso cells with K b -␣ and detected the ␣-chain with the polyclonal serum 997 (Fig. 1D,  left panel). Immunofluorescence double staining with the Golgi marker GM130 (Fig. 1D, middle panel) showed a significant amount of Fc⑀RI ␣-chain in a Golgi compartment in the absence of Fc⑀RI-␥ (Fig. 1D, overlay, right panel). Immunoprecipitation with IgE confirmed that the ␣-chain was properly modified in the Golgi in these cells (Fig. 1E). We confirmed by RT-PCR and immunoblot that neither 293T, HeLa, nor MelJuso express Fc⑀RI-␥ ( Fig. 1F and data not shown). In summary, this set of data demonstrated that the Fc⑀RI ␣-chain by itself can exit the ER and traffics to the Golgi where N-linked glycans are modified in the absence of Fc⑀RI-␥ transcripts.
Trafficking of Single Fc⑀RI ␣-Chain to the Cell Surface Depends on the Signal Peptide-We next investigated whether Fc⑀RI-␣ can reach the cell surface in the absence of Fc⑀RI-␥. FACS analysis for surface expression of endo-␣ and K b -␣ was performed in three different cell lines ( Fig. 2A). Regardless of the cell line, both ␣-chain constructs reached the cell surface without Fc⑀RI-␥. The extent of surface expression varied with the transfection efficiency as well as the cell line ( Fig. 2A). Importantly, we saw significant differences in surface expression levels depending on the nature of the signal peptide. Based on these observations, we speculated that efficiency of signal peptide processing is involved in the control of Fc⑀RI-␣ ER exit and subsequent transport to the cell surface.
To exclude that the differences in cell surface transport were influenced by different transfection efficiencies of the constructs, we next expressed endo-␣ and K b -␣ in a bicistronic IRES-eGFP vector system. This system allowed us to use eGFP as a surrogate marker for protein expression levels and to set gates for FACS analysis and sorting. We found a strong correlation between eGFP expression levels and surface expression of Fc⑀RI-␣ (Fig. 2B). The cells were analyzed after setting three

Construct name Description
Endo-␣ Human Fc⑀RI ␣-chain with endogenous signal peptide K b -␣ Human Fc⑀RI ␣-chain with signal peptide of murine major histocompatibility complex class I H2-K b Endo-␣ tail-minus Truncation mutant of endo-␣ lacking the cytosolic tail of the ␣-chain K b -␣ tail-minus Truncation mutant of K b -␣ lacking the cytosolic tail of the ␣-chain K b -␣ D/N Transmembrane mutant of K b -␣; the negatively charged aspartic acid in the transmembrane region was exchanged to the neutral asparagine Endo-␣ E/A Signal peptide mutant; the negatively charged glutamic acid at position 6 was exchanged to the neutral alanine Endo-␣ E/K Signal peptide mutant; the negatively charged glutamic acid at position 6 was exchanged to the positively charged lysine different analysis regions: eGFP-negative cells (Fig. 2B, upper  plot), low eGFP-expressing cells (eGPF low ), and high eGFP-expressing cells (eGFP high ). eGFP high cells expressed significantly more Fc⑀RI-␣ at the cell surface than eGPF low cells (i.e. 68% versus 15%, respectively).
To analyze cells with equal transfection efficiencies, we next sorted endo-␣ and K b -␣ transfected cells based on equal levels of eGFP expression and analyzed the protein characteristics of the Fc⑀RI ␣-chain by immunoblotting with the mAb 19-1 (Fig.  2C). This reagent reacts only with the properly folded IgE-bind- 293T cells were transfected with K b -␣ in pIRES2-eGFP. In the upper FACS dot plot eGFP expression is depicted. Three different regions were set corresponding to eGFP negative (R1), eGFP low (R2), and eGFP high (R3) cells. In the lower dot plots, ␣-chain expression at the cell surface was analyzed separately for these three different regions. C, 293T cells were transfected with endo-␣ and K b -␣ constructs in pIRES2-eGFP. Empty vector transfected cells (CTRL) or cells cotransfected with K b -␣ plus ␥-chains were used as controls. The cells expressing equal intensities of eGFP were FACS-sorted, and cell lysates were analyzed for the protein characteristics of Fc⑀RI-␣ by immunoblotting with the mAb 19-1. Detection of eGFP with a polyclonal anti-GFP serum was used as a loading control. D, Fc⑀RI-␣ mRNA of cells that were FACS-sorted for equal levels of eGFP expression was determined by quantitative RT-PCR. E, the H2-K b signal peptide drives surface expression of Fc⑀RI-␣ more efficiently than the endogenous signal peptide. Endo-␣ and K b -␣ transfectants were gated based on equal eGFP expression and analyzed for surface ␣-chain. Empty pIRES2-eGFP vector transfected cells (CTRL) were used as control. The bar diagram represents the means Ϯ S.E. of seven independent experiments.
