Cotranslational endoplasmic reticulum assembly of FcRI controls the formation of functional IgE-binding receptors
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《实验药学杂志》
1 Department of Pathology, Harvard Medical School, Boston, MA 02115
2 Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029
3 Laboratory of Allergy and Immunology, Beth Israel Deaconess Medical Center, Boston, MA 02215
CORRESPONDENCE Hidde Ploegh: ploegh@hms.harvard.edu
The human high affinity receptor for IgE (FcRI) is a cell surface structure critical for the pathology of allergic reactions. Human FcRI is expressed as a tetramer (?2) on basophils or mast cells and as trimeric (2) complex on antigen-presenting cells. Expression of the human subunit can be down-regulated by a splice variant of FcRI? (?var). We demonstrate that FcRI is the core subunit with which the other subunits assemble strictly cotranslationally. In addition to ?2 and 2, we demonstrate the presence of ? and ?var2 complexes that are stable in the detergent Brij 96. The role of individual FcRI subunits for the formation of functional, immunoglobulin E–binding FcRI complexes during endoplasmic reticulum (ER) assembly can be defined as follows: ? and support ER insertion, signal peptide cleavage and proper N-glycosylation of , whereas ?var allows accumulation of protein backbone. We show that assembly of FcRI in the ER is a key step for the regulation of surface expression of FcRI. The ER quality control system thus regulates the quantity of functional FcRI, which in turn controls onset and persistence of allergic reactions.
Abbreviations used: CC, U373; EndoH, endoglycosidase H; ITAM, immunoreceptor tyrosine–based signaling motifs.
A significant fraction of the population (20%) in the Western world is affected by allergies and the numbers of affected individuals is on the rise (1, 2). Convincing evidence exists that FcRI is one of the key molecules in the pathophysiology of all allergic reactions (3–6). As a member of the antigen receptor superfamily, FcRI shares the organizational principles of a ligand binding immunoglobulin-type protein associated with signaling subunits that regulate cellular activation via conserved immunoreceptor tyrosine–based signaling motifs (ITAMS; 7). BCR, TCR, and other Fc receptors fall in the same class (7–10). FcRI was initially described as a tetrameric receptor composed of a high-affinity ligand-binding chain, one ? chain, and a pair of disulphide-linked subunits (5, 9). The FcRI complexes on the surface of basophils and mast cells are tetrameric structures (?2). The ?2 is the only receptor isoform formed in rodents (5). Human antigen-presenting cells additionally display a trimeric form of FcRI that lacks the ? subunit (5, 11, 12). A new splice variant of FcRI? (?var, formerly referred to as ?T) exerts a dominant negative effect on ? function (13).
The structural integrity of FcRI is maintained by the noncovalent interactions of its various subunits. The extracellular domain of FcRI forms the binding site for the CH3 domain of IgE. It binds its ligand in 1:1 ratio, with an affinity of 1010 M–1. The ? chain contains four potential transmembrane spanning regions with both the NH2 and the COOH terminus protruding into the cytosol. FcRI forms a dimer and is a member of the gene family. IgE-dependent cross-linking of FcRI induces cellular activation regulated via ITAMs, which are present in one copy in the ? as well as in each of the chains (5, 9, 10). The subunit, when expressed in the absence of ? and , is retained in the ER. The ER retention signal of human can be overcome by the presence of alone. FcRI? was defined as an amplifier for chain signaling in vitro and in vivo (14, 15) and as a regulator for surface expression. The ?var subunit is a splice variant that has lost its ITAM (13). Therefore ?var-containing complexes must behave significantly differently from those that contain the conventional ? chain.
Multisubunit receptor complexes, like FcRI or the TCR, are assembled in the ER, from where they enter the secretory pathway (16, 17). The acquisition of the proper tertiary and quaternary structure in the ER is a carefully controlled sequence of events. Nascent polypeptides are subject to modifications, which often include signal–peptide cleavage, N-linked glycosylation and oligosaccharide trimming. Folding of proteins is guided by chaperones such as BiP and the lectins calnexin and calreticulin. Oxido-reductases control the formation of disulfide bonds between the correct pairs of cysteine residues to stabilize the folded structure (18). As a consequence of imperfections in protein folding, some polypeptides never attain their native conformation. Terminally misfolded proteins are singled out in the ER by a quality control process (19–21). However, their destruction takes place mostly in the cytosol. ER quality control substrates may cross the ER membrane before their degradation (22). In addition to the proper folding of the individual subunits, multimeric receptors like FcRI must assemble in a concerted fashion. Only with all players in place, can ER retention signals be overcome. The sequence of events in FcRI receptor assembly in its various configurations is interesting with respect to the functional differences of the receptor isoforms. These events all contribute to the control of receptor expression and thereby the outcome of allergic responses in vivo. The generally increased cell surface expression of FcRI in allergic individuals supports this hypothesis (23, 24).
Studies on the human receptor are hampered by a lack of cell lines that express FcRI irrespective of its isoforms. Primary human cells that express FcRI are difficult to obtain, even in small numbers. Because these cells shut down synthesis of the receptor immediately after isolation, they cannot be used to study complex formation and regulation of surface expression. We therefore used in vitro translation in membrane-supplemented rabbit reticulocyte lysates to study the early events of FcRI assembly in the ER. We translated the corresponding mRNAs of all FcRI subunits and performed studies on temporal aspects of protein–protein interaction and their consequences for receptor assembly. Our results show that ER assembly of the individual FcRI subunits is tightly controlled and indeed regulates the formation of properly formed receptors with IgE-binding epitopes.
Results
In vitro translation of FcRI
We used in vitro translation as a method to study FcRI receptor assembly in the ER (16, 25, 26), because aspects of multimeric receptor assembly cannot be studied in a time-resolved fashion in transfection experiments. First, we characterized the properties of the individual receptor subunits in this assembly system. The cDNAs for the human FcRI subunits allow the generation of the corresponding mRNAs for in vitro translations. The mRNAs were translated in the presence of microsomes from different sources. FcRI is a type I membrane protein and requires cleavage of its signal sequence before N-glycosylation, which is in turn required for the formation of functional IgE-binding sites (5, 9). FcRI cDNA equipped with its endogenous signal peptide translated poorly. Although we could detect the expected polypeptides reactive with anti– serum, cotranslation of the construct with ? and mRNAs would have rendered further assembly studies technically difficult (unpublished data). We therefore exchanged the signal sequence of the subunit for that of H2-Kb. The latter has proved efficient for translation as well as for adjustment of insertion efficiencies of different subunits during TCR assembly (16). This swap of signal peptides allowed efficient translation of FcRI (H2-KbFcRI, referred to as ; Fig. 1 A). The source of microsomes proved critical for the generation of chains with cleaved signal peptide (–sig, Fig. 1 A) as well as for N-glycosylation (Nglyc, Fig. 1 A), although no such effect was observed for FcRI or HLA-2A (unpublished data). Microsomes from the basophilic cell line KU812 allowed efficient translation, but yielded mostly as 1sig (Fig. 1 A). Microsomes derived from canine pancreas were successful in creating Nglyc, but a sizable fraction of the translated protein was present as 1sig (Fig. 1 A and unpublished data). Microsomes derived from the astrocytoma cell line U373 (CC) reproducibly generated endoglycosidase H (EndoH)-sensitive Nglyc efficiently (Fig. 1 A).
Figure 1. In vitro translation of IgE-binding FcRI. (A) Signal peptide cleavage and N-glycosylation of FcRI depends on the source of microsomes used during in vitro translation. Transcripts of Kb-FcRI were translated for 1 h at 30°C in the presence of microsomes derived from KU812, dog pancreas (dpm), or U373 (CC). After lysis of the microsomal pellets in 1% Brij96 lysis buffer, immunoprecipitates were obtained with polyclonal anti- serum and either mock digested or digested with Endo H. Subsequently immunoprecipitates were analyzed by 12.5% SDS-PAGE under reducing conditions. Conversion of +sig to Nglyc occurs most efficiently in CC microsomes. (B) Nglyc generated with CC microsomes expresses properly folded IgE-binding epitopes. IgE selectively precipitates the fully N-glycosylated form of FcRI. Representative experiments (n = 3).
We then asked whether we could generate FcRI with proper IgE-binding epitopes, using IgE as the bait to recover translation products from microsomal pellets (Fig. 1 B). The direct analysis of the microsomal extracts shows the presence of all forms of FcRI (1sig, 2sig, and Nglyc, Fig. 1 B), but only properly folded and N-glycosylated is recovered by IgE (Fig. 1 B).
In vitro translation of FcRI? and FcRI?var
The identity of the translation products from ? and ?var (13) was confirmed by immunoprecipitation with an anti-? serum generated against the NH2 terminus of ?, therefore reactive with both splice variants (Fig. 2 A). Indeed, both proteins migrate at their expected molecular weight of 28,000 and 22,000, respectively (Fig. 2 A; reference 13).
Figure 2. In vitro translation of FcRI? and FcRI?var. (A) Transcripts of FcRI? or FcRI?var were translated for 1 h at 30°C in the presence of CC microsomes. After lysis of the microsomal pellets in 1% Brij96 lysis buffer, immunoprecipitates were obtained with polyclonal anti-? serum and analyzed by 12.5% SDS-PAGE under reducing conditions. Representative experiments (n = 3).
In vitro translation of FcRI
The chain is a type I membrane protein (3, 8, 9). To achieve comparable translation efficiencies, we exchanged the signal sequence of that of H2-Kb (H2-KbFcRI, referred to as ; Fig. 3). Fig. 3 A shows the proper insertion and signal peptide cleavage of in the in vitro translation assay with a cysteine translation mix. The single methionine present in is removed upon insertion into the microsomes. However, labeling with the methionine translation mix (containing both Met and Cys) allows sufficient labeling as shown in anti- immunoprecipitations (Fig. 3 B). For reasons that remain unclear, in vitro translations using cysteine did not allow visualization of ?var (unpublished data). Thus, all subsequent translation assays were performed with the methionine translation mix. Under nonreducing conditions, runs as a dimer with both translation mixes (Fig. 3, C and D).
Figure 3. ER insertion and proper signal peptide cleavage of FcRI. (A) Proper insertion and signal peptide cleavage of Kb- in an in vitro translations with a cysteine translation mix. (B) In vitro translations of Kb- with a methionine translation mix. The single methionine of gets cleaved after insertion into the microsomes. Cysteine present in the methionine translation mix is sufficient for labeling of Kb-. In vitro translations were performed in the absence and presence of CC microsomes. Translation products were analyzed after immunoprecipitation from 1% Brij96 lysates of in vitro translations performed in the absence of microsomes (–) or lysates of microsomal pellets (+) and analyzed by 12.5% SDS-PAGE under reducing (A and B) or nonreducing (C and D) conditions. FcRI forms a dimer under nonreducing conditions. Representative experiments (n = 3).
Receptor assembly studies: complexes form cotranslationally
The next set of experiments addressed the existence of complexes and their assembly. Anti- immunoprecipitations from Brij 96 lysates of microsomal pellets demonstrate the presence of stable complexes (Fig. 4 A, lane 4). Such complexes arise only if both proteins are translated at the same time. Microsomal pellets from assay mixtures in which and RNA were translated consecutively are devoid of complexes, despite the presence of both proteins in the direct load of the microsomes (Fig. 4 A, lanes 1 and 3). Complexes of where absent when proteins were translated separately and then mixed before or after lysis (Fig. 4 B; and unpublished data). Direct loads of microsomes from translation mixtures in which and were present concurrently generally show more properly folded Nglyc when compared with samples in which was translated alone. This effect is even more pronounced when ? is cotranslated as well (unpublished data and see Fig. 6 A). Immunoprecipitations of complexes with serum directed against confirmed the association of 1sig, 2sig, and Nglyc with (Fig. 4 C).
