By
From the * International Center for Genetic Engineering and Biotechnology, Area Science Park,
Padriciano 99, 34012-Trieste, Italy; and Ospedale di Pediatria Burlo Garofalo, Via Dell'Istria 65, 34147-Trieste, Italy
The human C gene expresses two membrane IgE heavy chain mRNAs which differ in the sequence that encodes their extracellular membrane-proximal domain. In the long IgE isoform
(mLIgE), this domain contains a stretch of 52 amino acids which are absent in the short variant
(mSIgE). We have now generated B cell transfectoma cell lines that express these two isoforms
and show that both types of mIgE form functional B cell antigen receptors (BCR). Both receptors associate with the Ig-
/Ig-
heterodimer, as well as with protein kinases that are capable
of phosphorylating this complex. Upon their cross-linking, both receptors can activate protein
tyrosine kinases that phosphorylate the same substrate proteins. Both IgE receptors also associate with two novel proteins that do not bind to mIgM. Apart from these similarities, the two IgE-BCRs show several differences of which some are analogous to the differences between
the IgM- and IgD-BCRs. First, the mSIgE is transported to the cell surface at a higher rate than
the mLIgE. Second, the two IgE-BCRs associate with differently glycosylated Ig-
proteins,
the mLIgE associates with the completely glycosylated form, whereas the mSIgE associates with
an Ig-
glycoform that is partially sensitive to endoglycosidase H. Third, the kinetics of protein
tyrosine phosphorylation induced by receptor cross-linking is significantly different for the two
IgE-BCRs. Finally, cross-linking of the mSIgE-BCR leads to growth inhibition of the B cell
transfectoma, whereas signaling through the mLIgE-BCR does not affect the cellular proliferation. These data show that the two human membrane IgE isoforms assemble into functionally
distinct antigen receptors which can induce different cellular responses.
Antigen receptors on B lymphocytes are expressed on
the plasma membrane as a complex of disulfide-bonded
Ig heavy and light chains that are noncovalently associated
with at least two other glycoproteins, Ig- Depending on their developmental stage, B cells express
different classes of mIg. Immature B cells carry only the
IgM antigen receptor, whereas IgM and IgD are coexpressed at a later stage of differentiation (19, 20). After class
switching, B cells which express either IgG, IgA, or IgE
antigen receptors are generated. Engagement of the Ig receptors by antigen can lead to cell proliferation, differentiation into antibody-secreting plasma cells, anergy, or apoptosis (21).
The human Ig constant We have now investigated the expression and function
of the IgE molecules encoded by the two types of membrane
The long membrane transcript was initially described as
the predominant Cell Lines and Transfections.
The WEHI 231 B cell lymphoma
(provided by Dr. Roberto Sitia, Dipartimento di Ricerca Biologica e Tecnologica-San Raffaele Scientific Institute, Milan, Italy)
was maintained in DMEM supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), and 50 µM 2-mercaptoethanol. Transfections were performed by electroporation as
described elsewhere (30), except that 800 µg/ml of G418 (Geneticin; Life Technologies, Inc., Gaithersburg, MD) were used for
selection of the WEHI 231 clones. Cells were cloned by limiting
dilution, and positive clones were identified by immunofluorescence.
Construction of Vectors for the Expression of the Flow Cytometric Analysis.
The expression of surface IgE was
examined by flow cytometry on a FACScan®, (Becton Dickinson
Immunocytometry Sys., Mountain View, CA) using rabbit anti-
human IgE ( Immunoprecipitations.
Metabolic labeling and immunoprecipitations were performed as previously described (30). Briefly, 5 × 106 cells/ml were labeled with [35S]methionine (Amersham Intl.,
Buckinghamshire, England) at 100-250 µCi/ml (1 Ci = 37 GBq), and chased with cold methionine as indicated in the figures. Cell lysates were immunoprecipitated with rabbit Ig's to human IgE ( Surface Biotinylation and Immunoprecipitation of IgE and IgM
BCRs.