ing Fc⑀RI ␣-chain (25). We confirmed that K b -␣ transfectants contained significantly higher levels of Golgi-modified protein than endo-␣ transfectants (Fig. 2C). To control for the accuracy of the cell sorting, eGFP levels were determined (Fig. 2C). As an additional control we sorted cells with equal levels of eGFP and showed that those cells also expressed equal levels of Fc⑀RI-␣ mRNA by quantitative RT-PCR (Fig. 2D). We next studied surface expression efficiency of the single Fc⑀RI ␣-chain in the bicistronic expression system. 293T cells were transiently transfected with endo-␣, K b -␣, or the empty pIRES-eGFP vector as a control. Cells expressing equal levels of eGFP were gated and analyzed for cell surface expression of Fc⑀RI-␣ (Fig.  2E). Cells transfected with the endo-␣ construct showed low but significant surface expression levels compared with cells transfected with empty vector (6.0 Ϯ 1.5% versus 1.0 Ϯ 0.2%, respectively; means Ϯ S.E., n ϭ 7). Consistent with the more effective ER-to-Golgi transport (Fig. 2C), K b -␣ transfected cells showed a 3.45-fold increase in expression when compared with endo-␣ transfectants (20.7 Ϯ 2.9% versus 6.0 Ϯ 1.5%, respectively; means Ϯ S.E., n ϭ 7; Fig. 2E).
Because surface expression of Fc⑀RI-␣ was substantially higher using constructs with the H2-K b 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 Fc⑀RI.

The Signal Peptide Controls Surface Expression of Fc⑀RI-␣ in the Absence of Cytosolic ER Retention
Signals-ER retention/retrieval by dilysine motifs in the cytosolic tail of Fc⑀RI-␣ has been proposed as a key regulatory mechanism to prevent ER exit in the absence of the Fc⑀RI-␥ chain (15,16). Thus, we generated cytosolic tail truncations of endo-␣ and K b -␣ to dissect the contributions of the signal peptide from the ER retention signals of the cytosolic tail of the ␣-chain.
Endo-␣ and K b -␣ constructs that lack their cytosolic tails were generated by introducing stop codons following the transmembrane domain of Fc⑀RI-␣ at position serine 199 . These constructs are referred to as endo-␣ tail-minus and K b -␣ tail-minus . The full-length and tail-minus constructs were transiently transfected into 239T cells. Using bicistronic expression of eGFP as a marker, a gate was set for the analysis of cells with comparable transfection efficiencies (Fig. 3A, upper panel). Both the endo-␣ tail-minus and K b -␣ tail-minus reached the cell surface more efficiently than the full-length constructs (Fig. 3, A  and B). Importantly, K b -␣ tail-minus was exported far more efficiently than endo-␣ tail-minus (88 Ϯ 12% versus 37 Ϯ 16%, respectively; mean Ϯ S.D., n ϭ 4; Fig. 3B). Additionally, we sorted transfected cells based on their eGFP expression and performed immunoblot analysis with the mAb 19-1 (Fig. 3C). We detected, again, more Golgi form of Fc⑀RI-␣ in cells transfected with K b -␣ than endo-␣ (Fig. 3C, first and second lanes). Golgi-modified Fc⑀RI ␣-chains were also more abundant in cells trans- fected with K b -␣ tail-minus than endo-␣ tail-minus (Fig. 3C, third  and fourth lanes).
We also investigated whether the modification of the transmembrane ER retention signal Asp 192 (15) would have an effect on surface trafficking of K b -␣ and generated a K b -␣ D/N mutant. The removal of the transmembrane ER retention signal resulted in more efficient surface expression of K b -␣ D/N versus K b -␣ (Fig. 3D) at equal mRNA expression levels (Fig. 3E).
In summary, we showed that a single ␣-chain isoform of Fc⑀RI reached the cell surface alone. The K b -signal peptide chimeras trafficked more efficiently to the cell surface even in the absence of cytosolic ER retention motifs, indicating that the K b -signal peptide does not simply override ER retention. This set of data suggests that the signal peptide provides an additional regulatory element that controls Fc⑀RI ␣-chain trafficking in a concerted way with recently described ER retention signals.