Figure 4. FcRI complexes form cotranslationally. (A) Cotranslational formation of complexes. and RNA were translated consecutively (lanes 1 and 3, 1st 2nd) or at the same time (lane 2 and 4, + ) into CC microsomes. Direct loads of the microsomal pellets confirmed the presence of both translation products (lanes 1 and 2). complexes were only retrieved by anti- immunopreciptiation, if both proteins were translated at the same time (lane 4, + ). (B) and were translated separately and microsomal pellets were mixed before lysis. Direct loads of microsomes showed the presence of both proteins. No complexes were precipitated. (C) Immunoprecipitations of complexes with serum directed against .
Cotranslation of ? and chains resulted in efficient insertion of both proteins into microsomes (Fig. 5 A, lane 1). Neither serum coprecipitated the other protein. These experiments control for proper solubilization under the necessary mild lysis conditions and further show that ? complexes do not occur in the absence of (Fig. 5 A, lanes 2 and 3). We also failed to detect ? complexes when ? and mRNAs were translated separately and microsomes were mixed before lysis (Fig. 5 D and E, lanes 2) or when microsomal pellets of cotranslation experiments were lysed in 1% digitonin (unpublished data).
Figure 5. Association studies of different FcRI subunits. (A) Cotranslation of ? and chains results in efficient expression of both proteins in microsomes. Anti-? and - serum both failed to coprecipitate ? complexes. (B) Demonstration of ? complexes. Anti- serum precipitates stable ? complexes. The presence of ? is in the complex is confirmed by anti-? reimmunoprecipitations. (C) Confirmation of ? complexes in 293 cells transiently transfected with HA and GFP-? cDNA. (D and E) The ? and subunits associate with but not HLA-A2. FcRI complexes form only cotranslationally. Direct loads of microsomes (D) in which proteins were translated separately and mix before lysis (lanes 1–3) and microsomes from cotranslations (lanes 4–6) were compared with immunoprecipitations. Complexes were precipitated from 1% Brij96 lysates of in vitro translations from CC pellets and analyzed by 12.5% SDS-PAGE under reducing conditions. Representative experiments (n = 3).
These results fit with the assumption that the chain is the core of all FcRI complexes. This hypothesis also implies a direct interaction of with ?. We therefore attempted to demonstrate the existence of such complexes by in vitro translation. As shown in Fig. 5 B, the ? complex is stable in Brij96 and is generated only cotranslationally (Fig. 5, D and E, lanes 3). Due to its molecular weight, ? is difficult to distinguish from 1sig and 2sig. The anti-? reimmunoprecipitation unequivocally demonstrates the existance of ? complexes (Fig. 5 B, lane 2). We could also demonstrate the presence of these complexes on a cellular level by immunoprecipitations from 293 cells transiently transfected with and ? complexes (Fig. 5 C). The use of tagged versions of both proteins allowed the detection of the individual subunits by immunoblotting after immunoprecipitation. The ? complexes can be retrieved specifically with an anti-HA reagent via HA (Fig. 5 C, lanes 2–4), but not with a control Ab (Fig. 5 C, lane 1). A considerable amount of this protein becomes EndoH resistant, indicative of modifications of N-glycans in the Golgi apparatus (mod; Fig. 5 C, lane 3) and thus proper ER exit of ? complexes. Digestion of immunopreciptated ? complexes with PNGase was performed to provide further evidence for proper folding of . PNGase preferentially attacks improperly folded glycoproteins (27), and the resistance of to digestion by this enzyme thus supports our findings that is expressed as properly folded protein in ? complexes.
Cotranslation of HLA-A2 with ? and were performed as specificity controls (Fig. 5 D, lanes 4 and 6). As expected, HLA-A2 failed to form a complex with ? and (Fig. 5 E, lanes 4 and 6).
Immunoprecipitation of ? and ?var complexes
The ,?, , or ,?var, mRNAs were translated into microsomes, which were then solibilized in 1% Brij96 and subjected to immunoprecipitation. The anti- but not the control serum successfully precipitated ? complexes (Fig. 6 A). The anti- reagent also retrieved stable ?var complexes (Fig. 6 A). The chain in the latter complexes seemed to be underrepresented when compared with ?.
Figure 6. Cotranslational formation of tetrameric FcRI complexes. (A) ,?, , or ,?var, mRNAs were simultaneously translated into CC microsomes. Microsomal pellets were solibilized in 1% Brij96 lysis buffer and immunoprecipitations with anti- or control serum were performed. Stable ? as well as ?var complexes were retrieved by anti-. (B) ?var induces the accumulation of +sig. ?var mRNAs were cotranslated and direct loads of in vitro translations were compared with anti- immunoprecipitates. All translated proteins with a prominent band of 33 kD are present in direct loads of CC mircosomes translated with ?var . Anti- immunoprecipitation shows an overrepresentation of +sig when compared with translations of alone. Proteins were precipitated from 1% Brij96 lysates of in vitro translations from CC pellets and analyzed by 12.5% SDS-PAGE under reducing conditions. Representative experiments (n = 3).
?var induces the accumulation of +sig
We next examined the fate of when translated in the presence of ?var. For this purpose ?var mRNAs were cotranslated and direct loads of microsomal pellets were compared with anti- immunoprecipitates to assess more carefully all forms of present in the translation mix (Fig. 6 B). We detected the presence of all translated proteins, with a prominent band of 33 kD. Anti- immunoprecipitation confirmed the nature of this polypeptide as +sig (Fig. 6 B). The chain as well as Nglyc and –sig were coprecipitated. For unknown reasons, we were unable to directly demonstrate ?var in these precipitates. This finding might again reflect a decrease in the stability of ?var complexes, with ?var dissociating before , or equally likely, a more general problem of detection of ?var. As in cellular expression systems (13), ?var is rapidly lost from in vitro translation mixtures (unpublished data).
?var down-regulates surface IgE-binding epitopes
We subcloned ? and ?var into a bicistronic vector with EGFP (pIRES2-?-EGFP and pIRES2-?var-EGFP). Next, 293 cells were transiently transfected and treated with proteasome inhibitor for 2h. After SDS lysis, immunoblots with anti-? serum were performed to confirm the proper expression of both proteins (Fig. 7 A).
Figure 7. FcRI?var induces the accumulation of 1sig in vivo. (A) ?var down-regulates surface IgE-binding epitopes. ? and ?var were subcloned into a bicistronic vector expressing EGFP to control for equal expression levels (pIRES2-?-EGFP and pIRES2-?var-EGFP). 293 cells were transiently transfected and treated with proteasome inhibitor for 2 h. After SDS lysis, immunoblots with anti-? serum were preformed to demonstrate the expression of both proteins. (B) mAb 15–1 recognizes the IgE-binding epitope of FcRI. IgE binding capacity of CHO was assessed with biotinylated IgE (black). Preincubation with mAb 15–1 (reactivity shown in blue) inhibits subsequent IgE binding (red). Mean fluorescence is shown on the abscissa. (C) CHO? were transiently transfected with pIRES2-?-EGFP or pIRES2-?var-EGFP (? (red) and ?var(black)). Reactivity of mAb 15–1, which is specific for the IgE-binding epitope, was monitored on the surface of a cell population gated for EGFP-expression as a marker for expression of ? and ?var. pIRES2-?var-EGFP specifically down-regulates surface 15–1 reactivity. (D) ?var induces accumulation of unglycosylated protein backbone in vivo. A HA-tagged version of Kb- (HA) was transiently transfected into 293 cells in the presence of the indicated FcRI subunits. Transfection was followed by anti-HA immunoprecipitation and anti-HA immunoblotting. HA folds properly and becomes N-glycosylated (Nglyc) in the absence of other complex subunits (lane 1). Cotransfection of ? (lane 4) or ?var (lane 3) induces Golgi-associated glycosylation modification of HA (mod). Unglycosylated protein backbone accumulates specifically in the presence of ?var. (E) ?var induces the accumulation of +sig. Metabolic labeling of cells transiently transfected with HA, ? and followed by anti-HA immunoprecipitations and EndoH digestion. Most HA is rapidly transformed into its fully N-glycosylated modification (Nglyc). Susceptibility to EndoH treatment defines +sig.
We verified that mAb 15–1 recognizes the IgE-binding epitope of FcRI (13, 23, 28–30). IgE binding capacity of CHO was assessed by FACS with biotinylated IgE (Fig. 7 B, filled black). CHO show comparable reactivity when stained with 15–1 (Fig. 7 B, blue). Preincubation of cells with 15–1 inhibits subsequent IgE binding (Fig. 7 B, red). The mean fluorescence intensity (MFI) of IgE-reactivity drops from 370 to levels of the negative control (Fig. 7 B, black line, MFI=10). This result is in accordance with the literature (13, 23, 28–30) and confirms that 15–1 recognizes the IgE-binding site of FcRI. The fact that both reagents recognize the same epitope also accounts for the misinterpretation of cellular distribution patterns of FcRI in humans. Endogenous IgE bound to FcRI precludes recognition with mAb 15–1 or biotinylated IgE unless the natural ligand is removed by acid stripping (23, 28–30).
We show that our bicistronic constructs regulate the surface expression of IgE-binding epitopes as previously described (13). For this purpose, CHO? were transiently transfected with pIRES2-?-EGFP or pIRES2-?var-EGFP (Fig. 7 A, graph refers to ? and ?var ). Reactivity with mAb 15–1, which is specific for the IgE-binding epitope (5, 23), was monitored in a population gated for EGFP expression as a marker for successful transfection with ? or ?var. Although we observed surface expression of IgE-binding epitopes in cells transfected with pIRES2-?-EGFP, this surface marker was significantly down-regulated in cells transfected with pIRES2-?var-EGFP (Fig. 7 A, representative experiment). Transfections in CHO cells yield the same results (unpublished data). Our experiments confirm that ?var impairs formation of surface expressed IgE-binding epitopes in vivo and functions in a dominant way when coexpressed with ? in CHO cells (13).
?var induces accumulation of +sig in vivo
We next explored the mechanism by which ?var might interfere with the generation of IgE-binding epitopes. For this purpose we generated a COOH-terminally HA-tagged version of Kb- (HA) because the commonly used anti- reagents failed to detect the 30-kD protein backbone and yielded poor results when used for immunoprecipitation in pulse-chase experiments. Anti-HA immunoprecipitation followed by anti-HA immunoblotting on 293 cells transiently transfected with HA confirmed that HA is properly N-glycosylated in the absence of ? or subunits (Fig. 7 B, lane 1; 31). The presence of unglycosylated protein backbone was specific for the presence of ?var (Fig. 7 B, lane 3).
Metabolic labeling experiments were then performed to shown that the protein that accumulates in the presence of ?var is indeed +sig. Anti-HA immunoprecipitations followed by EndoH digestion were performed in cells transiently transfected with HA?var (Fig. 7 C). These experiments shown that most HA is transformed into its fully N-glycosylated modification irrespectively of the presence of the ?var subunits. Comparing its characteristic with EndoH-treated protein, the remaining HA protein can be identified as +sig (Fig. 7 C). We could thus confirm by both immunoblotting and by pulse labeling that ?var allows accumulation of +sig.