Twenty million cells were washed twice with PBS and
incubated in 1 ml of PBS containing 0.5 mg/ml of sulfo-NHSbiotin (Pierce Chem. Co., Rockford, IL) at room temperature
for 15 min. Free succinimide groups were blocked by the addition of 10 ml of nonsupplemented medium at room temperature
for 10 min. Cells were washed twice with PBS and resuspended
in 500 µl lysis buffer containing 1% digitonin (Sigma Chemical
Co., Steinheim, Germany), 50 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 5 mM EDTA, and the protease inhibitors PMSF (1 mM),
aprotinin (10 mg/ml), and leupeptin (10 mg/ml) (all from Sigma
Chemical Co.). Lysates were incubated on ice for 30 min and
centrifuged at 10,000 g for 30 min at 4°C. The supernatants were
precleared three times with 30 µl of protein A agarose beads (GIBCO BRL) at 4°C for 30 min. Immunoprecipitations were
performed by incubating the lysates with 6 µl of rabbit Igs to
human IgE ( In Vitro Kinase Assay.
Immunoprecipitates from digitonin lysates were resuspended in kinase buffer (50 mM Tris, pH 7.5, 10 mM
MnCl2, 1 mM EDTA, 1 mM sodium orthovanadate [Sigma Chemical Co.], 1% digitonin, plus inhibitors) containing 10 µCi [ Detection of Tyrosine Phosphorylated Proteins by Western Blotting.
Tyrosine phosphorylated proteins were detected as previously described (31). Briefly, 4 × 106 cells were resuspended in 0.5 ml of
DMEM and stimulated with either goat anti-human IgE ( Cellular Proliferation Assay.
Two B cell transfectomas that expressed comparable levels of the long IgE isoform (mLIgE) and
the short IgE isoform (mSIgE) were cultured in triplicate in 96well plates at a cell density of 2 × 104/well. The cells were incubated for 24 h with various amounts of goat anti-human IgE ( To investigate the properties of the two membrane IgE isoforms, we generated two chimeric mouse/human To investigate the assembly and transport of the mLIgE
and mSIgE isoforms, we perfomed pulse-chase studies in
the WEHI cell lines. Cells were given a 15 min pulse with
[35S]methionine and then chased for 1, 2, 3, and 4 h. mIgE
was immunoprecipitated from the WEHI-mSIgE or WEHImLIgE cell extracts and analyzed on a nonreducing 6%
SDS-PAGE (Fig. 2 a). Two major species of more than
240 kD were observed in both cell lines, demonstrating
that both membrane isoforms are assembled into H2L2 molecules. During the course of the chase, the quantity of immunoprecipitated proteins remained the same, indicating
that there is no intracellular degradation of either membrane isoform. The accumulation of higher molecular weight
species in the course of the chase suggested that both proteins are terminally glycosylated during their transport to the cell surface. This was confirmed by treatment of the
immunoprecipitated material with Endo H which cleaves
N-linked high mannose oligosaccharides of glycoproteins
before they arrive in the medial Golgi, but fails to cleave
terminally processed carbohydrate moieties. As shown in Fig.
2 b, after Endo H treatment two
To characterize the IgE-BCR, and to determine the mSIgE and mLIgE
associated proteins, we immunoprecipitated the IgE antigen receptor complex from digitonin lysates of surface biotinylated WEHI-mSIgE and WEHI-mLIgE cells. The immunoprecipitations were perfomed in duplicate using two
different anti-human IgE antibodies. As a control, the endogenous IgM-BCR was subsequently immunoprecipitated from the same cell extracts. The immunoprecipitated
material was resolved on a 10% SDS-PAGE under reducing conditions (Fig. 3 a). Analysis of the mIgM complex
yielded the expected characteristic pattern of an 80-kD
band corresponding to the µ heavy chain, a 25-kD band
corresponding to the light chain, and the 32- and 40-47-kD bands corresponding to Ig-
Two additional proteins of 37 and 41 kD were detected
in the mSIgE- and mLIgE-BCR immunoprecipitations (Fig.