Point Mutations in the Endogenous Signal Peptide Argue against Interference with Signal Particle Recognition as a Regulatory Mechanism for Cell Surface Transport-Signal peptides do not have a well defined consensus sequence; however, one common feature is a positively charged residue preceding the hydrophobic stretch. This N-terminal part of the signal peptide sequence is critical for recognition of nascent proteins by the signal recognition particle to initiate translocation across the ER membrane (32)(33)(34). The endogenous signal peptide of the Fc⑀RI ␣-chain does not contain this positively charged amino acid. Instead, a negatively charged glutamic acid is found in position 6 (Figs. 1A and 4A). The H2-K b signal peptide is devoid of any charged amino acids (Figs. 1A and 4A). To investigate whether the introduction of a positively charged amino acid or removal of the negative charged amino acid in the endogenous signal peptide of the ␣-chain could modulate intracellular trafficking, we generated point mutants where the glutamic acid (E) at position 6 was exchanged to lysine (K) or alanine (A). The generated constructs were termed endo-␣ E/K and endo-␣ E/A . Neither endo-␣ E/K nor endo-␣ E/A was able to reach the cell surface more efficiently than endo-␣ (Fig. 4B). We also compared the ER to Golgi transport and found no significant difference between endo-␣ and the mutants (Fig. 4C and data not shown). Thus, mutating the endogenous Fc⑀RI signal peptide to match the signal peptide consensus sequence or to resemble that of H2-K b did not affect trafficking. These results argue for signal peptide cleavage rather than ER insertion as the mechanism that regulates ER exit of the Fc⑀RI ␣-chain in the absence of the Fc⑀RI ␥-chains.
Single Fc⑀RI ␣-Chain Is Stabilized upon Monovalent Interaction with IgE-Multimeric isoforms of Fc⑀RI are stabilized at the cell surface by monovalent interactions with IgE (35). We next wanted to investigate whether the single-chain isoform of Fc⑀RI also shares this key feature of the multimeric Fc⑀RI complexes. We cultured endo-␣ and K b -␣ transfectants with or without IgE for 16 h. Interaction with IgE stabilized the single Fc⑀RI ␣-chain at the cell surface as measured by flow cytometry (Fig. 5). The degree of stabilization was comparable between both constructs (2.3-fold versus 1.7-fold; Fig.  5B). These data showed that single Fc⑀RI-␣ interacts with and is stabilized by its natural ligand IgE at the cell surface like multimeric receptor isoforms.
Single Fc⑀RI ␣-Chain Internalizes after Cross-linking-Crosslinking of multimeric Fc⑀RI isoforms (i.e. ␣␥ 2 and ␣␤␥ 2 ) induces signaling via the ITAM motifs in the ␥and ␤-chains as well as the internalization of receptor complexes from the cell surface (7,36). Next, we explored whether cross-linking of the single Fc⑀RI ␣-chain also induces internalization from the cell surface. We monitored ␣-chain internalization in HeLa cells. First, Fc⑀RI was loaded with the ␣-chain-specific antibody CRA1 at the cell surface. This incubation step does not result in receptor internalization (data not shown). Next, internalization of Fc⑀RI-␣ was induced by cross-linking with a fluorescently labeled secondary antibody. Irrespective of the presence of the ␥-chain, we found Fc⑀RI ␣-chain inside the cell within 30 min (Fig. 6A). HeLa cells stably transfected with a LAMP-1-eGFP reporter were used to study whether the single-chain receptor isoform traffics into endo/lysosomal compartments. We found that a single Fc⑀RI ␣-chain shuttles to a lysosomal compartment as characterized by LAMP-1 expression comparable with multimeric Fc⑀RI containing the common ␥-chain (Fig. 6B). This indicates that the Fc⑀RI signaling subunits are not essential for internalization and intracellular trafficking after Fc⑀RI cross-linking.

DISCUSSION
Fc⑀RI is an activating immune receptor complex that must accomplish the following tasks: assembling properly in the ER, reaching the cell surface, binding IgE, and inducing ITAMbased cell activation as well as internalization of receptor complexes upon antigen-mediated receptor cross-linking (schematic in Fig. 7A).
The fidelity of intracellular trafficking of Fc⑀RI complexes has so far been considered a function of cytosolic and transmembrane ER retention signals in the Fc⑀RI ␣-chain that are masked by association with the other receptor subunits. Here we demonstrated that the signal peptide of Fc⑀RI-␣ contains an additional regulatory element that contributes to the retention  of the ␣-chain in the ER. We found that human Fc⑀RI-␣ inefficiently reaches the cell surface by itself and that a swap of the endogenous signal peptide for that of H2-K b allows for significantly more efficient surface expression.
We show that the single Fc⑀RI ␣-chain binds IgE at the cell surface and stabilizes upon IgE binding. This observation supports an earlier report by Kubota et al. (35) describing that surface stabilization of Fc⑀RI is a function of the stalk region of the ␣-chain. This conclusion was derived from the analysis of chimeric ␣-chains in the presence of Fc⑀RI ␥-chains. Our experiments show unambiguously that the IgE-mediated surface stabilization affects wild type Fc⑀RI-␣ as a single-chain receptor. We further demonstrate that single Fc⑀RI ␣-chain is efficiently targeted into lysosomal compartments after receptor cross-linking. Thus, the single-chain isoform of the receptor could act as an IgE-receptor, which reacts to serum IgE levels and shuttles antigen into antigen presentation compartments. Removal of the cytosolic ER retention signals as well as mutating the transmembrane ER retention signal suggest that the signal peptide regulates ER retention in a concerted fashion with formerly described ER retention motifs (15,18). The fact that the regulatory influence of the signal peptide could still be observed when other ER retention signals were removed argues against a simple overexpression artifact.
