| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, October 23, 1995) From the
The B cell antigen receptor complex contains heterodimers of
Ig-
A B cell's response to antigen, whether it be
proliferation, differentiation, anergy, or deletion, is dependent upon
recognition of that antigen by the B cell antigen receptor
(BCR)( Many of the above studies utilized chimeras in which irrelevant
extracellular and transmembrane domains were fused to the single
cytoplasmic domain under study(18, 27, 28) .
Although this approach has yielded considerable insight into
ITAM-containing chains, it assumes that functions observed in the
isolated circumstance of a single chimera are reflective of the
function of that cytoplasmic domain within the intact receptor complex.
This might not be true since most ITAM-containing chains, such as
Ig- As
demonstrated in this report, Ig-
Figure 6:
Ig-
Figure 1:
A,
stimulation of PDGFR chimeras. Homodimers or predominantly heterodimers
were formed on singly (left) or doubly transfected (middle) cells, respectively, by first treating with PDGF-BB.
Complexes were then aggregated with anti-PDGFR
Figure 3:
Only
Ig-
Figure 2:
The pattern of total protein
phosphorylation in wild type (WT),
We engineered cDNAs encoding
for molecules in which either the cytoplasmic domains of Ig-
Figure 4:
Ig-
Figure 5:
A, Ig-
It is possible that the observed
differences in Ig-
Figure 7:
The Ig-
Herein we report that the ITAM-containing subunits within an
immune recognition receptor complex can cooperate to efficiently
initiate signal transduction cascades. Using a system that allowed us
to compare the signal transduction capacities and physical properties
of homo- and heterodimers of Ig- Our results reveal a new level of complexity in the
function of the BCR. Previous studies utilizing either fusion proteins,
peptides or chimeras, have sought to characterize the functional
capacities of individual cytoplasmic domains of the BCR
complex(18, 19, 27, 28) . This
reductionist approach assumes that each ITAM-containing domain is an
isolated signaling unit whose capacities can simply be added to those
of other domains to form an accurate picture of the whole receptor
complex. Our data suggest that this assumption is not entirely valid.
Rather, we would argue that while the study of each individual subunit
reveals what it can do, it is only in the context of other receptor
structures that one can elucidate what that subunit does do. We have
recently obtained data directly demonstrating that the coordinate
activities of Ig- Phosphorylation of the ITAM tyrosines within the BCR
cytoplasmic domains is a necessary and early event in the initiation of
tyrosine kinase activation. Previously, we and others have demonstrated
that members of the Src family of tyrosine kinases are constitutively
associated with the resting receptor complex and that it is probably
these kinases which mediate the phosphorylation of Ig- One
of the mechanisms by which Ig- Another
possibility is that Ig- Previously, models of antigen receptor-mediated signal transduction
have assumed that each ITAM-containing chain within a receptor complex
generates independent signals (Fig. 8, left). Some have
postulated that the signals generated are
redundant(12, 23, 24) , while others have
demonstrated that some chains are functionally distinct and capable of
preferentially coupling to selected secondary
effectors(10, 11, 19, 25, 26) .
However, our data support a model in which each chain within a
heterodimer can cross-modulate the phosphorylative state and, by
extension, the signal transducing capabilities of the other (Fig. 8, right). This leads to specialization of each
chain's function within the multimeric whole. In the particular
case of the BCR and tyrosine kinase activation, Ig-
Figure 8:
Model
of Ig-
Volume 271,
Number 9,
Issue of March 1, 1996 pp. 5158-5163
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
/
Heterodimer of the B
Cell Antigen Receptor Complex (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and Ig-
. The cytoplasmic tails of each of these chains
contain two conserved tyrosines, phosphorylation of which initiates the
signal transduction cascades activated by the receptor complex.