3 a). These proteins were not present in the IgM-BCR and
were immunoprecipitated with either of the two anti-IgE
Abs, indicating that they are specifically associated with
mIgE. The relative amount of these IgE-BCR associated proteins (further referred to as To further characterize the components of the IgEBCRs, two dimensional analysis of anti-IgE immunoprecipitated material was performed on digitonin lysates from
biotinylated WEHI-mSIgE and WEHI-mLIgE cells. The
analysis was done on a 10% SDS-PAGE under nonreducing conditions in the first dimension and reducing conditions in the second (Fig. 4). In addition to the bands corresponding to
To investigate if the IgE-BCRs contain protein kinases
capable of phosphorylating the Ig-
To analyze the activation of PTKs upon engagement of the IgE-BCRs, we used transfectant cell lines
(WEHI-mLIgE and WEHI-mSIgE) that expressed comparable levels of mLIgE and mSIgE (Fig. 6). Wild-type WEHI
cells were analyzed in parallel as control. The three cell
lines were incubated for different periods of time (1-40
min) in the presence of either anti-IgE or anti-IgM. The PTK activation in these cells was monitored by the increase
in tyrosine phosphorylation of PTK substrate proteins in
total Triton X-100 cell lysates. Cross-linking the IgM- or
the IgE-BCRs resulted in a similar pattern of phosphorylation, indicating that all three BCRs induce the phosphorylation of the same substrate proteins. However, the kinetics
of phosphorylation of the two IgE-BCRs was quite different. After cross-linking the mLIgE-BCR, the substrate phosphorylation reached its maximum in 1 min and drastically
declined within 5 min (Fig. 7). In contrast, in the mSIgEproducing transfectant, the substrate phosphorylation still
increased after 1 min of stimulation and reached its maximum after 5 min. Increased tyrosine phosphorylation was
still evident after 40 min, although for several polypeptides it had declined. These data showed that the kinetics of protein tyrosine phosphorylation induced by receptor crosslinking is significantly different for the two IgE-BCRs.
In subsequent experiments we observed that the kinetics
of tyrosine phosphorylation were independent of the amount
of IgE expressed on the cell surface. Analysis of WEHImSIgE and WEHI-mLIgE clones expressing different amounts
of IgE-BCR showed a difference only in the intensity of
the signal, whereas the kinetics of both receptors remained
the same (data not shown). Moreover, cross-linking of the
endogenous IgM-BCR in the two transfectants and in
wild-type WEHI cells showed the same kinetics of tyrosine
phosphorylation, and was consistent with published data (35).
These experiments excluded the possibility that the differences in signal transduction between the two IgE-BCRs
were due to peculiarities of the transfected cell lines rather
than the type of mIgE.
Cross-linking of the endogenous IgM-BCR
in WEHI cells leads to their growth inhibition (36). On the
other hand, cross-linking of the IgD-BCR in WEHI cells
transfected with a
The data presented in this paper are the first demonstration of the existence of two functional human IgE B cell
receptors that are generated by alternative splicing. Although both receptors were found to be correctly assembled, transported to the cell surface, and capable of signal
transduction upon receptor engagement, a number of differences were noted between them that could only be attributed to the different extracellular membrane proximal domains. The first difference was in the rate of their transport to the plasma membrane. The transit time of the
mLIgE was much longer, since only 20% of the molecules
were found to be terminally glycosylated within 3 h of
their synthesis, whereas almost all of the mSIgE molecules
were converted into the mature form during this period.
Interestingly, a different rate of transport has also been
shown for IgM and IgD with a shorter transit time for the
former (38). In our experiments the endogenous IgM was
transported at a similar rate as the mLIgE, indicating that
the transport of the mSIgE is extremely efficient.