To our knowledge, this is the first report showing that a signal peptide can regulate the surface expression of an Fc receptor. However, several reports in the literature have shown that signal peptides can function as more than just ER targeting sequences (14). A polymorphism in the luteinizing hormone receptor protein improves its signal peptide function and increases protein expression. The consequences of the higher expression levels of this luteinizing hormone receptor protein are unfavorable, and consequently, this polymorphism serves as a predictor for adverse outcome in breast cancer patients (37). Along this line, genetic polymorphism in the signal peptide of Fc⑀RI-␣ could influence IgE receptor expression levels and allergy. Whether such a polymorphism in the Fc⑀RI-␣ signal peptide indeed exists in humans needs to be addressed in future studies.
The ER assembly of Fc⑀RI complexes is regulated more tightly than that of other activating immune receptor complexes (19). Although other receptor complexes assemble in consecutive steps (4,5,20,30), assembly of Fc⑀RI complexes occurs strictly cotranslationally. It is also important to keep in mind that Fc⑀RI-␥ is a signaling subunit shared by multiple activating immune receptors. Therefore, several receptor complexes compete for this protein in the ER. Competition for limiting amounts of Fc⑀RI-␥ has been demonstrated in vivo. The absence of Fc⑀RI ␣-chain enhances Fc␥RIII-dependent mast cell degranulation and anaphylaxis (38). Fc⑀RI seems to be at a competitive disadvantage because it needs to fulfill its cotranslational assembly requirement. Therefore, the assembly machinery has to assure both temporal and spatial coordination of subunit translation. In this context, ER retention of Fc⑀RI-␣ by the signal peptide could facilitate this cotranslational event. One possibility for signal peptides to modulate ER retention is by regulating the activity of type I signal peptidase (39). Because signal peptide cleavage is a cotranslational event, a slow cleavage rate might impede folding of Fc⑀RI-␣ in a way that provides more time for assembly. Given that we were not successful in destroying the regulatory property of the endogenous signal peptide through a targeted mutation, however, we cannot formally rule out that the signal peptide controls ␣-chain trafficking via regulating its recognition and translocation into the ER.
We suggest that this additional regulatory sequence element is important to prevent the formation of an Fc⑀RI ␥-chain-free IgE receptor pool at the cell surface (schematic in Fig. 7B). In human antigen presenting cells, such as dendritic cells and macrophages, intracellular accumulation of Fc⑀RI-␣ is frequently found (21, 39 -41). This is probably because Fc⑀RI-␣ alone folds properly and forms IgE-binding sites (9) and is therefore not efficiently recognized by the ER-associated degradation machinery. A slow rate of signal peptide processing might however help the ER-associated degradation system to recognize some of this Fc⑀RI-␣ and target it for degradation. Although Fc⑀RI-␣ is mainly restricted to the ER in the absence of the ␥-chain, we show that some properly folded protein reaches the cell surface and forms a signaling-deficient IgE receptor isoform. This receptor pool lacks ITAM-based signaling modules yet retains its ability to bind IgE and to endocytose upon receptor activation. This single-chain IgE-receptor also reaches lysosomal compartments like tetrameric and trimeric Fc⑀RI (36). 3 Fc⑀RI shuttles IgE-antigen complexes into lysoso-3 B. Platzer, manuscript in preparation. mal compartments for antigen presentation, most likely to facilitate allergen presentation via major histocompatibility complex class II (36,42). We show here that this intracellular trafficking is not dependent on signaling subunits, which carry ITAM motifs in their cytosolic tails. We therefore conclude that signals via ITAMs in Fc⑀RI-␤ or Fc⑀RI-␥ are dispensable for intracellular trafficking of IgE-antigen complexes to lysosomal compartments.
In the context of allergy, this Fc⑀RI-␥ and ITAM-free receptor appears attractive. Such an IgE receptor could remove IgE or IgE-allergen complexes in an immunologically silent form, because cross-linking would not initiate cell activation via ITAMs (Fig. 7B). IgE-Fc⑀RI-mediated cell activation during allergy is, however, not the physiological function of IgE-mediated response. This pathway is highly active during helminth parasite infections and critical for the development of protective immunity. In this context, expression of an IgE-receptor that fails to signal is undesirable, because it could compromise the development of protective immunity. In summary, based on our findings, we propose that the endogenous signal peptide of Fc⑀RI-␣ represents a critical control element preventing unwanted expression of a signaling-deficient IgE receptor isoform.