Although the cytoplasmic domains of Ig- and Ig- have been
expressed individually and demonstrated to be competent signal
transduction units, we postulated that within the context of a
heterodimer, Ig- and Ig- could have new, complementary or
even synergistic functions. Therefore we developed a system to compare
the signal transducing capacities of dimers of Ig-/Ig-,
Ig-/Ig-, or Ig-/Ig-. This was done by fusing the
extracellular and transmembrane domains of either human
platelet-derived growth factor receptor (PDGFR) or to the
cytoplasmic tail of either Ig- or Ig-. Three cell lines
expressing PDGFR/Ig-, PDGFR/Ig-, or
PDGFR/Ig- together with PDGFR/Ig- were established
in the murine B cell line A20 IIA1.6. While aggregation of each dimer
by itself could induce the tyrosine phosphorylation of cellular
substrates, only aggregation of the heterodimer induced the
phosphorylation of substrates similar in range and intensity to that
induced by the endogenous B cell antigen receptor complex.
Interestingly, Ig- remarkably enhanced the rapidity (T
decreased from 5 to 1 min) and intensity
(greater than 10-fold enhancement) of Ig- phosphorylation.
Conversely, the phosphorylation of Ig- was reduced to undetectable
levels when co-aggregated with Ig-. The enhancement of Ig-
phosphorylation by Ig- correlated with a lowering of the
stimulation threshold for tyrosine kinase activation.
)(1, 2, 3) . The receptor is
a multimeric complex consisting of the antigen-recognition
substructure, membrane-bound immunoglobulin non-covalently associated
with heterodimer(s) of Ig- and
Ig-(4, 5, 6) . Present evidence
indicates that the cytoplasmic tails of Ig- and (7) translate antigen engagement into cytoplasmic signaling
events that initiate cellular
responses(8, 9, 10, 11, 12) .
Most proximally in the signaling cascade, one or more tyrosine kinases,
including Syk and members of the Src family, are
activated(13, 14, 15) . These in turn
activate a variety of pathways whose constituents include Ras,
phosphatidylinositol 3-kinase, and phospholipase C(1) .
Embedded within the cytoplasmic tails of both Ig- and is a
sequence common to other multichain immune recognition receptor (MIRR)
subunits including CD3, CD3
, TCR
, Fc
RIII
, and
Fc
RI
, termed the immunoreceptor tyrosine-based activation
motif (ITAM)(16, 17) . The motif contains two
tyrosines, both of which are critical for initiating tyrosine kinase
activation (10, 18) . Phosphorylation of these
tyrosines facilitates the recruitment and activation of tyrosine
kinases which contain SH2 domains, such as Syk and
Fyn(19, 20, 21, 22) . Substrates for
these kinases may also be recruited(22) . The presence of the
ITAM in all MIRR chains involved in signal transduction has led some to
suggest that apparently heterologous chains such as CD3
and
TCR
are functionally redundant and the presence of multiple ITAMs
within each MIRR serve to increase the strength of signal which can be
generated via the receptor. Evidence for this assertion has been
obtained in studies of both the B and T cell antigen
receptors(8, 12, 23, 24) . In
contrast, we and others have provided evidence indicating that each
heterologous ITAM containing chain has a distinct
function(10, 11, 19, 25, 26) .
and Ig-, are expressed on cell surfaces as
heterodimers(29, 30, 31, 32) .
Therefore, we postulated that within the context of a heterodimer,
Ig- and Ig- would have new, complementary or even synergistic
functions, not predicted from studies of single chain chimeras. and have new and
unpredicted functions in the context of a heterodimer. Using a novel
chimera system, which allowed us to form either hetero- or homodimers
of the cytoplasmic domains of Ig- and Ig-, we observed that
when Ig- is ligated independently it is able to activate tyrosine
kinases. However, when co-aggregated with Ig-, Ig- appears to
remarkably enhance Ig- phosphorylation. This in turn correlates
with an increase in the range and intensity of cellular substrates
phosphorylated by the heterodimer and a lowering of the stimulation
threshold for tyrosine kinase activation.