Both mIgE isoforms were found to associate into complete BCR complexes that contain the Ig- An unexpected observation in this study was the finding
that both IgE receptors associate with two proteins ( The most striking difference between the two IgEBCRs was related to the response elicited by their crosslinking. Both receptors appeared to activate the same
PTKs, as judged by the pattern of tyrosine phosphorylation. However, a clear difference was noted in the maximal
phosphorylation time point as well as in the duration of the
response. More specifically, the phosphorylation induced via the mLIgE-BCR reached its maximum after 1 min of
receptor engagement and declined within the following 5 min. In contrast, the signal induced by cross-linking of the
mSIgE-BCR reached its maximum after 5 min and remained for a prolonged period. Differences in the kinetics
of the response have also been observed for the IgM- and
IgD-BCRs (31). Experiments with these receptors or receptor chimeras have indicated that the extracellular membrane proximal and/or transmembrane domains influence
the duration of the response (31). Since in the IgE-BCRs
the difference resides only in the extracellular membrane-
proximal domain, it can be concluded that this region determines the kinetics of protein tyrosine phosphorylation
upon receptor engagement. The mechanisms through which
this could occur are unknown, but could be mediated through association and/or interaction with the different
Ig- The different kinetics of signal transduction through the
IgE-BCRs were associated with different cellular responses
of the WEHI transfectomas. Cross-linking of the mSIgEBCR led to growth inhibition, whereas signaling through
the mLIgE-BCR had no effect on cellular proliferation.
These cellular responses are analogous to those observed after signaling through the IgM- and IgD-BCRs in WEHI
cells (37). In this case the IgM-BCR, which, like mSIgE, has a short extracellular spacer, is capable of transmitting a growth inhibitory signal.
In conclusion, we have shown that both human mIgE
isoforms assemble into functional B cell receptors. When
expressed on immature B cells such as the WEHI lymphoma, the two IgE-BCRs transmit qualitatively distinct
signals after cross-linking. Work is in progress to determine the properties of these receptors in more mature B cells
which have undergone Ig H chain class switching. However, it should be noted here that B cells coexpressing IgM
and IgE have been detected in peripheral blood (41, 42).
Clearly, it would be of interest to determine the type of
mIgE expressed by these cells and whether the two mIgEBCRs can be coexpressed by the same cell. Such studies
should further delineate the relevance of having two receptors with identical specificity for the ligand and may allow
characterization of additional components of the BCR signal-transduction pathways.
(CD79a) and Ig-
(CD79b) (1). Ig-
and Ig-
are two glycosylated transmembrane proteins of the Ig superfamily that are encoded
by the B cell-specific genes mb-1 and B29, respectively (6, 7).
These proteins form a disulfide-linked heterodimer which
appears to be a prerequisite for the transport and cell-surface expression of the membrane-bound Igs (mIg)1 (2, 3,
8). While the mIg molecule serves as the antigen-binding component of the receptor, the noncovalently associated
Ig-
/Ig-
heterodimer has been shown to be the signal
transduction unit of the B cell antigen receptor (BCR) (9).
The Ig-
/Ig-
heterodimer is directly involved in the coupling of the BCR to several protein tyrosine kinases (PTKs)
expressed in B cells, such as the src-related PTKs Lyn, Fyn,
Lck, and Blk, and the cytoplasmic PTK Syk (13). Signal
transduction from the cross-linked BCR involves the
rapid activation of these enzymes which phosphorylate
several substrate proteins in B cells, including the Ig-
and
Ig-
components themselves (18).
gene (C
) appears to be peculiar in its capacity to produce a number of alternatively
spliced
mRNAs that encode two membrane-type and
several secretory-type IgE H chains (22). We have recently characterized the protein products of the secretory
transcripts and found that only two of them encode properly assembled and secreted IgE molecules (30). All other
isoforms were apparently aberrantly spliced byproducts which were retained and degraded by cellular posttranslational
quality control mechanisms (22).
transcripts. These two
mRNA species differ only
in the 5
part of the first membrane exon that encodes the
extracellular membrane proximal domain (Fig. 1 a). The
longer variant (
CH4-M1
-M2) contains 156 extra nucleotides as a consequence of alternative splicing between the
donor splice site at the 3
end of the CH4 exon and the upstream acceptor splice site in the M1 exon. Thus, the two
putative membrane proteins have the same constant
region and the same transmembrane and intracellular domains, but differ in a 52-amino acids segment which is
present only in the long membrane IgE variant (Fig. 1 b).