For more extended studies of the intracellular fate of , NH2-terminal EGFP fusion proteins of ? or ?var (GFP-? or GFP-?var) were generated. The fusion adds the expected 28 kD to the molecular mass but otherwise does not interfere with the molecular characteristics of either protein (reference 13; Fig. 8 A; and unpublished data). Pulse-chase analysis of GFP-? or GFP-?var demonstrates that both proteins are stabilized when inhibitors of the proteasome are present (Fig. 8 A). Pretreatment of cells with proteasome inhibitor ZL3VS (5 μm, 1 h; reference 32) and its presence throughout the pulse chase stabilize ? as well as ?var throughout the chase (Fig. 8 A). We infer that the ? subunits are subject to proteasomal proteolysis with ?var more susceptible to proteasomal degradation. GFP-? and GFP-?var should be informative reagents for the analysis of the fate of the subunit at the single cell level.
Figure 8. FcRI?var induces the accumulation of improperly folded FcRI in vivo. (A) NH2-terminal EGFP-fusion proteins of ? or ?var (GFP-? or GFP-?var) were transiently transfected into 293 cells with lipofectamine. The fusion adds the expected 28kD to the molecular weight but otherwise does not interfere with the molecular characteristics of ? or ?var. Pulse-chase analysis: cells were analyzed untreated or pretreated with proteasome inhibitor ZL3VS (5 μm, 2 h). Anti-GFP immunoprecipitates were obtained from 1% NP-40 lysates and analyzed by 12.5% SDS-PAGE under reducing conditions. Inhibition of the proteasome stabilizes GFP-?var. (B) CHO were transiently transfected with GFP-? and GFP-?var and analyzed by epifluorescence. Cells were treated with ZL3VS for 2 h, fixed and stained with mAb 15–1 to visualize IgE-binding epitopes. Staining with the anti- polyclonal serum was performed to visualize all forms of . Transfection with the GFP-? does not influence mAb 15–1 reactivity. Cells transfected with GFP-?var do not show mAb 15–1 reactivity but still remain positive with the anti- serum indicative for the presence of unfolded chain. Representative experiment (n = 4). (C, D) Pulse-chase experiments to confirm that FcRI not targeted to proteasomal degradation by ?var. FcRIHA was transiently transfected into 293 cells in the presence of ? or ?var cDNA. Transfection, immunoprecipitations and analysis were performed as described in (A). +sig is stabilized by the presence of ?var selectively (C). (D) Comparison of FcRI protein levels in ?var transfectants in the presence and absence of proteasome inhibitors. No alterations in the amount or expression pattern of FcRI in induced by the inhibitor.
To this end, CHO cells were transiently transfected with GFP-? and GFP-?var and analyzed by epifluorescence. Cells were treated with ZL3VS to inhibit proteasomal degradation for 2 h, fixed, and stained with mAb 15–1 to visualize the IgE-binding form of as previously described (23, 33). Staining with the anti- polyclonal serum was performed to visualize all forms of . CHO transfected with GFP-? are positive for both mAb 15–1 and the anti- serum (Fig. 8 B). In contrast, CHO transfected with GFP-?var do not stain with mAb 15–1 but still remain positive with the anti- serum (Fig. 8 B). Experiments performed with CHO? cells show identical results (unpublished data). It is important to note that inclusion of the proteasome inhibitor did not rescue the expression of IgE-binding epitopes. In agreement with our in vitro translation results, this experiment demonstrates that expression of GFP-?var interferes with proper folding and the formation of IgE-binding epitopes on the subunit. Cells transfected with GFP-?var still express readily detectable unfolded chain, suggestive of the mechanism by which ?var and GFP-?var down-regulate IgE-binding epitopes. In experiments without inhibition of proteasomal activity, GFP-?var is more difficult to detect but can still be visualized. Such cells also contain unfolded chain but are devoid of IgE-binding epitopes, as visualized by staining with anti- serum or 15–1, respectively (unpublished data).
Additionally we performed a set of pulse-chase experiments to confirm on the cellular level, that FcRI is indeed not targeted to proteasomal degradation by ?var. FcRIHA was transiently transfected into 293 cells in the presence of ? or ?var cDNA (Fig. 8 C). Immunoprecipitations were performed with anti-HA in 1% NP-40 lysis buffer to assure access to the total cellular pool of FcRI. No enhanced degradation of any form of FcRI was observed in cells transfected with ?var. In correlation with the results presented earlier in this study, the only significant difference was the persistence of +sig in the presence of ?var. The slight and progressive decrease in the molecular weight of Nglyc observed in all conditions is attributable to mannose trimming (32, 34). We failed to detect mod in this experimental setting. Next we compared FcRI protein levels in ?var transfectants in the presence and absence of proteasome inhibitors (Fig. 8 D). Although proteasomal inhibition stabilizes ?var (reference 13; Fig. 8 A; and unpublished data), we do not detect alterations in the amount or expression pattern of FcRI. Because inclusion of proteasome inhibitor and consequent stabilization of ?var do not change the fate of FcRI is not targeted to proteasomal degradation. We consider it unlikely that the short half-life of ?var is of functional importance for this mechanism.
Discussion
Allergen- and IgE-dependent cross-linking of FcRI is responsible for the immediate as well as the chronic inflammatory responses observed in atopic patients (5, 6). Surface expression of FcRI critically determines the sensitivity to an allergic stimulus and is therefore pivotal for the ensuing clinical response. The only defined extracellular regulator of FcRI surface expression defined so far is IgE, its natural ligand (5). With regard to intracellular regulation of receptor expression, FcRI? was described as an amplifier for chain signaling as well as for receptor surface expression (14, 15). In contrast, ?var has been shown to down-regulate surface expression of (13). The mechanistic basis of these regulatory events is poorly understood. Glycosylation-mediated quality control regulates ER export of FcRI (31). Here we show the efficiency of ER assembly is controlled by the presence of the different subunits. We were able to define the IgE-binding chain as the core of the receptor that pairs with the other subunits in a strictly cotranslationally regulated assembly event. These experiments establish ER assembly as a rate-limiting step in the expression of functional surface receptors, with obvious consequences for the onset of allergic diseases. In addition to the well-described FcRI isoforms, we were also able to demonstrate the existence of ?var and ? complexes, not previously documented.
Protein synthesis and folding in the ER are not always efficient: improperly folded structures are cleared from the ER and directed toward degradation (19–21). In addition to the proper folding of the individual subunits, multimeric receptors must assemble in a concerted fashion. Receptors such as FcRI or the TCR assemble in the ER and maintain their integrity by noncovalent interaction of the various subunits. Only with all players in place can ER retention signals be overcome (5, 9). For the TCR this process is well established and occurs in three consecutive assembly steps (17). Although FcRI and TCR share the same principal structure of ligand-binding and signal-transducing units and can even use the same chain for signaling (5), we show that their assembly is regulated differently. FcRI complexes form strictly cotranslationally. The presence of ? and clearly favors a conversion of into its IgE-binding form when compared with translation in the presence of alone. The ?var, on the other hand, slows down this conversion and induces the accumulation of unglycosylated with the signal peptide still in place.
Not all FcRI receptor subunit RNAs are generated in the same quantities in primary cells. When compared with , ? is always underrepresented (5). These observations were also confirmed at the protein level (11, 23). The demands of receptor stochiometry make cotranslational assembly of the receptor a key step that controls expression of functional IgE-binding epitopes at the cell surface. It may thereby affect the susceptibility to allergic stimuli in vivo. Receptor subunits can still fold properly, but do so at a less efficient rate when compared with folding in the presence of ? and . Such chains lack the necessary partners for complex formation during translation and consequently for ER exit. Our finding thus provides an explanation for the intracellular accumulation of in cell types that express high levels of , which in principle should allow surface expression (5). It is possible that in such cells, translation of and might not occur in synchrony and thus ER exit may be compromised. In addition to its critical role for FcRI signaling and ER exit, FcRI is a subunit of Fc receptors (35). Functional association of FcRI? with CD16, FcRIII, has also been demonstrated (36, 37). Both Fc receptor types might thus compete for and ? subunits, which adds an additional level of complexity and importance to assembly control. Because various Fc receptor subtypes and FcRI are expressed simultaneously in a variety of cell types (e.g., monocytes, dendritic cells, Langerhans cells, mast cells), this event is of physiological importance. Antigen presenting cells and monocytes of all individuals express Fc receptors. The proper assembly of Fc receptors could indirectly control the expression of FcRI by depleting the pool of receptor units that are required for FcRI ER exit. The functional consequences of ?var expression for cotranslational assembly need to be interpreted carefully, in particular with regard to the fact that so far transcripts of ?var were not depicted in absence of the classic ? chain.
Improperly assembled FcRI subunits are subjected to the ER quality control system and must be directed toward degradation. FcRI?var is a natural substrate for proteasomal degradation and is itself rapidly destroyed, irrespective of the presence of FcRI's other subunits (13). Whereas the subunit is also degraded by the proteasome, we found no evidence for a redirection of toward proteasomal degradation by ?var (unpublished data). When expressed alone, Nglyc is a rather stable protein. It remains to be established whether primary cells such as Langerhans cells, eosinophils, or monocytes of allergic patients have a mechanism to direct preformed pools of toward the cell surface. In view of our results, this possibility appears at present unlikely.
Our study establishes cotranslational assembly of FcRI as the first quality control mechanism for the generation of functional IgE-binding FcRI. The dysregulation of this quality control process might contribute to the expression of high FcRI levels in allergic patients. We show that the ER quality control system regulates quantities of functional FcRI. It thereby controls onset and persistence of allergic reactions and might thus be a target for therapeutic interventions.
Materials and Methods
DNA constructs
The cDNAs encoding FcRI, FcRI?, FcRI?var, and FcRI were provided by the laboratory of J.-P. Kinet (Laboratory of Allergy and Immunology, Beth Israel Deaconess Medical Center, Boston, MA) (13). Signal peptide encoding sequences of and were exchanged for the signal peptide of mouse class I heavy chain H2-Kb (16). All cDNAs were cloned into pcDNA3.1 under the control of the T7 promotor. ? and ?var were also subcloned into pIRES2-EGFP (CLONTECH Laboratories, Inc.). NH2-terminal EGFP fusion proteins of ? and ?var were generated in pEGFP-C1 (CLONTECH Laboratories, Inc.). A COOH-terminal Kb-HA fusion protein was generated and expressed in pcDNA3.1.
Antibodies and antisera
cIgE (Serotec) and mAb 15–1 recognize the IgE-binding epitope of and were used for the detection of properly folded and N-glycosylated form of the protein (11, 12). Polyclonal rabbit anti- recognizes all forms of the protein irrespective of its folding status. Antisera against , ?, and are published and were used as described in the literature (13). GFP was generated by immunizing rabbits with the bacterially expressed GFP and used as previously described (32). HA-tagged proteins were precipitated with mAb 12CA5 and detected with HRP-conjugated rat mAb 3F10 (Roche).
Cell lines and transient transfections
293, CHO? and CHO cells were maintained in DMEM as previously described (13, 23). Various constructs of ? and ?var were expressed in 293, CHO, or CHO? cells by transient transfection, using a liposome-mediated transfection protocol (5–10 μg of DNA, 20 μl of lipofectamine, 10 cm dish; Lipofectamine; GIBCO BRL) as previously described (33). Cells were analyzed between 24 and 48 h after transfection.
