© Rockefeller University Press, 0022-1007 $8.00
JEM, Volume 201, Number 2, 267-277
Cotranslational endoplasmic reticulum assembly of Fc
RI controls the formation of functional IgE-binding receptors
Edda Fiebiger1,
Domenico Tortorella2,
Marie-Helene Jouvin3,
Jean-Pierre Kinet3, and
Hidde L. Ploegh1
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{at}hms.harvard.edu
The human high affinity receptor for IgE (Fc
RI) is a cell surface structure critical for the pathology of allergic reactions. Human Fc
RI is expressed as a tetramer (
ß
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 Fc
RIß (ßvar). We demonstrate that Fc
RI
is the core subunit with which the other subunits assemble strictly cotranslationally. In addition to
ß
2 and 
2, we demonstrate the presence of
ß and
ßvar
2 complexes that are stable in the detergent Brij 96. The role of individual Fc
RI subunits for the formation of functional, immunoglobulin Ebinding Fc
RI complexes during endoplasmic reticulum (ER) assembly can be defined as follows: ß and
support ER insertion, signal peptide cleavage and proper N-glycosylation of
, whereas ßvar allows accumulation of
protein backbone. We show that assembly of Fc
RI in the ER is a key step for the regulation of surface expression of Fc
RI. The ER quality control system thus regulates the quantity of functional Fc
RI, which in turn controls onset and persistence of allergic reactions.
Abbreviations used: CC, U373; EndoH, endoglycosidase H; ITAM, immunoreceptor tyrosinebased 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 Fc
RI is one of the key molecules in the pathophysiology of all allergic reactions (36). As a member of the antigen receptor superfamily, Fc
RI shares the organizational principles of a ligand binding immunoglobulin-type protein associated with signaling subunits that regulate cellular activation via conserved immunoreceptor tyrosinebased signaling motifs (ITAMS; 7). BCR, TCR, and other Fc receptors fall in the same class (710). Fc
RI was initially described as a tetrameric receptor composed of a high-affinity ligand-binding
chain, one ß chain, and a pair of disulphide-linked
subunits (5, 9). The Fc
RI complexes on the surface of basophils and mast cells are tetrameric structures (
ß
2). The
ß
2 is the only receptor isoform formed in rodents (5). Human antigen-presenting cells additionally display a trimeric form of Fc
RI that lacks the ß subunit (5, 11, 12). A new splice variant of Fc
RIß (ßvar, formerly referred to as ßT) exerts a dominant negative effect on ß function (13).
The structural integrity of Fc
RI is maintained by the noncovalent interactions of its various subunits. The extracellular domain of Fc
RI
forms the binding site for the CH3 domain of IgE. It binds its ligand in 1:1 ratio, with an affinity of
1010 M1. The ß chain contains four potential transmembrane spanning regions with both the NH2 and the COOH terminus protruding into the cytosol. Fc
RI
forms a dimer and is a member of the
gene family. IgE-dependent cross-linking of Fc
RI induces cellular activation regulated via ITAMs, which are present in one copy in the ß as well as in each of the
chains (5, 9, 10). The
subunit, when expressed in the absence of ß and
, is retained in the ER. The ER retention signal of human
can be overcome by the presence of
alone. Fc
RIß was defined as an amplifier for
chain signaling in vitro and in vivo (14, 15) and as a regulator for surface expression. The ßvar subunit is a splice variant that has lost its ITAM (13). Therefore ßvar-containing complexes must behave significantly differently from those that contain the conventional ß chain.
Multisubunit receptor complexes, like Fc
RI 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 signalpeptide 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 (1921). 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 Fc
RI must assemble in a concerted fashion. Only with all players in place, can ER retention signals be overcome. The sequence of events in Fc
RI 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 Fc
RI in allergic individuals supports this hypothesis (23, 24).