Fig. 1.
Schematic representation of the two membrane IgE H chain
isoforms. (a) Diagram of the 3 part of the C
and the pattern of alternative splicing that generates the
CH4-M1-M2 and
CH4-M1
-M2 membrane transcripts. Open boxes represent coding sequences and shadowed
boxes represent 3
untranslated regions; stop codons are indicated by asterisks. (b) Amino acid sequence of the COOH terminus of the short
(
CH4-M1-M2) and long (
CH4-M1
-M2)
chains. The extra 52 amino
acids present in the extracellular membrane-proximal domain of the long
chain are underlined. (c) Diagram of the chimeric mouse VH-NIP/C
membrane gene constructs. The 3
ends of the two membrane
isoforms
were cloned in their cDNA form (starting from the CH3 exon) into
pCIG-C
, using the indicated restriction enzyme sites.
[View Larger Version of this Image (33K GIF file)]
mRNA species in humans because it was
found at significantly higher levels than the short species in
IL-4 plus anti-CD40-stimulated PBL and in IgE-producing myeloma cell lines (27, 29). However, we have recently shown that the short membrane transcript (
CH4M1-M2), which is homologous to the murine membrane
transcript, is predominantly expressed by unstimulated PBL,
indicating that the expression of the two membrane
mRNAs may depend on the stage of B cell differentiation (23).
CH4-M1-M2 and
CH4-M1
-M2 H Chains.
The construction of the pCIG-C
CH4M1
-M2 has been described in detail previously (22). The pCIGC
CH4-M1-M2 vector was similarly constructed by replacing
the BglII/KpnI fragment of pCIG-C
CH4-M1
-M2 with a corresponding fragment containing the M1 instead of the M1
exon
(Fig. 1 c).
chain) (Dako Corp., Carpinteria, CA), and FITCconjugated swine anti-rabbit IgG (Dako Corp.).
chains) or rabbit Igs to mouse IgM (µ-chains) (Dako
Corp.) and purified by protein A-Sepharose. The samples were
analyzed by SDS-PAGE in the presence or absence of mercaptoethanol, as indicated in the figure legends. Treatments of labeled
supernatants with recombinant N-glycosidase F (PNGase F) and
endoglycosidase H (Endo H) were performed according to the
protocols provided by the manufacturer (New England Biolabs
Inc., Beverly, MA).
chains) (Dako Corp.), rabbit Igs to mouse IgM (µ chains) (Dako Corp.), or goat Igs to human IgE (
chains)
(Kirkegaard & Perry Labs., Inc., Gaithersburg, MD), at 4°C for
60 min. 20 µl of protein A agarose were then added and incubated for a further 30 min at 4°C. The Ig-
/Ig-
heterodimer
was immunoprecipitated using a polyclonal antibody against Ig-
(provided by Dr. J.C. Cambier, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO). Beads were
preincubated with 10 mg/ml bovine serum albumin for 20 min
and washed three times with lysis buffer before use. The beads
were pelleted by centrifugation (the supernatants were saved for
reprecipitation), washed twice with high salt lysis buffer (50 mM
Tris, pH 7.5, 500 mM NaCl, 5 mM EDTA, 0.2% digitonin, plus
inhibitors), and twice with low salt buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.2% digitonin, plus inhibitors). For reprecipitation, supernatants were precleared twice with 30 µl
beads before adding a different primary antibody.
32P]
(Amersham Intl.), and incubated at room temperature for 5 min. The reaction was terminated by addition of kinase buffer containing 50 mM EDTA, and the pellets were collected by centrifugation before resuspending in SDS-PAGE reducing sample buffer.