Construction of PDGFR/Ig-
The construction and expression of the chimeras has
been described in detail elsewhere. (/
Chimeras
)Briefly, cDNAs
encoding the PDGFR and chains (gift of A. Kazlauskas,
National Jewish Center, Denver, CO) were mutated to introduce BamHI and EcoRI sites immediately after that portion
of each cDNA which encodes the transmembrane domain. The introduction
of these sites facilitated the insertion of cDNA fragments encoding the
cytoplasmic domains of Ig- and Ig-(25) . These
fragments were assembled with an EcoRI/XhoI-flanked
cDNA fragment containing multiple stop codons in pSK (Stratagene, La
Jolla, CA), which were then subcloned into the expression vector
pCB6/muTk (gift of H. Singh, University of Chicago,
Chicago, IL) which contains a neomycin resistance gene, an IgM enhancer
and a thymidine kinase promoter. The cDNAs encoding the chimeric
constructs PDGFR
/Ig- and PDGFR/Ig- were transfected
either separately or together into A20 IIA1.6 (33) by
electroporation. Clones were derived by selection with G-418 and
stained with anti-PDGFR and anti-PDGFR antibodies (Genzyme,
Cambridge, MA) then FITC-conjugated anti-IgG (Zymed, San
Francisco, CA). They were analyzed by flow cytometry (FACScan, Becton
Dickinson, Bedford, MA).Reagents
Polyclonal anti-Ig- and
anti-Ig- antibodies were made by immunizing rabbits (HTI
Bioproducts, Ramona, CA) with glutathione S-transferase fusion
proteins containing the cytoplasmic domains of murine Ig- or
Ig-(25) . The serum of rabbits immunized with the Ig-
fusion protein was purified over a column (CNBr-activated Sepharose,
Pharmacia Biotech Inc.) coupled to a peptide corresponding to the
murine Ig- cytoplasmic tail ITAM (amino acid residues
177-196)(34) , whereas the polyclonal anti-Ig-
antibody was purified over a column containing the immunizing Ig-
fusion protein. The anti-phosphotyrosine monoclonal antibodies FB2 and
Ab2 were obtained from ATCC (Rockville, MD) and Oncogene Sciences
(Uniondale, NY), respectively.Cell Growth and Stimulation
For most experiments,
cells were grown in Iscove's modified Dulbecco's medium
(Sigma) supplemented with 10% fetal calf serum (HyClone, Logan UT), 2
mM glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin at 37 °C in 7.5% CO. For the experiments
described in Fig. 6, cells were serum-starved (0.5% fetal calf
serum) for 18 h before initiation of each experiment. For all the
stimulation experiments described, aliquots of 10 
10
cells were suspended in 300 µl of Iscove's modified
Dulbecco's medium and incubated at 37 °C for 5 min. To
stimulate cells via the endogenous BCR, cells were incubated with a
rabbit polyclonal anti-IgG antibody (Jackson Immunoresearch, West
Grove, PA) at 15 µg/ml for the times indicated in each experiment.
To stimulate transfectants through the chimera, cells were incubated
with PDGF-BB ligand (100 ng/ml, except where noted) (Sigma) for 5 min,
followed by anti-PDGFR antibody (5 µg/ml, except where noted)
(Genzyme) for 3 min, then anti-IgG antibody (5 µg/ml)
(Jackson Immunoresearch).
phosphorylation was enhanced when
co-ligated with Ig-. 10 10 cells/sample of
/Ig-///Ig- were stimulated through chimeras with
and without PDGF-BB. Cells were then lysed in 1% Nonidet P-40 at
different time points after stimulation. The cell lysates were
immunoprecipitated with a combination of anti-Ig- and
anti-Ig- antibodies. The immunoprecipitates were resolved by 7.5%
SDS-PAGE, transferred, and probed with Ab2. The immunoblot was then
stripped and reprobed with a combination of anti-Ig- and
anti-Ig- antibodies.
Immunoprecipitation and Immunoblotting
Aliquots of
stimulated or unstimulated cells were lysed on ice with an equal volume
of 2% Nonidet P-40 lysis buffer(19) . After lysis, insoluble
material was removed by centrifugation. Supernatants were incubated
with FB2 (3 µg), anti-Ig- antibody (3 µg), and/or
anti-Ig- antibody and then with protein A-Sepharose beads
(Pharmacia). Washed immunoprecipitates were boiled in SDS sample
buffer, resolved by 7.5% or 10% SDS-PAGE, transferred to nylon membrane
(Immobilon-P, Millipore, Bedford, MA), and finally immunoblotted with
anti-Ig- (1 µg/ml), anti-Ig- (1 µg/ml), or Ab2 (1
µg/ml) antibodies in 3% bovine serum albumin in 10 mM Tris
(pH 8.0), 150 mM NaCl, 0.1% Triton X-100 (TBST). After washing
in TBST, blots were incubated in either horseradish-conjugated goat
anti-mouse IgG or goat anti-rabbit IgG antibodies (Amersham Corp.),
washed in TBST, and then visualized by chemiluminescence (ECL,
Amersham).