Studies on the human receptor are hampered by a lack of cell lines that express Fc
RI irrespective of its isoforms. Primary human cells that express Fc
RI 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 Fc
RI assembly in the ER. We translated the corresponding mRNAs of all Fc
RI subunits and performed studies on temporal aspects of proteinprotein interaction and their consequences for receptor assembly. Our results show that ER assembly of the individual Fc
RI subunits is tightly controlled and indeed regulates the formation of properly formed receptors with IgE-binding epitopes.
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Results
|
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In vitro translation of Fc
RI
We used in vitro translation as a method to study Fc
RI 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 Fc
RI subunits allow the generation of the corresponding mRNAs for in vitro translations. The mRNAs were translated in the presence of microsomes from different sources. Fc
RI
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). Fc
RI
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 ß 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 Fc
RI
(H2-KbFc
RI
, 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 Fc
RI
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).
We then asked whether we could generate Fc
RI
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 Fc
RI
(
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 Fc
RIß and Fc
RIßvar
The identity of the translation products from ß and ßvar (13) was confirmed by immunoprecipitation with an anti-ß serum generated against the NH2 terminus of ß, 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).
In vitro translation of Fc
RI
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-KbFc
RI
, 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 [35S]cysteine translation mix. The single methionine present in
is removed upon insertion into the microsomes. However, labeling with the [35S]methionine translation mix (containing both [35S]Met and [35S]Cys) allows sufficient labeling as shown in anti-
immunoprecipitations (Fig. 3 B). For reasons that remain unclear, in vitro translations using [35S]cysteine did not allow visualization of ßvar (unpublished data). Thus, all subsequent translation assays were performed with the [35S]methionine translation mix. Under nonreducing conditions,
runs as a dimer with both translation mixes (Fig. 3, C and D).
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 ß 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).
Cotranslation of ß 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 ß
complexes do not occur in the absence of
(Fig. 5 A, lanes 2 and 3). We also failed to detect ß
complexes when ß 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).
These results fit with the assumption that the
chain is the core of all Fc
RI complexes. This hypothesis also implies a direct interaction of
with ß. We therefore attempted to demonstrate the existence of such complexes by in vitro translation. As shown in Fig. 5 B, the
ß complex is stable in Brij96 and is generated only cotranslationally (Fig. 5, D and E, lanes 3). Due to its molecular weight, ß is difficult to distinguish from
1sig and
2sig. The anti-ß reimmunoprecipitation unequivocally demonstrates the existance of
ß 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 ß complexes (Fig. 5 C). The use of tagged versions of both proteins allowed the detection of the individual subunits by immunoblotting after immunoprecipitation. The
ß complexes can be retrieved specifically with an anti-HA reagent via
HA (Fig. 5 C, lanes 24), 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
ß complexes. Digestion of immunopreciptated
ß 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
ß complexes.
Cotranslation of HLA-A2 with ß and
were performed as specificity controls (Fig. 5 D, lanes 4 and 6). As expected, HLA-A2 failed to form a complex with ß and
(Fig. 5 E, lanes 4 and 6).
Immunoprecipitation of
ß
and
ßvar
complexes
The
,ß,
, or
,ßvar,
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
ß
complexes (Fig. 6 A). The anti-
reagent also retrieved stable
ßvar
complexes (Fig. 6 A). The
chain in the latter complexes seemed to be underrepresented when compared with
ß
.
ßvar induces the accumulation of
+sig
We next examined the fate of
when translated in the presence of ßvar. For this purpose
ßvar
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 ßvar in these precipitates. This finding might again reflect a decrease in the stability of
ßvar
complexes, with ßvar dissociating before
, or equally likely, a more general problem of detection of ßvar. As in cellular expression systems (13), ßvar is rapidly lost from in vitro translation mixtures (unpublished data).
ßvar down-regulates surface IgE-binding epitopes
We subcloned ß and ßvar into a bicistronic vector with EGFP (pIRES2-ß-EGFP and pIRES2-ßvar-EGFP). Next, 293 cells were transiently transfected and treated with proteasome inhibitor for 2h. After SDS lysis, immunoblots with anti-ß serum were performed to confirm the proper expression of both proteins (Fig. 7 A).