The samples were boiled and electrophoresed on a 10% SDSPAGE. The gel was dried without fixing and autoradiographed at
70°C.
chain)
(Kirkegaard & Perry Labs., Inc.) (20 µg/ml) or anti-mouse IgM
(5 µg/ml) at 37°C for the indicated periods of time. After washing twice with ice-cold PBS containing 1 mM sodium orthovanadate, cells were lysed with 100 µl of Triton X-100 lysis buffer
containing 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, plus inhibitors, and 1 mM
sodium orthovanadate. After incubation for 10 min on ice, the
supernatants were cleared by centrifugation at 10,000 g for 15 min
at 4°C. Subsequently, 30 µl of supernatant was subjected to 10%
SDS-PAGE and Western blotting. The PVDF membrane (Amersham Intl.) was incubated with the antiphosphotyrosine monoclonal antibody PY20 (Transduction Laboratories, Lexington,
KY), followed by horseradish peroxidase-coupled anti-mouse Ig
antibody (Dako Corp.). The bound antibodies were visualized by
the enhanced chemiluminescence detection system (Amersham Intl.).
chain) (Kirkegaard & Perry Labs., Inc.) in 0.2 ml medium. Proliferation was determined by incorporation of [methyl-3H]thymidine (1 µCi/well, 94.0 Ci/mmol; Amersham Intl.). After 14 h of
incubation, cells were harvested and levels of incorporated [3H]thymidine were measured by scintillation counting.
Expression, Assembly, and Transport of mSIgE and mLIgE.
-chain
gene constructs which contained the mouse heavy chain variable region segment from an Ab with anti-4-hydroxy5-iodo-3-nitro phenacetyl (NIP) specificity and the human
C
region with a COOH terminus corresponding to the
mLIgE or the mSIgE isoform. A schematic diagram of the two constructs is depicted in Fig. 1 c. The two constructs
were independently transfected into WEHI-231 cells, and
G418 resistant transfectants were selected for the expression
of mIgE by immunofluorescence.
species were observed in
each case (a) a 60-(mSIgE) or 65-(mLIgE) kD species corresponding to Endo H-sensitive
chains that carried unprocessed high mannose carbohydrates and (b) an 87-(mSIgE) or
90-(mLIgE) kD species corresponding to mature
chains
that bore processed N-linked carbohydrates which were
resistant to the enzyme. In the WEHI-mLIgE transfectoma, only 20-30% of the newly synthesized membrane
chains
were of the mature processed type after 4 h chase (Fig. 2).
This was similar to the rate of transport observed for the
endogenous mouse mIgM. Interestingly, three to four
times more mSIgE H chains were present in the mature
form after the same period of time. This clearly demonstrated that the mSIgE is more efficiently transported to the
cell surface than the mLIgE, or even the endogenous
mIgM.
Fig. 2.
Assembly and transport of mLIgE
and mSIgE. WEHI-mSIgE, WEHI-mLIgE, and
wild-type WEHI cells were pulse labeled with
[35S]methionine for 15 min and chased for the
indicated times in the presence of excess cold
methionine. Cellular extracts were immunoprecipitated with the indicated antibodies and
(a) analyzed on a nonreducing 6% SDS-PAGE
or (b) treated with Endo H and separated on reducing 10% SDS-PAGE.
[View Larger Version of this Image (61K GIF file)]
and Ig-
polypeptides, respectively (18). A similar pattern was observed after immunoprecipitation of the mSIgE- and mLIgE-BCRs, indicating that Ig-
and Ig-
chains are also present in these
receptors. This was confirmed by analysis of the material
that was dissociated from the immunoprecipitated IgEBCRs by treatment with NP-40. Reprecipitation of this
material with an anti-Ig-
specific antiserum produced
only the Ig-
and Ig-
subunits (Fig. 3 b).The Ig-
subunit
was of the same molecular mass in the three BCRs,
whereas the Ig-
of the mLIgE (mLIgE-
) showed a slower
mobility than the IgM-
and mSIgE-
(Fig. 3). Slower
mobility of the Ig-
chain has also been observed in the case of the IgD-BCR. This difference has been shown to
be the consequence of different terminal glycosylation of
the two N-glycosylation sites in the polypeptide (8, 32,
33). To establish if this is the case with the mLIgE-
, immunopreciptated materials were treated with endoglycosidases. Fig. 3 a shows that Endo H treatment decreased the
molecular weight of the IgM- and mSIgE-associated Ig-
polypeptides. On the other hand, the mLIgE-
remained at the same position, indicating that both glycosylation sites
are terminally glycosylated. Treatment with PNGase, which
cleaves all sugar residues, eliminated the difference in size
between the different Ig-
polypeptides.
