(Received for publication, October 23, 1995)
From the
The B cell antigen receptor complex contains heterodimers of
Ig- 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.
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)()(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) .
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- 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.
As
demonstrated in this report, Ig- 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.
Figure 6:
Ig- 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.
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 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).
Figure 3:
Only
Ig- 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.
Figure 2:
The pattern of total protein
phosphorylation in wild type (WT), /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.
We engineered cDNAs encoding
for molecules in which either the cytoplasmic domains of Ig- 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 Fc
RII, 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).
Figure 4:
Ig- 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).
Figure 5:
A, Ig- 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.
It is possible that the observed
differences in Ig- 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).
Figure 7:
The Ig-/
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.
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- 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.
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- 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.
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- 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.
One
of the mechanisms by which Ig- 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.
Another
possibility is that Ig- 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.
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-'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.
Figure 8:
Model
of Ig-/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).