We verified that mAb 151 recognizes the IgE-binding epitope of Fc
RI
(13, 23, 2830). IgE binding capacity of CHO
was assessed by FACS with biotinylated IgE (Fig. 7 B, filled black). CHO
show comparable reactivity when stained with 151 (Fig. 7 B, blue). Preincubation of cells with 151 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, 2830) and confirms that 151 recognizes the IgE-binding site of Fc
RI
. The fact that both reagents recognize the same epitope also accounts for the misinterpretation of cellular distribution patterns of Fc
RI
in humans. Endogenous IgE bound to Fc
RI
precludes recognition with mAb 151 or biotinylated IgE unless the natural ligand is removed by acid stripping (23, 2830).
We show that our bicistronic constructs regulate the surface expression of IgE-binding epitopes as previously described (13). For this purpose, CHO
ß
were transiently transfected with pIRES2-ß-EGFP or pIRES2-ßvar-EGFP (Fig. 7 A, graph refers to ß [red] and ßvar [black]). Reactivity with mAb 151, 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 ß or ßvar. Although we observed surface expression of IgE-binding epitopes in cells transfected with pIRES2-ß-EGFP, this surface marker was significantly down-regulated in cells transfected with pIRES2-ßvar-EGFP (Fig. 7 A, representative experiment). Transfections in CHO
cells yield the same results (unpublished data). Our experiments confirm that ßvar impairs formation of surface expressed IgE-binding epitopes in vivo and functions in a dominant way when coexpressed with ß in CHO
cells (13).
ßvar induces accumulation of
+sig in vivo
We next explored the mechanism by which ßvar 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 ß or
subunits (Fig. 7 B, lane 1; 31). The presence of unglycosylated
protein backbone was specific for the presence of ßvar (Fig. 7 B, lane 3).
Metabolic labeling experiments were then performed to shown that the
protein that accumulates in the presence of ßvar is indeed
+sig. Anti-HA immunoprecipitations followed by EndoH digestion were performed in cells transiently transfected with
HAßvar
(Fig. 7 C). These experiments shown that most
HA is transformed into its fully N-glycosylated modification irrespectively of the presence of the ßvar 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 ßvar allows accumulation of
+sig.
For more extended studies of the intracellular fate of
, NH2-terminal EGFP fusion proteins of ß or ßvar (GFP-ß or GFP-ßvar) 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-ß or GFP-ßvar 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 ß as well as ßvar throughout the chase (Fig. 8 A). We infer that the ß subunits are subject to proteasomal proteolysis with ßvar more susceptible to proteasomal degradation. GFP-ß and GFP-ßvar should be informative reagents for the analysis of the fate of the
subunit at the single cell level.
To this end, CHO
cells were transiently transfected with GFP-ß and GFP-ßvar and analyzed by epifluorescence. Cells were treated with ZL3VS to inhibit proteasomal degradation for 2 h, fixed, and stained with mAb 151 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-ß are positive for both mAb 151 and the anti-
serum (Fig. 8 B). In contrast, CHO
transfected with GFP-ßvar do not stain with mAb 151 but still remain positive with the anti-
serum (Fig. 8 B). Experiments performed with CHO
ß
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-ßvar interferes with proper folding and the formation of IgE-binding epitopes on the
subunit. Cells transfected with GFP-ßvar still express readily detectable unfolded
chain, suggestive of the mechanism by which ßvar and GFP-ßvar down-regulate IgE-binding epitopes. In experiments without inhibition of proteasomal activity, GFP-ßvar 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 151, respectively (unpublished data).