Fig. 3.
Immunoprecipitation of BCRs from biotin-labeled cell-surface proteins. WEHI-mSIgE and WEHI-mLIgE were surface biotinylated and
treated with digitonin lysis buffer to preserve the BCR complexes. (a) Lysates were treated first with anti-IgE, and then with anti-IgM sera to immunoprecipitate the IgE-BCR and endogenous IgM-BCR complexes, respectively. Immunoprecipitated material was treated with Endo H or PNGase as indicated, and analyzed by reducing 10% SDS-PAGE. Arrowheads indicate the position of the Ig- polypeptide after removal of one of the two N-linked
carbohydrate moieties by Endo H. Ig-
(N
2) and Ig-
(N
3) correspond to the Ig-
and Ig-
polypeptides after removal by PNGase of the two or three N-linked carbohydrate moieties, respectively. (b) Reprecipitation of the Ig-
/Ig-
heterodimer dissociated by NP-40 from the mSIgE-BCR and mLIgEBCR. The immunoprecipitation was done with an anti Ig-
serum and analyzed on a 10% SDS-PAGE under reducing conditions.
[View Larger Version of this Image (41K GIF file)]
BAP37 and
BAP41) was
different in the two IgE-BCRs, with significantly lower
quantities of
BAP41 in the mLIgE-BCR. These proteins
were not immunoprecipitated with the anti-Ig-
antiserum, indicating that they are not covalently associated to
Ig-
or antigenically related to this polypeptide (Fig. 3 b).
2L2 and
L, a complex of ~75 kD was present
in the nonreducing PAGE analysis of the mSIgE-BCR. A
slightly higher mol wt complex (80 kD) was detected in
the mLIgE-BCR. In the second dimension, these complexes dissociated into free Ig-
and Ig-
chains. This experiment also indicated that the
BAP37 and
BAP41 proteins form a disulfide-bound complex (
BAP37/41) which,
in the nonreducing PAGE analysis of the mSIgE-BCR, migrated distinctly from the Ig-
/Ig-
heterodimer. In the
mLIgE-BCR the
BAP37 apparently formed a homodimer which comigrated with the Ig-
/Ig-
complex.
Fig. 4.
Bidimensional analysis of surface-biotinylated IgEBCRs. Anti-IgE immunoprecipitates from the two transfectomas
were analyzed by bidimensional
(nonreducing/reducing) PAGE.
The migration of the same material run only under reducing
conditions or nonreducing conditions is shown in the left lane
of each gel, or as a separate lane
on top of each gel, respectively.
Arrowheads indicate the position
of the dissociated BAP37 and
BAP41 proteins. The other components of the IgE-BCRs
are indicated in the figure.
[View Larger Version of this Image (47K GIF file)]
/Ig-
heterodimers as
shown for the IgM-, IgD- and IgG-BCRs (34), we performed in vitro [
32P]ATP labeling of immunoprecipitated
BCRs. The endogenous IgM-BCR from both transfectomas was analyzed as an internal control. As shown in
Fig. 5, bands corresponding to the Ig-
and Ig-
proteins were found to be phosphorylated when incubated with
[
32P]ATP in all three BCRs. The identity of these bands
was again confirmed by treatment with Endo H and PNGase F. The two newly identified
BAPs were not detected in this in vitro assay, suggesting that they are not
substrates for the BCR-associated protein kinases.
Fig. 5.
In vitro phosphorylation of BCR associated proteins. IgE-
and IgM-BCR from the WEHI-mSIgE and WEHI-mLIgE transfectomas
were immunoprecipitated, incubated with [32P]ATP, and analyzed on
reducing 10% SDS-PAGE. Treatment with Endo H or PNGase is indicated on top of each gel. Arrowheads, Ig-
(N
2), and Ig-
(N
3) as in
legend to Fig. 3.