Construction, Expression, and Stimulation of PDGFR
Chimeras
To examine if Ig- and Ig- may function
together to initiate pathways of cellular activation, we designed a
system using the human PDGFRs, which allowed us to form either homo- or
heterodimers of Ig- and . Two forms of PDGFR exist, and
, each of which is recognized by specific monoclonal antibodies.
Furthermore, although distinct, each has an equal affinity for the
naturally occurring ligand, PDGF-BB(35) . Therefore, each chain
can be expressed independently, yet made to form homodimers or
predominantly heterodimers on singly or doubly transfected cells,
respectively, by the addition of PDGF-BB. These dimers, which are
representative of the resting complex (PDGF-BB does not induce tyrosine
kinase activation; data not shown), can then be activated by specific
antibodies (Fig. 1A).
antibodies,
followed by goat anti-mouse antibodies. Omission of PDGF-BB before
stimulating doubly transfected cells led to the aggregation of
PDGFR/Ig- alone (right panel). B, schematic
representation of PDGFR chimeras. cDNAs encoding the PDGFR and
chains were mutated to introduce BamHI and EcoRI sites immediately after that portion of each cDNA
encoding the transmembrane domain (Tm). The introduction of
these sites facilitated the insertion of cDNA fragments encoding the
cytoplasmic domains of Ig- and Ig-. Assembled cDNAs were
cloned into the expression vector pCB6+/muTk, which contains a
neomycin resistance gene, an IgM enhancer and a thymidine kinase
promoter. C, flow cytometric analysis of A20 IIA1.6 cells
expressing PDGFR chimeras. Expression of either surface IgG, PDGFR
or PDGFR on wild type, /Ig-, /Ig-, and
/Ig-///Ig- cells. To stain for surface IgG, 2
10
cells/sample from each cell line were incubated
with FITC-conjugated anti-IgG at 4 °C. For chimera staining, 2
10 cells/sample from each cell line were incubated
with anti-PDGFR or anti-PDGFR antibodies and subsequently
FITC-conjugated anti-IgG1 at 4 °C. Also shown is staining of each
cell line without primary antibody. As demonstrated by
immunoprecipitation and immunoblotting, cells with equal staining
intensity with anti-PDGFR and anti-PDGFR antibodies expressed
approximately equal amounts of each protein (Fig. 3).
was phosphorylated upon stimulation through the chimeras in
/Ig-///Ig-. 10 10 cells/sample
of the wild type (WT) or /Ig-///Ig- cells
were either left unstimulated or were stimulated through the chimeras
as in Fig. 2. Lysates from these cells were immunoprecipitated
with FB2, anti-Ig-, or anti-Ig- antibodies.
Immunoprecipitates were resolved by 7.5% SDS-PAGE, transferred to nylon
membrane, and then probed with Ab2, anti-Ig-, or anti-Ig-
antibodies.
/Ig-,
/Ig-, and /Ig-///Ig- cells upon
stimulation through endogenous receptor or through the chimeras. 10
10 cells/sample from each cell line were left
unstimulated (us) or were stimulated through the endogenous
receptor (Ig) or through the chimeras (Ch). Cells
were then lysed in 1% Nonidet P-40 lysis buffer and cell lysates were
immunoprecipitated with anti-phosphotyrosine antibodies (FB2).