In vitro transcription and translation
Both methods were essentially performed as previously described (16, 25, 26). In vitro transcriptions were performed using T7 polymerase (Promega). All cDNAs were subcloned into pcDNA3.1. After linearization, T7 polymerase was used for in vitro transcription (Promega). RNA was caped as previously described and stored as alcohol precipitates at –80°C. Before translation, RNA was decaped. Optimal amount of the individual RNAs was determined empirically for each individual receptor subunit and each stock of RNA. RNAs were stored as alcohol precipitates at –80°C. The optimal reaction time of the in vitro translation was determined empirically as 1 h. Reticulocyte lysate was purchased from Promega. Microsomes were prepared from various cell lines as previously described and pelleted after in vitro translations for further analysis as previously described (26). Complex precipitations of FcRI were performed in 1% Brij 96 lysates as previously described (11).
Metabolic labeling of cell, pulse-chase experiments, immunoprecipitation, enzymatic digestion, and immunoblotting 293 cells were detached, followed by starvation in methionine-/cysteine-free DME for 60 min at 37°C. Cells were metabolically labeled with 500 μCi of methionine/cysteine (1,200 Ci/mM; NEN)/ml at 37°C for the times indicated. Pulse-chase experiments, cell lysis, and immunoprecipitations were performed as previously described (33). 1% Brij96 lysis buffer was used to maintain the integrity of FcRI complexes as previously described (11). The immunoprecipitates were analyzed by SDS-PAGE followed by fluorography (38). Endo H (New England Biolabs, Inc.) digestions was performed as described by the manufacture. HA immunoblots were performed with SDS lysates under nonreducing conditions (23).
Flow cytometry analysis
Quantitative flow cytometry analysis of cells expressing constructs in pIRES2-EGFP in living cells was performed by FACS? (FACS?Calibur; BD, Mountain View, CA) supported by CellQuest software (BD). IgE-binding epitopes of FcRI were stained with mAb15-1 or biotinylated IgE as previously described (13, 23, 28–30).
Immunostaining and epifluorescence microscopy
Immunofluorescence experiments were performed essentially as previously described (39) with minor modifications as follows. Cells were allowed to attach to slides overnight before inhibitor incubation (ZL3VS; reference 40; 4 h 10 μM final from a DMSO stock). DMSO was used as solvent control. After fixation with 3.7% paraformaldehyde for 20 min at room temperature immunohistochemistry was performed in a 0.5% saponin/3% BSA/PBS solution. mAbs 15–1 was used to define chains that exhibit properly folded IgE-binding epitopes (13, 23). Polyclonal anti- serum was used to show all forms of irrespective of their folding or glycosylation status (13). Anti-mouse Alexa Fluor 568 (Molecular Probes) and anti-rabbit Alexa Fluor 568 (Molecular Probes) were used as the fluorescent probe. Further analysis was performed with a Bio-Rad epifluorescence microscope as previously described (39).
Acknowledgments
This study was supported by the Sandler Program for Asthma Research. During the course of this study Edda Fiebiger was supported by the APART Program of the Austrian Academy of Sciences and the Charles A. King Trust, Fleet National Bank, a Bank of America Company, Co-Trustee (Boston, MA).
References
Sutton, B.J., and H.J. Gould. 1993. The human IgE network. Nature. 366:421–428.
Cookson, W.O., and M.F. Moffatt. 1997. Asthma: an epidemic in the absence of infection? Science. 275:41–42.
Metzger, H. 1991. The high affinity receptor for IgE on mast cells. Clin. Exp. Allergy. 21:269–279.
Mudde, G.C., I.G. Reischl, N. Corvaia, A. Hren, and E.M. Poellabauer. 1996. Antigen presentation in allergic sensitization. Immunol. Cell Biol. 74:167–173.
Kinet, J.P. 1999. The high-affinity IgE receptor (Fc epsilon RI): from physiology to pathology. Annu. Rev. Immunol. 17:931–972.
Kinet, J.P. 1999. Atopic allergy and other hypersensitivities. Curr. Opin. Immunol. 11:603–605.
Reth, M. 1989. Antigen receptor tail clue. Nature. 338:383–384.
Ravetch, J.V., and J.-P. Kinet. 1991. Fc Receptors. Annu. Rev. Immunol. 9:457–492.
Kinet, J.-P., M.-H. Jouvin, R. Paolini, R. Numerof, and A. Scharenberg. 1996. IgE receptor (FcRI) and signal transduction. Eur. Respir. J. 9:116s–118s.
Turner, H., and J.P. Kinet. 1999. Signalling through the high-affinity IgE receptor Fc epsilonRI. Nature. 402:B24–B30.
Maurer, D., E. Fiebiger, C. Ebner, B. Reininger, G.F. Fischer, S. Wichlas, M.-H. Jouvin, M. Schmitt-Egenolf, D. Kraft, J.-P. Kinet, and G. Stingl. 1996. Peripheral blood dendritic cells express FcRI as a complex composed of FcRI- and FcRI-chains and can use this receptor for IgE-mediated allergen presentation. J. Immunol. 157:607–613.
Maurer, D., C. Ebner, B. Reininger, E. Fiebiger, D. Kraft, J.-P. Kinet, and G. Stingl. 1995. The high affinity IgE receptor (FcRI) mediates IgE-dependent allergen presentation. J. Immunol. 154:6285–6290.
Donnadieu, E., M.H. Jouvin, S. Rana, M.F. Moffatt, E.H. Mockford, W.O. Cookson, and J.P. Kinet. 2003. Competing functions encoded in the allergy-associated F(c)epsilonRIbeta gene. Immunity. 18:665–674.
Donnadieu, E., M.H. Jouvin, and J.P. Kinet. 2000. A second amplifier function for the allergy-associated Fc(epsilon)RI-beta subunit. Immunity. 12:515–523.
Dombrowicz, D., S. Lin, V. Flamand, A.T. Brini, B.H. Koller, and J.P. Kinet. 1998. Allergy-associated FcRbeta is a molecular amplifier of IgE- and IgG-mediated in vivo responses. Immunity. 8:517–529.
Huppa, J.B., and H.L. Ploegh. 1997. In vitro translation and assembly of a complete T cell receptor-CD3 complex. J. Exp. Med. 186:393–403.
Call, M.E., J. Pyrdol, M. Wiedmann, and K.W. Wucherpfennig. 2002. The organizing principle in the formation of the T cell receptor-CD3 complex. Cell. 111:967–979.
Jahn-Schmid, B., U. Wiedermann, B. Bohle, A. Repa, D. Kraft, and C. Ebner. 1999. Oligodeoxynucleotides containing CpG motifs modulate the allergic TH2 response of BALB/c mice to Bet v 1, the major birch pollen allergen. J. Allergy Clin. Immunol. 104:1015–1023.
Dobson, C.M. 2004. Principles of protein folding, misfolding and aggregation. Semin. Cell Dev. Biol. 15:3–16.
Goldberg, A.L. 2003. Protein degradation and protection against misfolded or damaged proteins. Nature. 426:895–899.
Sitia, R., and I. Braakman. 2003. Quality control in the endoplasmic reticulum protein factory. Nature. 426:891–894.
Benaroudj, N., E. Tarcsa, P. Cascio, and A.L. Goldberg. 2001. The unfolding of substrates and ubiquitin-independent protein degradation by proteasomes. Biochimie. 83:311–318.
Maurer, D., E. Fiebiger, B. Reininger, B. Wolff-Winiski, M.-H. Jouvin, O. Kilgus, J.-P. Kinet, and G. Stingl. 1994. Expression of functional high affinity immunoglobulin E receptors (FcRI) on monocytes of atopic individuals. J. Exp. Med. 179:745–750.
Bieber, T. 1994. FcRI on human Langerhans cells: a receptor in search of new functions. Immunol. Today. 15:52–53.
Bijlmakers, M.J., P. Benaroch, and H.L. Ploegh. 1994. Assembly of HLA DR1 molecules translated in vitro: binding of peptide in the endoplasmic reticulum precludes association with invariant chain. EMBO J. 13:2699–2707.
Furman, M.H., H.L. Ploegh, and D. Tortorella. 2002. Membrane-specific, host-derived factors are required for US2- and US11-mediated degradation of major histocompatibility complex class I molecules. J. Biol. Chem. 277:3258–3267.
Hirsch, C., S. Misaghi, D. Blom, M.E. Pacold, and H.L. Ploegh. 2004. Yeast N-glycanase distinguishes between native and non-native glycoproteins. EMBO Rep. 5:201–206.
Wang, B., A. Rieger, O. Kilgus, K. Ochiai, D. Maurer, D. Fodinger, J.P. Kinet, and G. Stingl. 1992. Epidermal Langerhans cells from normal human skin bind monomeric IgE via Fc epsilon RI. J. Exp. Med. 175:1353–1365.
Osterhoff, B., K. Rappersberger, B. Wang, F. Koszik, K. Ochiai, J.P. Kinet, and G. Stingl. 1994. Immunomorphologic characterization of Fc epsilon RI-bearing cells within the human dermis. J. Invest. Dermatol. 102:315–320.
Klubal, R., B. Osterhoff, B. Wang, J.P. Kinet, D. Maurer, and G. Stingl. 1997. The high-affinity receptor for IgE is the predominant IgE-binding structure in lesional skin of atopic dermatitis patients. J. Invest. Dermatol. 108:336–342.
Albrecht, B., M. Woisetschlager, and M.W. Robertson. 2000. Export of the high affinity IgE receptor from the endoplasmic reticulum depends on a glycosylation-mediated quality control mechanism. J. Immunol. 165:5686–5694.
Fiebiger, E., C. Story, H.L. Ploegh, and D. Tortorella. 2002. Visualization of the ER-to-cytosol dislocation reaction of a type I membrane protein. EMBO J. 21:1041–1053.
Fiebiger, E., C. Hirsch, J.M. Vyas, E. Gordon, H.L. Ploegh, and D. Tortorella. 2004. Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin. Mol. Biol. Cell. 15:1635–1646.
Tokunaga, F., C. Brostrom, T. Koide, and P. Arvan. 2000. Endoplasmic reticulum (ER)-associated degradation of misfolded N-linked glycoproteins is suppressed upon inhibition of ER mannosidase I. J. Biol. Chem. 275:40757–40764.
Ra, C., M.H. Jouvin, U. Blank, and J.P. Kinet. 1989. A macrophage Fc gamma receptor and the mast cell receptor for IgE share an identical subunit. Nature. 341:752–754.
Kurosaki, T., I. Gander, U. Wirthmueller, and J.V. Ravetch. 1992. The beta subunit of the Fc epsilon RI is associated with the Fc gamma RIII on mast cells. J. Exp. Med. 175:447–451.
Dombrowicz, D., V. Flamand, I. Miyajima, J.V. Ravetch, S.J. Galli, and J.P. Kinet. 1997. Absence of Fc epsilonRI alpha chain results in upregulation of Fc gammaRIII-dependent mast cell degranulation and anaphylaxis. Evidence of competition between Fc epsilonRI and Fc gammaRIII for limiting amounts of FcR beta and gamma chains. J. Clin. Invest. 99:915–925.
Ploegh, H.L. 1995. Current Protocols in Protein Science. Wiley, New York. 10.12.11–10.12.18.
Bertho, N., J. Cerny, Y.M. Kim, E. Fiebiger, H. Ploegh, and M. Boes. 2003. Requirements for T cell-polarized tubulation of class II+ compartments in dendritic cells. J. Immunol. 171:5689–5696.