[View Larger Version of this Image (65K GIF file)]
Fig. 6.
Quantitation of
mSIgE- and mLIgE-BCR levels
on the surface of transfected
WEHI cells. The level of surface
IgE was determined in each cell
line by fluorescent flow cytometry analysis with a rabbit anti-
human IgE antibody and FITCconjugated swine anti-rabbit IgG.
[View Larger Version of this Image (11K GIF file)]
Fig. 7.
Kinetics of PTK
substrate phosphorylation upon
cross-linking of the mSIgE-,
mLIgE-, and IgM-BCR. Wildtype WEHI, WEHI-mLIgE, or
WEHI-mSIgE cells were incubated for various times with antiIgE or anti-IgM antibodies, as
indicated on top of each gel. Cellular extracts were then prepared
and tyrosine phosphorylated proteins detected by immunoblotting with an anti-phosphotyrosine mAb.
[View Larger Version of this Image (57K GIF file)]
chain does not affect their proliferation
(37). To investigate whether signaling through the IgE-BCRs
has an effect on cellular proliferation, WEHI-mSIgE and
WEHI-mLIgE cells were grown for 24 h in the presence of
various concentrations of anti-IgE Ab and were subsequently incubated for 14 h with [3H]thymidine. A marked
inhibition in proliferation was observed only in the WEHImSIgE transfectoma; cross-linking of the mLIgE had no effect (Fig. 8). The above results indicate that different cellular responses can occur after IgE-BCR signaling, depending on the type of mIgE expressed on the cell surface.
Fig. 8.
Proliferation of WEHI-mLIgE and WEHI-mSIgE cells after
cross-linking of mIgE. 2 × 104 cells were incubated with 0, 1, 5, 25, or
50 µg/ml of polyclonal anti-human IgE anti-serum. Results show one
representative of three separate experiments with similar results. Each
point represents an average of triplicate cultures (SEM <5%).
[View Larger Version of this Image (15K GIF file)]
/Ig-
heterodimer as well as the PTKs involved in their phosphorylation. However, a difference was also noted in the pattern
of glycosylation of the associated Ig-
; when associated
with mSIgE, the Ig-
polypeptide had only one of the sites
terminally glycosylated and resistant to Endo H cleavage,
whereas, when associated with mLIgE, both sites were resistant to the enzyme. This is identical to what has been reported for IgM and IgD, which associate with partially or
completely processed Ig-
, respectively (8, 32). Several
possibilities have been considered to explain the difference
in glycosylation of the Ig-
polypeptide associated with
IgD, including the length of the extracellular membrane
proximal domain, the presence of interchain disulfide bonds
in this region, and the transit time of the complex through
the Golgi (33). These possibilities could also account for the
different processing of the IgE-associated Ig-
polypeptides,
since, analogous to IgD, the mLIgE contains a much longer
extracellular domain than the mSIgE, has three extra cysteines in this region, and also has a lower rate of transport to the cell surface.
BAP37
and
BAP41) that were not present in the endogenous
IgM-BCR of the WEHI lymphoma. Their association with
the IgE receptors appeared to be very specific, since they
were coprecipitated with two different anti-IgE antisera. These proteins obviously differ from other BCR-associated
proteins, such as BAP32 and BAP37 which are exclusively
associated with IgM (39), and BAP29 and BAP31 which
are preferentially associated with IgD molecules (40), for
the following reasons: (a) they have an extracellular domain
which was labeled in the cell-surface biotinylation experiment, (b) both proteins were glycosylated, and (c) they
form disulfide-bonded complexes. The
BAP41 appeared to be underrepresented in the mLIgE-BCR, suggesting that
the longer extracellular membrane-proximal domain affects
the association of
BAP41 to the BCR or its accessibility to
biotinylation.
glycoforms or other proteins of the BCR complex.
Address correspondence to Dr. Oscar R. Burrone, ICGEB, Area Science Park, Padriciano, 99, 34012, Trieste, Italy.
Received for publication 21 August 1996
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