Immunoprecipitates were resolved by 10% SDS-PAGE, transferred to nylon
membrane, and then probed with the anti-phosphotyrosine antibody
Ab2.
or
Ig- were fused to the extracellular and transmembrane domains of
either PDGFR or (Fig. 1B). These cDNAs were
expressed singly or in combination in A20 IIA1.6, a B cell lymphoma
that lacks FcRII, to yield three cell lines expressing
approximately equal levels of each chimera (
/Ig- (expressing
PDGFR/Ig-), /Ig- and
/Ig-///Ig-) (Fig. 1C and Fig. 3).The Cytoplasmic Domains of Both Ig-
We first asked if the cytoplasmic tails of Ig- and Ig- Are
Needed to Induce the Efficient Tyrosine Phosphorylation of Cellular
Proteins or
Ig- alone, or the two chains together, were capable of inducing
the tyrosine phosphorylation of cellular proteins in a manner similar
to that induced via the endogenous BCR. Chimeras were stimulated by
sequential incubation with PDGF-BB, followed by anti-PDGFR
antibody and rabbit anti-mouse IgG1. In parallel samples, the
endogenous BCR on each transfectant was stimulated with polyclonal
antibodies to IgG. After stimulation, cells were lysed and
phosphotyrosine immunoprecipitates (with FB2) from each lysate were
resolved by SDS-PAGE and analyzed by blotting with anti-phosphotyrosine
antibodies (Ab2). As shown in Fig. 2, stimulation of the BCR on
wild type and transfected cells induced a similar spectrum and
intensity of tyrosine phosphorylation. In contrast, only in
/Ig-///Ig- cells did stimulation of chimeras induce
tyrosine phosphorylation that was similar in distribution and intensity
to that induced by the endogenous antigen receptor. In /Ig-
or /Ig- cells, stimulation of the chimeras could only induce
the strong tyrosine phosphorylation of a subset of proteins. In related
experiments, truncated co-expressed versions of each chimera which
lacked cytoplasmic domains, PDGFR/- and PDGFR/-,
were incapable of inducing any detectable tyrosine
phosphorylation. However, differences were observed in the
induction via the chimeras in /Ig-///Ig- cells and
by the endogenous BCR. Stimulation of /Ig-///Ig-
failed to induce the tyrosine phosphorylation of proteins of 32 and 40
kDa. Subsequent immunoblotting revealed that the 32-kDa protein was
Ig- (data not shown). The 40-kDa protein appears to be a novel
molecule which is associated with the endogenous Ig-/
heterodimer. (
)These observations suggest that the chimeras
do not utilize the BCR, or associated structures, to initiate tyrosine
phosphorylation. Finally, at least one protein of 120-130 kDa was
phosphorylated strongly in /Ig-///Ig-, weakly in
/Ig- and /Ig-, and not at all by stimulation of the
BCR. Since the chimeras are predicted to have molecular masses of
approximately this size, we examined if this protein was a chimeric
molecule.Stimulation of Chimeric Heterodimers Induces the Tyrosine
Phosphorylation of PDGFR
The wild type A20 IIA1.6 and
/Ig- but Not
PDGFR/Ig-/Ig-///Ig- cells were treated with PDGF-BB and then
stimulated with anti-receptor antibodies as above.
Anti-phosphotyrosine, anti-Ig-, or anti-Ig-
immunoprecipitates were resolved by SDS-PAGE and probed with antibodies
of the same specificity in various combinations. Immunoblotting of the
Ig- and Ig- immunoprecipitates with the same antibodies
confirmed that /Ig-///Ig- cells but not wild type
cells expressed PDGFR/Ig- (135 kDa) and PDGFR/Ig-
(125 kDa) (Fig. 3). Although both chimeras were expressed in
readily detectable amounts, only PDGFR/Ig- was observed to be
phosphorylated following stimulation. The phosphorylation of
PDGFR/Ig- was detected in both Ig- and Ig-
immunoprecipitations, the latter presumably being a result of
co-ligation during stimulation. The PDGFR/Ig- chimera
co-migrated with a prominent tyrosine phosphoprotein precipitated from
the lysates of stimulated cells. No tyrosine phosphoprotein
corresponding to the PDGFR/Ig- chimera was observed, even on
overexposed gels (data not shown). From these results, it is readily
apparent that PDGFR/Ig-, but not PDGFR/Ig-, was
inducibly phosphorylated in /Ig-///Ig- cells.