Wiertz, E.J., T.R. Jones, L. Sun, M. Bogyo, H.J. Geuze, and H.L. Ploegh. 1996. The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell. 84:769–779.(Edda Fiebiger1, Domenico )
2 Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029
3 Laboratory of Allergy and Immunology, Beth Israel Deaconess Medical Center, Boston, MA 02215
CORRESPONDENCE Hidde Ploegh: ploegh@hms.harvard.edu
The human high affinity receptor for IgE (FcRI) is a cell surface structure critical for the pathology of allergic reactions. Human FcRI is expressed as a tetramer (?2) on basophils or mast cells and as trimeric (2) complex on antigen-presenting cells. Expression of the human subunit can be down-regulated by a splice variant of FcRI? (?var). We demonstrate that FcRI is the core subunit with which the other subunits assemble strictly cotranslationally. In addition to ?2 and 2, we demonstrate the presence of ? and ?var2 complexes that are stable in the detergent Brij 96. The role of individual FcRI subunits for the formation of functional, immunoglobulin E–binding FcRI complexes during endoplasmic reticulum (ER) assembly can be defined as follows: ? and support ER insertion, signal peptide cleavage and proper N-glycosylation of , whereas ?var allows accumulation of protein backbone. We show that assembly of FcRI in the ER is a key step for the regulation of surface expression of FcRI. The ER quality control system thus regulates the quantity of functional FcRI, which in turn controls onset and persistence of allergic reactions.
Abbreviations used: CC, U373; EndoH, endoglycosidase H; ITAM, immunoreceptor tyrosine–based signaling motifs.
A significant fraction of the population (20%) in the Western world is affected by allergies and the numbers of affected individuals is on the rise (1, 2). Convincing evidence exists that FcRI is one of the key molecules in the pathophysiology of all allergic reactions (3–6). As a member of the antigen receptor superfamily, FcRI shares the organizational principles of a ligand binding immunoglobulin-type protein associated with signaling subunits that regulate cellular activation via conserved immunoreceptor tyrosine–based signaling motifs (ITAMS; 7). BCR, TCR, and other Fc receptors fall in the same class (7–10). FcRI was initially described as a tetrameric receptor composed of a high-affinity ligand-binding chain, one ? chain, and a pair of disulphide-linked subunits (5, 9). The FcRI complexes on the surface of basophils and mast cells are tetrameric structures (?2). The ?2 is the only receptor isoform formed in rodents (5). Human antigen-presenting cells additionally display a trimeric form of FcRI that lacks the ? subunit (5, 11, 12). A new splice variant of FcRI? (?var, formerly referred to as ?T) exerts a dominant negative effect on ? function (13).
The structural integrity of FcRI is maintained by the noncovalent interactions of its various subunits. The extracellular domain of FcRI forms the binding site for the CH3 domain of IgE. It binds its ligand in 1:1 ratio, with an affinity of 1010 M–1. The ? chain contains four potential transmembrane spanning regions with both the NH2 and the COOH terminus protruding into the cytosol. FcRI forms a dimer and is a member of the gene family. IgE-dependent cross-linking of FcRI induces cellular activation regulated via ITAMs, which are present in one copy in the ? as well as in each of the chains (5, 9, 10). The subunit, when expressed in the absence of ? and , is retained in the ER. The ER retention signal of human can be overcome by the presence of alone. FcRI? was defined as an amplifier for chain signaling in vitro and in vivo (14, 15) and as a regulator for surface expression. The ?var subunit is a splice variant that has lost its ITAM (13). Therefore ?var-containing complexes must behave significantly differently from those that contain the conventional ? chain.
Multisubunit receptor complexes, like FcRI or the TCR, are assembled in the ER, from where they enter the secretory pathway (16, 17). The acquisition of the proper tertiary and quaternary structure in the ER is a carefully controlled sequence of events. Nascent polypeptides are subject to modifications, which often include signal–peptide cleavage, N-linked glycosylation and oligosaccharide trimming. Folding of proteins is guided by chaperones such as BiP and the lectins calnexin and calreticulin. Oxido-reductases control the formation of disulfide bonds between the correct pairs of cysteine residues to stabilize the folded structure (18). As a consequence of imperfections in protein folding, some polypeptides never attain their native conformation. Terminally misfolded proteins are singled out in the ER by a quality control process (19–21). However, their destruction takes place mostly in the cytosol. ER quality control substrates may cross the ER membrane before their degradation (22). In addition to the proper folding of the individual subunits, multimeric receptors like FcRI must assemble in a concerted fashion. Only with all players in place, can ER retention signals be overcome. The sequence of events in FcRI receptor assembly in its various configurations is interesting with respect to the functional differences of the receptor isoforms. These events all contribute to the control of receptor expression and thereby the outcome of allergic responses in vivo. The generally increased cell surface expression of FcRI in allergic individuals supports this hypothesis (23, 24).
Studies on the human receptor are hampered by a lack of cell lines that express FcRI irrespective of its isoforms. Primary human cells that express FcRI are difficult to obtain, even in small numbers. Because these cells shut down synthesis of the receptor immediately after isolation, they cannot be used to study complex formation and regulation of surface expression. We therefore used in vitro translation in membrane-supplemented rabbit reticulocyte lysates to study the early events of FcRI assembly in the ER. We translated the corresponding mRNAs of all FcRI subunits and performed studies on temporal aspects of protein–protein interaction and their consequences for receptor assembly. Our results show that ER assembly of the individual FcRI subunits is tightly controlled and indeed regulates the formation of properly formed receptors with IgE-binding epitopes.
Results
In vitro translation of FcRI
We used in vitro translation as a method to study FcRI receptor assembly in the ER (16, 25, 26), because aspects of multimeric receptor assembly cannot be studied in a time-resolved fashion in transfection experiments. First, we characterized the properties of the individual receptor subunits in this assembly system. The cDNAs for the human FcRI subunits allow the generation of the corresponding mRNAs for in vitro translations. The mRNAs were translated in the presence of microsomes from different sources. FcRI is a type I membrane protein and requires cleavage of its signal sequence before N-glycosylation, which is in turn required for the formation of functional IgE-binding sites (5, 9). FcRI cDNA equipped with its endogenous signal peptide translated poorly. Although we could detect the expected polypeptides reactive with anti– serum, cotranslation of the construct with ? and mRNAs would have rendered further assembly studies technically difficult (unpublished data). We therefore exchanged the signal sequence of the subunit for that of H2-Kb. The latter has proved efficient for translation as well as for adjustment of insertion efficiencies of different subunits during TCR assembly (16). This swap of signal peptides allowed efficient translation of FcRI (H2-KbFcRI, referred to as ; Fig. 1 A). The source of microsomes proved critical for the generation of chains with cleaved signal peptide (–sig, Fig. 1 A) as well as for N-glycosylation (Nglyc, Fig. 1 A), although no such effect was observed for FcRI or HLA-2A (unpublished data). Microsomes from the basophilic cell line KU812 allowed efficient translation, but yielded mostly as 1sig (Fig. 1 A). Microsomes derived from canine pancreas were successful in creating Nglyc, but a sizable fraction of the translated protein was present as 1sig (Fig. 1 A and unpublished data). Microsomes derived from the astrocytoma cell line U373 (CC) reproducibly generated endoglycosidase H (EndoH)-sensitive Nglyc efficiently (Fig. 1 A).
Figure 1. In vitro translation of IgE-binding FcRI. (A) Signal peptide cleavage and N-glycosylation of FcRI depends on the source of microsomes used during in vitro translation. Transcripts of Kb-FcRI were translated for 1 h at 30°C in the presence of microsomes derived from KU812, dog pancreas (dpm), or U373 (CC). After lysis of the microsomal pellets in 1% Brij96 lysis buffer, immunoprecipitates were obtained with polyclonal anti- serum and either mock digested or digested with Endo H. Subsequently immunoprecipitates were analyzed by 12.5% SDS-PAGE under reducing conditions. Conversion of +sig to Nglyc occurs most efficiently in CC microsomes. (B) Nglyc generated with CC microsomes expresses properly folded IgE-binding epitopes. IgE selectively precipitates the fully N-glycosylated form of FcRI. Representative experiments (n = 3).
We then asked whether we could generate FcRI with proper IgE-binding epitopes, using IgE as the bait to recover translation products from microsomal pellets (Fig. 1 B). The direct analysis of the microsomal extracts shows the presence of all forms of FcRI (1sig, 2sig, and Nglyc, Fig. 1 B), but only properly folded and N-glycosylated is recovered by IgE (Fig. 1 B).
In vitro translation of FcRI? and FcRI?var
The identity of the translation products from ? and ?var (13) was confirmed by immunoprecipitation with an anti-? serum generated against the NH2 terminus of ?, therefore reactive with both splice variants (Fig. 2 A). Indeed, both proteins migrate at their expected molecular weight of 28,000 and 22,000, respectively (Fig. 2 A; reference 13).
Figure 2. In vitro translation of FcRI? and FcRI?var. (A) Transcripts of FcRI? or FcRI?var were translated for 1 h at 30°C in the presence of CC microsomes. After lysis of the microsomal pellets in 1% Brij96 lysis buffer, immunoprecipitates were obtained with polyclonal anti-? serum and analyzed by 12.5% SDS-PAGE under reducing conditions. Representative experiments (n = 3).
In vitro translation of FcRI
The chain is a type I membrane protein (3, 8, 9). To achieve comparable translation efficiencies, we exchanged the signal sequence of that of H2-Kb (H2-KbFcRI, referred to as ; Fig. 3). Fig. 3 A shows the proper insertion and signal peptide cleavage of in the in vitro translation assay with a cysteine translation mix. The single methionine present in is removed upon insertion into the microsomes. However, labeling with the methionine translation mix (containing both Met and Cys) allows sufficient labeling as shown in anti- immunoprecipitations (Fig. 3 B). For reasons that remain unclear, in vitro translations using cysteine did not allow visualization of ?var (unpublished data). Thus, all subsequent translation assays were performed with the methionine translation mix. Under nonreducing conditions, runs as a dimer with both translation mixes (Fig. 3, C and D).
Figure 3. ER insertion and proper signal peptide cleavage of FcRI. (A) Proper insertion and signal peptide cleavage of Kb- in an in vitro translations with a cysteine translation mix. (B) In vitro translations of Kb- with a methionine translation mix. The single methionine of gets cleaved after insertion into the microsomes. Cysteine present in the methionine translation mix is sufficient for labeling of Kb-. In vitro translations were performed in the absence and presence of CC microsomes. Translation products were analyzed after immunoprecipitation from 1% Brij96 lysates of in vitro translations performed in the absence of microsomes (–) or lysates of microsomal pellets (+) and analyzed by 12.5% SDS-PAGE under reducing (A and B) or nonreducing (C and D) conditions. FcRI forms a dimer under nonreducing conditions. Representative experiments (n = 3).
Receptor assembly studies: complexes form cotranslationally
The next set of experiments addressed the existence of complexes and their assembly. Anti- immunoprecipitations from Brij 96 lysates of microsomal pellets demonstrate the presence of stable complexes (Fig. 4 A, lane 4). Such complexes arise only if both proteins are translated at the same time. Microsomal pellets from assay mixtures in which and RNA were translated consecutively are devoid of complexes, despite the presence of both proteins in the direct load of the microsomes (Fig. 4 A, lanes 1 and 3). Complexes of where absent when proteins were translated separately and then mixed before or after lysis (Fig. 4 B; and unpublished data). Direct loads of microsomes from translation mixtures in which and were present concurrently generally show more properly folded Nglyc when compared with samples in which was translated alone. This effect is even more pronounced when ? is cotranslated as well (unpublished data and see Fig. 6 A). Immunoprecipitations of complexes with serum directed against confirmed the association of 1sig, 2sig, and Nglyc with (Fig. 4 C).