Additionally we performed a set of pulse-chase experiments to confirm on the cellular level, that Fc
RI
is indeed not targeted to proteasomal degradation by ßvar. Fc
RI
HA was transiently transfected into 293 cells in the presence of ß
or ßvar
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 Fc
RI
. No enhanced degradation of any form of Fc
RI
was observed in cells transfected with
ßvar
. In correlation with the results presented earlier in this study, the only significant difference was the persistence of
+sig in the presence of ßvar. 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 Fc
RI
protein levels in
ßvar
transfectants in the presence and absence of proteasome inhibitors (Fig. 8 D). Although proteasomal inhibition stabilizes ßvar (reference 13; Fig. 8 A; and unpublished data), we do not detect alterations in the amount or expression pattern of Fc
RI
. Because inclusion of proteasome inhibitor and consequent stabilization of ßvar do not change the fate of Fc
RI
is not targeted to proteasomal degradation. We consider it unlikely that the short half-life of ßvar is of functional importance for this mechanism.
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Discussion
|
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Allergen- and IgE-dependent cross-linking of Fc
RI is responsible for the immediate as well as the chronic inflammatory responses observed in atopic patients (5, 6). Surface expression of Fc
RI critically determines the sensitivity to an allergic stimulus and is therefore pivotal for the ensuing clinical response. The only defined extracellular regulator of Fc
RI surface expression defined so far is IgE, its natural ligand (5). With regard to intracellular regulation of receptor expression, Fc
RIß was described as an amplifier for
chain signaling as well as for receptor surface expression (14, 15). In contrast, ßvar 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 Fc
RI (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 Fc
RI isoforms, we were also able to demonstrate the existence of
ßvar
and
ß 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 (1921). In addition to the proper folding of the individual subunits, multimeric receptors must assemble in a concerted fashion. Receptors such as Fc
RI 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 Fc
RI 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. Fc
RI complexes form strictly cotranslationally. The presence of ß and
clearly favors a conversion of
into its IgE-binding form when compared with translation in the presence of
alone. The ßvar, on the other hand, slows down this conversion and induces the accumulation of unglycosylated
with the signal peptide still in place.
Not all Fc
RI receptor subunit RNAs are generated in the same quantities in primary cells. When compared with
, ß 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 ß 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 Fc
RI signaling and ER exit, Fc
RI
is a subunit of Fc
receptors (35). Functional association of Fc
RIß with CD16, Fc
RIII, has also been demonstrated (36, 37). Both Fc receptor types might thus compete for
and ß subunits, which adds an additional level of complexity and importance to assembly control. Because various Fc
receptor subtypes and Fc
RI 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 Fc
RI by depleting the pool of receptor units that are required for Fc
RI ER exit. The functional consequences of ßvar expression for cotranslational assembly need to be interpreted carefully, in particular with regard to the fact that so far transcripts of ßvar were not depicted in absence of the classic ß chain.
Improperly assembled Fc
RI subunits are subjected to the ER quality control system and must be directed toward degradation. Fc
RIßvar is a natural substrate for proteasomal degradation and is itself rapidly destroyed, irrespective of the presence of Fc
RI'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 ßvar (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 Fc
RI as the first quality control mechanism for the generation of functional IgE-binding Fc
RI. The dysregulation of this quality control process might contribute to the expression of high Fc
RI levels in allergic patients. We show that the ER quality control system regulates quantities of functional Fc
RI. It thereby controls onset and persistence of allergic reactions and might thus be a target for therapeutic interventions.
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Materials and Methods
|
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DNA constructs
The cDNAs encoding Fc
RI
, Fc
RIß, Fc
RIßvar, and Fc
RI
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. ß and ßvar were also subcloned into pIRES2-EGFP (CLONTECH Laboratories, Inc.). NH2-terminal EGFP fusion proteins of ß and ßvar 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 151 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
, ß, 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
ß
and CHO
cells were maintained in DMEM as previously described (13, 23). Various constructs of ß and ßvar were expressed in 293, CHO
, or CHO
ß
cells by transient transfection, using a liposome-mediated transfection protocol (510 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 Fc
RI 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 [35S]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 Fc
RI 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 Fc
RI
were stained with mAb15-1 or biotinylated IgE as previously described (13, 23, 2830).
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 151 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).
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Acknowledgments
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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).
The authors have no conflicting financial interests.
Submitted: 12 July 2004
Accepted: 8 December 2004
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