However, it is not clear if the differences in phosphorylation observed
are due to differences intrinsic to each chain or due to interactions
between the chains. To differentiate between these possibilities, we
compared PDGFR/Ig- and PDGFR/Ig- phosphorylation in
singly and doubly transfected cells.The Phosphorylation of Ig-
We first examined the influence of Ig- Was Extinguished in the
Presence of Ig-
on Ig- phosphorylation by comparing PDGFR/Ig- phosphorylation
in /Ig- and /Ig-///Ig-. The chimeras were
precipitated from the lysates of unstimulated cells (us) or
cells stimulated with PDGF-BB ligand and anti-PDGFR antibody
followed by rabbit anti-mouse IgG for 1, 2, or 5 min. A
representative experiment is shown in Fig. 4(n = 4). In the /Ig- cell line, stimulation of the
chimera induced its own phosphorylation, which was maximal at 2 min and
transient (Fig. 4, upper panel). In contrast,
phosphorylation of PDGFR/Ig- was not detected in
/Ig-///Ig- cells, even though more
PDGFR/Ig- protein was immunoprecipitated (Fig. 4, lower panel). These results suggest that when expressed in
isolation, Ig- can be phosphorylated. However, when ligated in the
presence of Ig-, its own phosphorylation is inhibited. These
observations are in accordance with the minimal inducible tyrosine
phosphorylation of Ig- observed following BCR ligation in A20 (Fig. 5C).
phosphorylation was extinguished
to undetectable levels in /Ig-///Ig-. 10
10 cells/sample of /Ig-, /Ig- and
/Ig-///Ig- were stimulated through the chimeras as
before and then lysed at different time points after stimulation. Cell
lysates were immunoprecipitated with a combination of anti-Ig- and
anti-Ig- antibodies. Immunoprecipitates were resolved by 7.5%
SDS-PAGE, transferred to nylon membrane, and blotted with Ab2 (upper panel). The immunoblot was then stripped and reprobed
with a combination of anti-Ig- and anti-Ig- antibodies (lower panel).
enhanced Ig-
phosphorylation. 10 10 cells/sample of
/Ig- and /Ig-///Ig- cells were stimulated
through their respective chimeras and then lysed at different time
points after stimulation. Cell lysates were immunoprecipitated with
combination of anti-Ig- and anti-Ig- antibodies.
Immunoprecipitates were resolved by 7.5% SDS-PAGE, transferred to nylon
membrane, and probed with Ab2. The immunoblot was then stripped and
reprobed with combination of anti-Ig- and anti-Ig-
antibodies. B, quantitation of the enhancement of Ig-
phosphorylation by Ig-. Immunoreactivities from the immunoblot in A were quantitated densiometrically. The specific tyrosine
phosphorylation of Ig- was calculated as: (immunoreactivity of
sample to Ab2/immunoreactivity of sample to anti-Ig-) 100.
This value was plotted as a function of time. C, the induction
of Ig- and Ig- tyrosine phosphorylation following BCR
stimulation in wild type A20 IIA1.6. 10 10 cells/sample of wild type cells were stimulated with rabbit
anti-mouse antibodies. Cells were then lysed, and the lysates were
immunoprecipitated with FB2. The immunoprecipitates were resolved by
10% SDS-PAGE, transferred to nylon membrane, and probed with Ab2. The
position of Ig- and Ig-, which was determined by probing
parallel samples with antibodies to these molecules (data not shown) is
indicated.
Ig-
In Fig. 3and 4, it is apparent that only
PDGFR Enhances the Phosphorylation of
Ig-/Ig- is tyrosine-phosphorylated following chimera
stimulation and its phosphorylation is strong only when co-ligated with
PDGFR/Ig-. To examine this further, we compared the inductive
tyrosine phosphorylation of PDGFR/Ig- when expressed alone
(/Ig-) or with PDGFR/Ig-
(/Ig-///Ig-). The cell lines /Ig- and
/Ig-///Ig- were stimulated via the chimeras, lysed,
and then immunoprecipitated with a combination of anti-Ig- and
anti-Ig- antibodies. Shown in Fig. 5are the results of a
representative experiment (n = 3). Immunoprecipitates
were resolved by SDS-PAGE, and after transfer to membrane, probed first
with Ab2 (Fig. 5A, upper panel), then stripped
and reprobed with a combination of anti-Ig- and Ig-
antibodies (lower panel). There were remarkable differences in
both the degree and kinetics of PDGFR/Ig- phosphorylation
with and without PDGFR/Ig- co-ligation. In the absence of
PDGFR/Ig-, the tyrosine phosphorylation of PDGFR/Ig- was
weak at 1 min, increased to a maximum at 5 min, and was essentially
absent at 30 min. In contrast, in the presence of PDGFR/Ig-,
PDGFR/Ig- phosphorylation was maximal at 1 min and thereafter
decreased, being almost undetectable at 30 min. To analyze this data
further, we densitometrically quantitated the immunoreactivity of each
sample with Ab2 and anti-Ig- antibodies and then plotted the ratio
of these values as a function of time. As seen in Fig. 5B, co-ligation of PDGFR/Ig- enhanced the
phosphorylation of PDGFR/Ig- approximately 12-fold at 1 min.