Figure 4. FcRI complexes form cotranslationally. (A) Cotranslational formation of complexes. and RNA were translated consecutively (lanes 1 and 3, 1st 2nd) or at the same time (lane 2 and 4, + ) into CC microsomes. Direct loads of the microsomal pellets confirmed the presence of both translation products (lanes 1 and 2). complexes were only retrieved by anti- immunopreciptiation, if both proteins were translated at the same time (lane 4, + ). (B) and were translated separately and microsomal pellets were mixed before lysis. Direct loads of microsomes showed the presence of both proteins. No complexes were precipitated. (C) Immunoprecipitations of complexes with serum directed against .
Cotranslation of ? and chains resulted in efficient insertion of both proteins into microsomes (Fig. 5 A, lane 1). Neither serum coprecipitated the other protein. These experiments control for proper solubilization under the necessary mild lysis conditions and further show that ? complexes do not occur in the absence of (Fig. 5 A, lanes 2 and 3). We also failed to detect ? complexes when ? and mRNAs were translated separately and microsomes were mixed before lysis (Fig. 5 D and E, lanes 2) or when microsomal pellets of cotranslation experiments were lysed in 1% digitonin (unpublished data).
Figure 5. Association studies of different FcRI subunits. (A) Cotranslation of ? and chains results in efficient expression of both proteins in microsomes. Anti-? and - serum both failed to coprecipitate ? complexes. (B) Demonstration of ? complexes. Anti- serum precipitates stable ? complexes. The presence of ? is in the complex is confirmed by anti-? reimmunoprecipitations. (C) Confirmation of ? complexes in 293 cells transiently transfected with HA and GFP-? cDNA. (D and E) The ? and subunits associate with but not HLA-A2. FcRI complexes form only cotranslationally. Direct loads of microsomes (D) in which proteins were translated separately and mix before lysis (lanes 1–3) and microsomes from cotranslations (lanes 4–6) were compared with immunoprecipitations. Complexes were precipitated from 1% Brij96 lysates of in vitro translations from CC pellets and analyzed by 12.5% SDS-PAGE under reducing conditions. Representative experiments (n = 3).
These results fit with the assumption that the chain is the core of all FcRI complexes. This hypothesis also implies a direct interaction of with ?. We therefore attempted to demonstrate the existence of such complexes by in vitro translation. As shown in Fig. 5 B, the ? complex is stable in Brij96 and is generated only cotranslationally (Fig. 5, D and E, lanes 3). Due to its molecular weight, ? is difficult to distinguish from 1sig and 2sig. The anti-? reimmunoprecipitation unequivocally demonstrates the existance of ? complexes (Fig. 5 B, lane 2). We could also demonstrate the presence of these complexes on a cellular level by immunoprecipitations from 293 cells transiently transfected with and ? complexes (Fig. 5 C). The use of tagged versions of both proteins allowed the detection of the individual subunits by immunoblotting after immunoprecipitation. The ? complexes can be retrieved specifically with an anti-HA reagent via HA (Fig. 5 C, lanes 2–4), but not with a control Ab (Fig. 5 C, lane 1). A considerable amount of this protein becomes EndoH resistant, indicative of modifications of N-glycans in the Golgi apparatus (mod; Fig. 5 C, lane 3) and thus proper ER exit of ? complexes. Digestion of immunopreciptated ? complexes with PNGase was performed to provide further evidence for proper folding of . PNGase preferentially attacks improperly folded glycoproteins (27), and the resistance of to digestion by this enzyme thus supports our findings that is expressed as properly folded protein in ? complexes.
Cotranslation of HLA-A2 with ? and were performed as specificity controls (Fig. 5 D, lanes 4 and 6). As expected, HLA-A2 failed to form a complex with ? and (Fig. 5 E, lanes 4 and 6).
Immunoprecipitation of ? and ?var complexes
The ,?, , or ,?var, mRNAs were translated into microsomes, which were then solibilized in 1% Brij96 and subjected to immunoprecipitation. The anti- but not the control serum successfully precipitated ? complexes (Fig. 6 A). The anti- reagent also retrieved stable ?var complexes (Fig. 6 A). The chain in the latter complexes seemed to be underrepresented when compared with ?.
Figure 6. Cotranslational formation of tetrameric FcRI complexes. (A) ,?, , or ,?var, mRNAs were simultaneously translated into CC microsomes. Microsomal pellets were solibilized in 1% Brij96 lysis buffer and immunoprecipitations with anti- or control serum were performed. Stable ? as well as ?var complexes were retrieved by anti-. (B) ?var induces the accumulation of +sig. ?var mRNAs were cotranslated and direct loads of in vitro translations were compared with anti- immunoprecipitates. All translated proteins with a prominent band of 33 kD are present in direct loads of CC mircosomes translated with ?var . Anti- immunoprecipitation shows an overrepresentation of +sig when compared with translations of alone. Proteins were precipitated from 1% Brij96 lysates of in vitro translations from CC pellets and analyzed by 12.5% SDS-PAGE under reducing conditions. Representative experiments (n = 3).
?var induces the accumulation of +sig
We next examined the fate of when translated in the presence of ?var. For this purpose ?var mRNAs were cotranslated and direct loads of microsomal pellets were compared with anti- immunoprecipitates to assess more carefully all forms of present in the translation mix (Fig. 6 B). We detected the presence of all translated proteins, with a prominent band of 33 kD. Anti- immunoprecipitation confirmed the nature of this polypeptide as +sig (Fig. 6 B). The chain as well as Nglyc and –sig were coprecipitated. For unknown reasons, we were unable to directly demonstrate ?var in these precipitates. This finding might again reflect a decrease in the stability of ?var complexes, with ?var dissociating before , or equally likely, a more general problem of detection of ?var. As in cellular expression systems (13), ?var is rapidly lost from in vitro translation mixtures (unpublished data).
?var down-regulates surface IgE-binding epitopes
We subcloned ? and ?var into a bicistronic vector with EGFP (pIRES2-?-EGFP and pIRES2-?var-EGFP). Next, 293 cells were transiently transfected and treated with proteasome inhibitor for 2h. After SDS lysis, immunoblots with anti-? serum were performed to confirm the proper expression of both proteins (Fig. 7 A).
Figure 7. FcRI?var induces the accumulation of 1sig in vivo. (A) ?var down-regulates surface IgE-binding epitopes. ? and ?var were subcloned into a bicistronic vector expressing EGFP to control for equal expression levels (pIRES2-?-EGFP and pIRES2-?var-EGFP). 293 cells were transiently transfected and treated with proteasome inhibitor for 2 h. After SDS lysis, immunoblots with anti-? serum were preformed to demonstrate the expression of both proteins. (B) mAb 15–1 recognizes the IgE-binding epitope of FcRI. IgE binding capacity of CHO was assessed with biotinylated IgE (black). Preincubation with mAb 15–1 (reactivity shown in blue) inhibits subsequent IgE binding (red). Mean fluorescence is shown on the abscissa. (C) CHO? were transiently transfected with pIRES2-?-EGFP or pIRES2-?var-EGFP (? (red) and ?var(black)). Reactivity of mAb 15–1, which is specific for the IgE-binding epitope, was monitored on the surface of a cell population gated for EGFP-expression as a marker for expression of ? and ?var. pIRES2-?var-EGFP specifically down-regulates surface 15–1 reactivity. (D) ?var induces accumulation of unglycosylated protein backbone in vivo. A HA-tagged version of Kb- (HA) was transiently transfected into 293 cells in the presence of the indicated FcRI subunits. Transfection was followed by anti-HA immunoprecipitation and anti-HA immunoblotting. HA folds properly and becomes N-glycosylated (Nglyc) in the absence of other complex subunits (lane 1). Cotransfection of ? (lane 4) or ?var (lane 3) induces Golgi-associated glycosylation modification of HA (mod). Unglycosylated protein backbone accumulates specifically in the presence of ?var. (E) ?var induces the accumulation of +sig. Metabolic labeling of cells transiently transfected with HA, ? and followed by anti-HA immunoprecipitations and EndoH digestion. Most HA is rapidly transformed into its fully N-glycosylated modification (Nglyc). Susceptibility to EndoH treatment defines +sig.
We verified that mAb 15–1 recognizes the IgE-binding epitope of FcRI (13, 23, 28–30). IgE binding capacity of CHO was assessed by FACS with biotinylated IgE (Fig. 7 B, filled black). CHO show comparable reactivity when stained with 15–1 (Fig. 7 B, blue). Preincubation of cells with 15–1 inhibits subsequent IgE binding (Fig. 7 B, red). The mean fluorescence intensity (MFI) of IgE-reactivity drops from 370 to levels of the negative control (Fig. 7 B, black line, MFI=10). This result is in accordance with the literature (13, 23, 28–30) and confirms that 15–1 recognizes the IgE-binding site of FcRI. The fact that both reagents recognize the same epitope also accounts for the misinterpretation of cellular distribution patterns of FcRI in humans. Endogenous IgE bound to FcRI precludes recognition with mAb 15–1 or biotinylated IgE unless the natural ligand is removed by acid stripping (23, 28–30).
We show that our bicistronic constructs regulate the surface expression of IgE-binding epitopes as previously described (13). For this purpose, CHO? were transiently transfected with pIRES2-?-EGFP or pIRES2-?var-EGFP (Fig. 7 A, graph refers to ? and ?var ). Reactivity with mAb 15–1, which is specific for the IgE-binding epitope (5, 23), was monitored in a population gated for EGFP expression as a marker for successful transfection with ? or ?var. Although we observed surface expression of IgE-binding epitopes in cells transfected with pIRES2-?-EGFP, this surface marker was significantly down-regulated in cells transfected with pIRES2-?var-EGFP (Fig. 7 A, representative experiment). Transfections in CHO cells yield the same results (unpublished data). Our experiments confirm that ?var impairs formation of surface expressed IgE-binding epitopes in vivo and functions in a dominant way when coexpressed with ? in CHO cells (13).
?var induces accumulation of +sig in vivo
We next explored the mechanism by which ?var might interfere with the generation of IgE-binding epitopes. For this purpose we generated a COOH-terminally HA-tagged version of Kb- (HA) because the commonly used anti- reagents failed to detect the 30-kD protein backbone and yielded poor results when used for immunoprecipitation in pulse-chase experiments. Anti-HA immunoprecipitation followed by anti-HA immunoblotting on 293 cells transiently transfected with HA confirmed that HA is properly N-glycosylated in the absence of ? or subunits (Fig. 7 B, lane 1; 31). The presence of unglycosylated protein backbone was specific for the presence of ?var (Fig. 7 B, lane 3).
Metabolic labeling experiments were then performed to shown that the protein that accumulates in the presence of ?var is indeed +sig. Anti-HA immunoprecipitations followed by EndoH digestion were performed in cells transiently transfected with HA?var (Fig. 7 C). These experiments shown that most HA is transformed into its fully N-glycosylated modification irrespectively of the presence of the ?var subunits. Comparing its characteristic with EndoH-treated protein, the remaining HA protein can be identified as +sig (Fig. 7 C). We could thus confirm by both immunoblotting and by pulse labeling that ?var allows accumulation of +sig.