This intensity and rapidity of tyrosine phosphorylation is similar to
what is observed for endogenous Ig- following ligation of the BCR (Fig. 5C). phosphorylation with and without
co-cross-linking Ig- were due to differences intrinsic to those
cells in which both chains were expressed. To address this possibility,
we examined if the degree of phosphorylation of PDGFR/Ig- was
influenced by PDGFR/Ig- co-cross-linking on the same cell
line. Therefore, we studied /Ig-///Ig- cells under
two different stimulating conditions. First, following the stimulating
protocol described above, which includes PDGF-BB, heterodimers were
formed and then aggregated in /Ig-///Ig-. In
parallel, cells were stimulated without PDGF-BB, where only
PDGFR/Ig- would have been aggregated. As predicted, the
degree of PDGFR/Ig- phosphorylation was remarkably enhanced
when heterodimers were first formed with PDGF-BB. The results from a
representative experiment are shown in Fig. 6(n = 3).Co-aggregation of PDGFR
Given the remarkable enhancement of Ig-/Ig- and
PDGFR/Ig- Lowered the Threshold for Tyrosine Kinase
Activation
phosphorylation by Ig-, we postulated that the heterodimeric
complex would be more efficient in its ability to activate tyrosine
kinases than a homodimeric complex. Therefore, we compared the
stimulation threshold for the induction of tyrosine phosphorylation in
/Ig-///Ig- and /Ig- by titering out the
primary stimulating antibody. Cells were otherwise stimulated and
analyzed as in Fig. 2. As can be seen in Fig. 7,
stimulating both transfectants with high concentrations of primary
stimulating antibody induced the tyrosine phosphorylation of cellular
proteins. However, the tyrosine phosphorylation of cellular proteins in
/Ig- diminished significantly after the first 4-fold dilution
of anti-PDGFR antibody. In contrast to
/Ig-///Ig-, the tyrosine phosphorylation was still
detected after two dilutions of anti-PDGFR antibody. Similar
results were obtained when /Ig- was compared to
/Ig-///Ig- (data not shown). These results suggest
that the heterodimeric structure of the BCR complex facilitates B cell
responses to low doses of antigen.
/ heterodimer had a lower
threshold of stimulation than the Ig-/ homodimer. 10
10 cells/sample of /Ig- or
/Ig-///Ig- were stimulated through the chimeras as
before except that the indicated decreasing concentrations of
anti-PDGFR antibody (serial 4-fold dilutions) were used. After
stimulation cells were lysed in 1% Nonidet P-40 lysis buffer. Cell
lysates were immunoprecipitated with FB2, and these were resolved by
10% SDS-PAGE, transferred, and probed with
Ab2.
and Ig-, we observed that
the Ig-/ heterodimer induced the phosphorylation of a wider
range of substrates at a lower threshold of stimulation than either
homodimer. This synergy correlated with the ability of Ig- to
enhance the tyrosine phosphorylation of Ig- by more than 10-fold.