For more extended studies of the intracellular fate of , NH2-terminal EGFP fusion proteins of ? or ?var (GFP-? or GFP-?var) were generated. The fusion adds the expected 28 kD to the molecular mass but otherwise does not interfere with the molecular characteristics of either protein (reference 13; Fig. 8 A; and unpublished data). Pulse-chase analysis of GFP-? or GFP-?var demonstrates that both proteins are stabilized when inhibitors of the proteasome are present (Fig. 8 A). Pretreatment of cells with proteasome inhibitor ZL3VS (5 μm, 1 h; reference 32) and its presence throughout the pulse chase stabilize ? as well as ?var throughout the chase (Fig. 8 A). We infer that the ? subunits are subject to proteasomal proteolysis with ?var more susceptible to proteasomal degradation. GFP-? and GFP-?var should be informative reagents for the analysis of the fate of the subunit at the single cell level.
Figure 8. FcRI?var induces the accumulation of improperly folded FcRI in vivo. (A) NH2-terminal EGFP-fusion proteins of ? or ?var (GFP-? or GFP-?var) were transiently transfected into 293 cells with lipofectamine. The fusion adds the expected 28kD to the molecular weight but otherwise does not interfere with the molecular characteristics of ? or ?var. Pulse-chase analysis: cells were analyzed untreated or pretreated with proteasome inhibitor ZL3VS (5 μm, 2 h). Anti-GFP immunoprecipitates were obtained from 1% NP-40 lysates and analyzed by 12.5% SDS-PAGE under reducing conditions. Inhibition of the proteasome stabilizes GFP-?var. (B) CHO were transiently transfected with GFP-? and GFP-?var and analyzed by epifluorescence. Cells were treated with ZL3VS for 2 h, fixed and stained with mAb 15–1 to visualize IgE-binding epitopes. Staining with the anti- polyclonal serum was performed to visualize all forms of . Transfection with the GFP-? does not influence mAb 15–1 reactivity. Cells transfected with GFP-?var do not show mAb 15–1 reactivity but still remain positive with the anti- serum indicative for the presence of unfolded chain. Representative experiment (n = 4). (C, D) Pulse-chase experiments to confirm that FcRI not targeted to proteasomal degradation by ?var. FcRIHA was transiently transfected into 293 cells in the presence of ? or ?var cDNA. Transfection, immunoprecipitations and analysis were performed as described in (A). +sig is stabilized by the presence of ?var selectively (C). (D) Comparison of FcRI protein levels in ?var transfectants in the presence and absence of proteasome inhibitors. No alterations in the amount or expression pattern of FcRI in induced by the inhibitor.
To this end, CHO cells were transiently transfected with GFP-? and GFP-?var and analyzed by epifluorescence. Cells were treated with ZL3VS to inhibit proteasomal degradation for 2 h, fixed, and stained with mAb 15–1 to visualize the IgE-binding form of as previously described (23, 33). Staining with the anti- polyclonal serum was performed to visualize all forms of . CHO transfected with GFP-? are positive for both mAb 15–1 and the anti- serum (Fig. 8 B). In contrast, CHO transfected with GFP-?var do not stain with mAb 15–1 but still remain positive with the anti- serum (Fig. 8 B). Experiments performed with CHO? cells show identical results (unpublished data). It is important to note that inclusion of the proteasome inhibitor did not rescue the expression of IgE-binding epitopes. In agreement with our in vitro translation results, this experiment demonstrates that expression of GFP-?var interferes with proper folding and the formation of IgE-binding epitopes on the subunit. Cells transfected with GFP-?var still express readily detectable unfolded chain, suggestive of the mechanism by which ?var and GFP-?var down-regulate IgE-binding epitopes. In experiments without inhibition of proteasomal activity, GFP-?var is more difficult to detect but can still be visualized. Such cells also contain unfolded chain but are devoid of IgE-binding epitopes, as visualized by staining with anti- serum or 15–1, respectively (unpublished data).
Additionally we performed a set of pulse-chase experiments to confirm on the cellular level, that FcRI is indeed not targeted to proteasomal degradation by ?var. FcRIHA was transiently transfected into 293 cells in the presence of ? or ?var cDNA (Fig. 8 C). Immunoprecipitations were performed with anti-HA in 1% NP-40 lysis buffer to assure access to the total cellular pool of FcRI. No enhanced degradation of any form of FcRI was observed in cells transfected with ?var. In correlation with the results presented earlier in this study, the only significant difference was the persistence of +sig in the presence of ?var. The slight and progressive decrease in the molecular weight of Nglyc observed in all conditions is attributable to mannose trimming (32, 34). We failed to detect mod in this experimental setting. Next we compared FcRI protein levels in ?var transfectants in the presence and absence of proteasome inhibitors (Fig. 8 D). Although proteasomal inhibition stabilizes ?var (reference 13; Fig. 8 A; and unpublished data), we do not detect alterations in the amount or expression pattern of FcRI. Because inclusion of proteasome inhibitor and consequent stabilization of ?var do not change the fate of FcRI is not targeted to proteasomal degradation. We consider it unlikely that the short half-life of ?var is of functional importance for this mechanism.
Discussion
Allergen- and IgE-dependent cross-linking of FcRI is responsible for the immediate as well as the chronic inflammatory responses observed in atopic patients (5, 6). Surface expression of FcRI critically determines the sensitivity to an allergic stimulus and is therefore pivotal for the ensuing clinical response. The only defined extracellular regulator of FcRI surface expression defined so far is IgE, its natural ligand (5). With regard to intracellular regulation of receptor expression, FcRI? was described as an amplifier for chain signaling as well as for receptor surface expression (14, 15). In contrast, ?var has been shown to down-regulate surface expression of (13). The mechanistic basis of these regulatory events is poorly understood. Glycosylation-mediated quality control regulates ER export of FcRI (31). Here we show the efficiency of ER assembly is controlled by the presence of the different subunits. We were able to define the IgE-binding chain as the core of the receptor that pairs with the other subunits in a strictly cotranslationally regulated assembly event. These experiments establish ER assembly as a rate-limiting step in the expression of functional surface receptors, with obvious consequences for the onset of allergic diseases. In addition to the well-described FcRI isoforms, we were also able to demonstrate the existence of ?var and ? complexes, not previously documented.
Protein synthesis and folding in the ER are not always efficient: improperly folded structures are cleared from the ER and directed toward degradation (19–21). In addition to the proper folding of the individual subunits, multimeric receptors must assemble in a concerted fashion. Receptors such as FcRI or the TCR assemble in the ER and maintain their integrity by noncovalent interaction of the various subunits. Only with all players in place can ER retention signals be overcome (5, 9). For the TCR this process is well established and occurs in three consecutive assembly steps (17). Although FcRI and TCR share the same principal structure of ligand-binding and signal-transducing units and can even use the same chain for signaling (5), we show that their assembly is regulated differently. FcRI complexes form strictly cotranslationally. The presence of ? and clearly favors a conversion of into its IgE-binding form when compared with translation in the presence of alone. The ?var, on the other hand, slows down this conversion and induces the accumulation of unglycosylated with the signal peptide still in place.
Not all FcRI receptor subunit RNAs are generated in the same quantities in primary cells. When compared with , ? is always underrepresented (5). These observations were also confirmed at the protein level (11, 23). The demands of receptor stochiometry make cotranslational assembly of the receptor a key step that controls expression of functional IgE-binding epitopes at the cell surface. It may thereby affect the susceptibility to allergic stimuli in vivo. Receptor subunits can still fold properly, but do so at a less efficient rate when compared with folding in the presence of ? and . Such chains lack the necessary partners for complex formation during translation and consequently for ER exit. Our finding thus provides an explanation for the intracellular accumulation of in cell types that express high levels of , which in principle should allow surface expression (5). It is possible that in such cells, translation of and might not occur in synchrony and thus ER exit may be compromised. In addition to its critical role for FcRI signaling and ER exit, FcRI is a subunit of Fc receptors (35). Functional association of FcRI? with CD16, FcRIII, has also been demonstrated (36, 37). Both Fc receptor types might thus compete for and ? subunits, which adds an additional level of complexity and importance to assembly control. Because various Fc receptor subtypes and FcRI are expressed simultaneously in a variety of cell types (e.g., monocytes, dendritic cells, Langerhans cells, mast cells), this event is of physiological importance. Antigen presenting cells and monocytes of all individuals express Fc receptors. The proper assembly of Fc receptors could indirectly control the expression of FcRI by depleting the pool of receptor units that are required for FcRI ER exit. The functional consequences of ?var expression for cotranslational assembly need to be interpreted carefully, in particular with regard to the fact that so far transcripts of ?var were not depicted in absence of the classic ? chain.
Improperly assembled FcRI subunits are subjected to the ER quality control system and must be directed toward degradation. FcRI?var is a natural substrate for proteasomal degradation and is itself rapidly destroyed, irrespective of the presence of FcRI's other subunits (13). Whereas the subunit is also degraded by the proteasome, we found no evidence for a redirection of toward proteasomal degradation by ?var (unpublished data). When expressed alone, Nglyc is a rather stable protein. It remains to be established whether primary cells such as Langerhans cells, eosinophils, or monocytes of allergic patients have a mechanism to direct preformed pools of toward the cell surface. In view of our results, this possibility appears at present unlikely.
Our study establishes cotranslational assembly of FcRI as the first quality control mechanism for the generation of functional IgE-binding FcRI. The dysregulation of this quality control process might contribute to the expression of high FcRI levels in allergic patients. We show that the ER quality control system regulates quantities of functional FcRI. It thereby controls onset and persistence of allergic reactions and might thus be a target for therapeutic interventions.
Materials and Methods
DNA constructs
The cDNAs encoding FcRI, FcRI?, FcRI?var, and FcRI were provided by the laboratory of J.-P. Kinet (Laboratory of Allergy and Immunology, Beth Israel Deaconess Medical Center, Boston, MA) (13). Signal peptide encoding sequences of and were exchanged for the signal peptide of mouse class I heavy chain H2-Kb (16). All cDNAs were cloned into pcDNA3.1 under the control of the T7 promotor. ? and ?var were also subcloned into pIRES2-EGFP (CLONTECH Laboratories, Inc.). NH2-terminal EGFP fusion proteins of ? and ?var were generated in pEGFP-C1 (CLONTECH Laboratories, Inc.). A COOH-terminal Kb-HA fusion protein was generated and expressed in pcDNA3.1.
Antibodies and antisera
cIgE (Serotec) and mAb 15–1 recognize the IgE-binding epitope of and were used for the detection of properly folded and N-glycosylated form of the protein (11, 12). Polyclonal rabbit anti- recognizes all forms of the protein irrespective of its folding status. Antisera against , ?, and are published and were used as described in the literature (13). GFP was generated by immunizing rabbits with the bacterially expressed GFP and used as previously described (32). HA-tagged proteins were precipitated with mAb 12CA5 and detected with HRP-conjugated rat mAb 3F10 (Roche).
Cell lines and transient transfections
293, CHO? and CHO cells were maintained in DMEM as previously described (13, 23). Various constructs of ? and ?var were expressed in 293, CHO, or CHO? cells by transient transfection, using a liposome-mediated transfection protocol (5–10 μg of DNA, 20 μl of lipofectamine, 10 cm dish; Lipofectamine; GIBCO BRL) as previously described (33). Cells were analyzed between 24 and 48 h after transfection.
In vitro transcription and translation
Both methods were essentially performed as previously described (16, 25, 26). In vitro transcriptions were performed using T7 polymerase (Promega). All cDNAs were subcloned into pcDNA3.1. After linearization, T7 polymerase was used for in vitro transcription (Promega). RNA was caped as previously described and stored as alcohol precipitates at –80°C. Before translation, RNA was decaped. Optimal amount of the individual RNAs was determined empirically for each individual receptor subunit and each stock of RNA. RNAs were stored as alcohol precipitates at –80°C. The optimal reaction time of the in vitro translation was determined empirically as 1 h. Reticulocyte lysate was purchased from Promega. Microsomes were prepared from various cell lines as previously described and pelleted after in vitro translations for further analysis as previously described (26). Complex precipitations of FcRI were performed in 1% Brij 96 lysates as previously described (11).
Metabolic labeling of cell, pulse-chase experiments, immunoprecipitation, enzymatic digestion, and immunoblotting 293 cells were detached, followed by starvation in methionine-/cysteine-free DME for 60 min at 37°C. Cells were metabolically labeled with 500 μCi of methionine/cysteine (1,200 Ci/mM; NEN)/ml at 37°C for the times indicated. Pulse-chase experiments, cell lysis, and immunoprecipitations were performed as previously described (33). 1% Brij96 lysis buffer was used to maintain the integrity of FcRI complexes as previously described (11). The immunoprecipitates were analyzed by SDS-PAGE followed by fluorography (38). Endo H (New England Biolabs, Inc.) digestions was performed as described by the manufacture. HA immunoblots were performed with SDS lysates under nonreducing conditions (23).
Flow cytometry analysis
Quantitative flow cytometry analysis of cells expressing constructs in pIRES2-EGFP in living cells was performed by FACS? (FACS?Calibur; BD, Mountain View, CA) supported by CellQuest software (BD). IgE-binding epitopes of FcRI were stained with mAb15-1 or biotinylated IgE as previously described (13, 23, 28–30).
Immunostaining and epifluorescence microscopy
Immunofluorescence experiments were performed essentially as previously described (39) with minor modifications as follows. Cells were allowed to attach to slides overnight before inhibitor incubation (ZL3VS; reference 40; 4 h 10 μM final from a DMSO stock). DMSO was used as solvent control. After fixation with 3.7% paraformaldehyde for 20 min at room temperature immunohistochemistry was performed in a 0.5% saponin/3% BSA/PBS solution. mAbs 15–1 was used to define chains that exhibit properly folded IgE-binding epitopes (13, 23). Polyclonal anti- serum was used to show all forms of irrespective of their folding or glycosylation status (13). Anti-mouse Alexa Fluor 568 (Molecular Probes) and anti-rabbit Alexa Fluor 568 (Molecular Probes) were used as the fluorescent probe. Further analysis was performed with a Bio-Rad epifluorescence microscope as previously described (39).
Acknowledgments
This study was supported by the Sandler Program for Asthma Research. During the course of this study Edda Fiebiger was supported by the APART Program of the Austrian Academy of Sciences and the Charles A. King Trust, Fleet National Bank, a Bank of America Company, Co-Trustee (Boston, MA).
References
Sutton, B.J., and H.J. Gould. 1993. The human IgE network. Nature. 366:421–428.
Cookson, W.O., and M.F. Moffatt. 1997. Asthma: an epidemic in the absence of infection? Science. 275:41–42.
Metzger, H. 1991. The high affinity receptor for IgE on mast cells. Clin. Exp. Allergy. 21:269–279.
Mudde, G.C., I.G. Reischl, N. Corvaia, A. Hren, and E.M. Poellabauer. 1996. Antigen presentation in allergic sensitization. Immunol. Cell Biol. 74:167–173.
Kinet, J.P. 1999. The high-affinity IgE receptor (Fc epsilon RI): from physiology to pathology. Annu. Rev. Immunol. 17:931–972.
Kinet, J.P. 1999. Atopic allergy and other hypersensitivities. Curr. Opin. Immunol. 11:603–605.
Reth, M. 1989. Antigen receptor tail clue. Nature. 338:383–384.
Ravetch, J.V., and J.-P. Kinet. 1991. Fc Receptors. Annu. Rev. Immunol. 9:457–492.
Kinet, J.-P., M.-H. Jouvin, R. Paolini, R. Numerof, and A. Scharenberg. 1996. IgE receptor (FcRI) and signal transduction. Eur. Respir. J. 9:116s–118s.
Turner, H., and J.P. Kinet. 1999. Signalling through the high-affinity IgE receptor Fc epsilonRI. Nature. 402:B24–B30.
Maurer, D., E. Fiebiger, C. Ebner, B. Reininger, G.F. Fischer, S. Wichlas, M.-H. Jouvin, M. Schmitt-Egenolf, D. Kraft, J.-P. Kinet, and G. Stingl. 1996. Peripheral blood dendritic cells express FcRI as a complex composed of FcRI- and FcRI-chains and can use this receptor for IgE-mediated allergen presentation. J. Immunol. 157:607–613.
Maurer, D., C. Ebner, B. Reininger, E. Fiebiger, D. Kraft, J.-P. Kinet, and G. Stingl. 1995. The high affinity IgE receptor (FcRI) mediates IgE-dependent allergen presentation. J. Immunol. 154:6285–6290.
Donnadieu, E., M.H. Jouvin, S. Rana, M.F. Moffatt, E.H. Mockford, W.O. Cookson, and J.P. Kinet. 2003. Competing functions encoded in the allergy-associated F(c)epsilonRIbeta gene. Immunity. 18:665–674.
Donnadieu, E., M.H. Jouvin, and J.P. Kinet. 2000. A second amplifier function for the allergy-associated Fc(epsilon)RI-beta subunit. Immunity. 12:515–523.
Dombrowicz, D., S. Lin, V. Flamand, A.T. Brini, B.H. Koller, and J.P. Kinet. 1998. Allergy-associated FcRbeta is a molecular amplifier of IgE- and IgG-mediated in vivo responses. Immunity. 8:517–529.
Huppa, J.B., and H.L. Ploegh. 1997. In vitro translation and assembly of a complete T cell receptor-CD3 complex. J. Exp. Med. 186:393–403.
Call, M.E., J. Pyrdol, M. Wiedmann, and K.W. Wucherpfennig. 2002. The organizing principle in the formation of the T cell receptor-CD3 complex. Cell. 111:967–979.
Jahn-Schmid, B., U. Wiedermann, B. Bohle, A. Repa, D. Kraft, and C. Ebner. 1999. Oligodeoxynucleotides containing CpG motifs modulate the allergic TH2 response of BALB/c mice to Bet v 1, the major birch pollen allergen. J. Allergy Clin. Immunol. 104:1015–1023.
Dobson, C.M. 2004. Principles of protein folding, misfolding and aggregation. Semin. Cell Dev. Biol. 15:3–16.
Goldberg, A.L. 2003. Protein degradation and protection against misfolded or damaged proteins. Nature. 426:895–899.
Sitia, R., and I. Braakman. 2003. Quality control in the endoplasmic reticulum protein factory. Nature. 426:891–894.
Benaroudj, N., E. Tarcsa, P. Cascio, and A.L. Goldberg. 2001. The unfolding of substrates and ubiquitin-independent protein degradation by proteasomes. Biochimie. 83:311–318.
Maurer, D., E. Fiebiger, B. Reininger, B. Wolff-Winiski, M.-H. Jouvin, O. Kilgus, J.-P. Kinet, and G. Stingl. 1994. Expression of functional high affinity immunoglobulin E receptors (FcRI) on monocytes of atopic individuals. J. Exp. Med. 179:745–750.
Bieber, T. 1994. FcRI on human Langerhans cells: a receptor in search of new functions. Immunol. Today. 15:52–53.
Bijlmakers, M.J., P. Benaroch, and H.L. Ploegh. 1994. Assembly of HLA DR1 molecules translated in vitro: binding of peptide in the endoplasmic reticulum precludes association with invariant chain. EMBO J. 13:2699–2707.
Furman, M.H., H.L. Ploegh, and D. Tortorella. 2002. Membrane-specific, host-derived factors are required for US2- and US11-mediated degradation of major histocompatibility complex class I molecules. J. Biol. Chem. 277:3258–3267.
Hirsch, C., S. Misaghi, D. Blom, M.E. Pacold, and H.L. Ploegh. 2004. Yeast N-glycanase distinguishes between native and non-native glycoproteins. EMBO Rep. 5:201–206.
Wang, B., A. Rieger, O. Kilgus, K. Ochiai, D. Maurer, D. Fodinger, J.P. Kinet, and G. Stingl. 1992. Epidermal Langerhans cells from normal human skin bind monomeric IgE via Fc epsilon RI. J. Exp. Med. 175:1353–1365.
Osterhoff, B., K. Rappersberger, B. Wang, F. Koszik, K. Ochiai, J.P. Kinet, and G. Stingl. 1994. Immunomorphologic characterization of Fc epsilon RI-bearing cells within the human dermis. J. Invest. Dermatol. 102:315–320.
Klubal, R., B. Osterhoff, B. Wang, J.P. Kinet, D. Maurer, and G. Stingl. 1997. The high-affinity receptor for IgE is the predominant IgE-binding structure in lesional skin of atopic dermatitis patients. J. Invest. Dermatol. 108:336–342.
Albrecht, B., M. Woisetschlager, and M.W. Robertson. 2000. Export of the high affinity IgE receptor from the endoplasmic reticulum depends on a glycosylation-mediated quality control mechanism. J. Immunol. 165:5686–5694.
Fiebiger, E., C. Story, H.L. Ploegh, and D. Tortorella. 2002. Visualization of the ER-to-cytosol dislocation reaction of a type I membrane protein. EMBO J. 21:1041–1053.
Fiebiger, E., C. Hirsch, J.M. Vyas, E. Gordon, H.L. Ploegh, and D. Tortorella. 2004. Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin. Mol. Biol. Cell. 15:1635–1646.
Tokunaga, F., C. Brostrom, T. Koide, and P. Arvan. 2000. Endoplasmic reticulum (ER)-associated degradation of misfolded N-linked glycoproteins is suppressed upon inhibition of ER mannosidase I. J. Biol. Chem. 275:40757–40764.
Ra, C., M.H. Jouvin, U. Blank, and J.P. Kinet. 1989. A macrophage Fc gamma receptor and the mast cell receptor for IgE share an identical subunit. Nature. 341:752–754.
Kurosaki, T., I. Gander, U. Wirthmueller, and J.V. Ravetch. 1992. The beta subunit of the Fc epsilon RI is associated with the Fc gamma RIII on mast cells. J. Exp. Med. 175:447–451.
Dombrowicz, D., V. Flamand, I. Miyajima, J.V. Ravetch, S.J. Galli, and J.P. Kinet. 1997. Absence of Fc epsilonRI alpha chain results in upregulation of Fc gammaRIII-dependent mast cell degranulation and anaphylaxis. Evidence of competition between Fc epsilonRI and Fc gammaRIII for limiting amounts of FcR beta and gamma chains. J. Clin. Invest. 99:915–925.
Ploegh, H.L. 1995. Current Protocols in Protein Science. Wiley, New York. 10.12.11–10.12.18.
Bertho, N., J. Cerny, Y.M. Kim, E. Fiebiger, H. Ploegh, and M. Boes. 2003. Requirements for T cell-polarized tubulation of class II+ compartments in dendritic cells. J. Immunol. 171:5689–5696.
Wiertz, E.J., T.R. Jones, L. Sun, M. Bogyo, H.J. Geuze, and H.L. Ploegh. 1996. The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell. 84:769–779.(Edda Fiebiger1, Domenico )