Conversely, in the presence of Ig-, Ig- phosphorylation was
extinguished to undetectable levels. These data suggest that one of the
major functions of Ig- is to enhance the phosphorylation and,
therefore, the signal transducing capability of Ig-. Furthermore,
these data suggest that significant ``cross-talk'' or
cross-modulation occurs between the subunits of the B cell antigen
receptor. and Ig- is of biological significance. We
have established clones of WEHI 231, an immature B cell sensitive to
apoptosis, expressing similar combinations of the chimeras described
here. When the chimeras in these transfectants were stimulated, we
observed that the induction of apoptosis required the cytoplasmic tails
of both Ig- and Ig-. In those experiments, as in the
experiments described in this report, only the heterodimerized chimeras
induced tyrosine kinase activation efficiently. and thereby
initiate signaling by recruiting and activating SH2 domain containing
secondary effectors(2, 13, 19, 36) .
Which effectors are recruited by the receptor complex would be
determined, in part, by which of the four tyrosine(s) (34, 37) in the cytoplasmic domain of Ig- are
phosphorylated upon receptor engagement(38) . From our data it
is not clear if Ig- merely enhances the phosphorylation at
previously modified tyrosines or directs the phosphorylation of new
sites. This distinction is of potential significance because if
Ig- directs the phosphorylation of Ig-, the spectrum of
substrates activated by the receptor complex would be altered. could augment Ig-
phosphorylation would be to recruit novel kinases to the receptor
complex. Previously, we found that phosphoproteins of 40 and 42 kDa
bind in vitro to the cytoplasmic domain of Ig- via a
phosphotyrosine-independent QTAT sequence embedded within the Ig-
ITAM(19) . Recently, we have demonstrated that similar
molecules are inducibly tyrosine-phosphorylated by BCR engagement and
are associated with the native Ig-/ heterodimer. Alternatively, it is possible that kinases are constitutively
associated with Ig- because their SH2 domains bind a small
subpopulation of phosphorylated Ig- tails. While we did not
observe any phosphorylation of Ig- in our heterodimerized
chimeras, there was a low level of Ig- phosphorylation in the
native BCR, which minimally increased following receptor engagement (Fig. 5C). It is possible that such phosphorylation of
PDGFR/Ig- occurred at a level too low to detect. recruits SH2 domain-containing proteins,
other than kinases, which bind to and protect Ig- tyrosines from
dephosphorylation. This is a plausible alternative since the tyrosine
phosphatase CD45 is associated with the receptor complex and its
function is necessary for BCR-mediated signal transduction. 's
function appears to be to facilitate the phosphorylation of Ig-,
which in turn allows the efficient activation of tyrosine kinases and
possibly the recruitment of their substrates. The proof of this model
will require an understanding of the mechanisms whereby Ig-
enhances Ig- phosphorylation.
/Ig--mediated signal transduction. Previously, models
of antigen receptor signal transduction have assumed that the
cytoplasmic domains of each receptor chain was an independent signal
transduction unit (left). In contrast, our data support a
model in which cooperation between receptor components leads to the
enhancement of signals initiated by particular chains (right).
)))
We express our appreciation to Frank Fitch and Jean
McGuire for their careful reading of this manuscript and helpful
comments.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Zhang, M. Veselits, S. O'Neill, P. Hou, A. L. Reddi, I. Berlin, M. Ikeda, P. D. Nash, R. Longnecker, H. Band, et al. Ubiquitinylation of Igbeta Dictates the Endocytic Fate of the B Cell Antigen Receptor J. Immunol., October 1, 2007; 179(7): 4435 - 4443. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Reichlin, A. Gazumyan, H. Nagaoka, K. H. Kirsch, M. Kraus, K. Rajewsky, and M. C. Nussenzweig A B Cell Receptor with Two Ig{alpha} Cytoplasmic Domains Supports Development of Mature But Anergic B Cells J. Exp. Med., March 15, 2004; 199(6): 855 - 865. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. D. Wang, J. Lopes, A. B. Cooper, M. Dang-Lawson, L. Matsuuchi, and M. R. Clark Selection of B lymphocytes in the periphery is determined by the functional capacity of the B cell antigen receptor PNAS, January 27, 2004; 101(4): 1027 - 1032. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Li, K. Siemasko, M. R. Clark, and W. Song Cooperative interaction of Ig{alpha} and Ig{beta} of the BCR regulates the kinetics and specificity of antigen targeting Int. Immunol., October 1, 2002; 14(10): 1179 - 1191. [Abstract] |
