(Received for publication, April 28, 1995; and in revised form, September 26, 1995)
From the
Engagement of the T cell receptor (TCR)CD3 complex results
in the induction of multiple intracellular events, with protein
tyrosine kinases playing a pivotal role in their initiation.
Biochemical studies also exist suggesting the involvement of
heterotrimeric GTP-binding proteins (G proteins); however, the
functional consequence of this participation in TCR
CD3-mediated
signaling is unresolved. Here, we report TCR
CD3-mediated guanine
nucleotide exchange among the 42-kDa G protein
subunits of the
G
q/11 family, their physical association with CD3
, and the
G
11-dependent activation of phospholipase C
. Protein
tyrosine kinase inhibitors, however, abrogate TCR
CD3-mediated G
protein activation. Quite interesting is the observation that cells
transfected with a function-deficient mutant of G
11 display
diminished tyrosine phosphorylation of TCR
CD3
and
chains, as well as ZAP-70, upon anti-CD3 antibody triggering. These
data indicate the involvement of the G
q/11 family in TCR
CD3
signaling at a step proximal to the receptor and suggest a reciprocal
regulation between tyrosine kinases and G proteins in T cells.
T lymphocytes acquire antigenic peptide/MHC recognition via
their multicomponent T cell receptor (TCR) ()complex
consisting of a polymorphic
heterodimer and associated
,
, and
chains, termed CD3(1) . Engagement of
the TCR
CD3 complex results in a range of cellular responses from
proliferation and clonal expansion to induction of tolerance and, in
some instances, apoptotic cell death(2, 3) . The type
of response is determined by the developmental stage of the T cell, the
affinity of the TCR-antigen interaction, and whether or not there is
simultaneous engagement of costimulatory
receptors(2, 3) .
Upon recognition of antigen, a
complex and intricately choreographed set of signals is transmitted to
the interior of the T cell. The initial early events observed include
tyrosine phosphorylation of a number of cellular substrates, and
phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol
bisphosphate(4) . The latter yields inositol
1,4,5-trisphosphate (IP) and diacylglycerol, second
messengers responsible for mobilization of intracellular calcium and
activation of protein kinase C, respectively (4) .
Of the
PLC isoforms expressed in T cells, only 1 has been shown to
require tyrosine phosphorylation for its activation(5) , while
the
isoforms are activated as a result of interaction with the
GTP-bound
subunit of the heterotrimeric class of G proteins
belonging to the Gq family(6, 7) . Phosphorylation and
subsequent activation of PLC-
1 by tyrosine kinases has been
identified as one of the early events following engagement of the
TCR
CD3 complex(8) . Even though treatment with tyrosine
kinase inhibitors prevents normal PLC-mediated
events(9, 10) , addition of agents known to activate G
protein
subunits restores a substantial portion of this
activity(10) . Since the current literature indicates an
essential role for tyrosine kinase involvement in T cell signal
transduction and places it in the pathway between receptor engagement
and PLC activation(8, 9, 10) , the
involvement of G proteins in T cell signaling remains unclear. However,
considerable evidence does exist supporting the contention that G
proteins are involved in T cell activation(11) . A number of
biochemical events triggered by TCR
CD3-induced activation are
ablated by agents that modulate the action of G proteins. Pertinent to
this is the ability of cholera toxin to inhibit the cellular
proliferation and intracellular Ca
mobilization that
is mediated by anti-CD3 antibody treatment of T
cells(12, 13) . The G protein competitive inhibitor
GDP
S, can impede the extent of inositol phosphates generated upon
stimulation in peripheral T lymphocytes(14, 15) .
Nonhydrolyzable analogs of GTP, such as GTP
S, or other agents such
as ALF
that activate G proteins by circumventing the
need for receptor engagement, can result in T cell
activation(14, 15) .
In the present investigation,
we have attempted to better, and more directly, define the role of G
proteins in T cell activation by studying the involvement of the
G11 G protein, a member of the Gq family(6) , in T cell
signaling via the TCR
CD3 complex. Our results reveal 1)
TCR
CD3-mediated GTP exchange within G
q/11, 2) physical
association between G
q/11 and CD3, 3) tyrosine kinase
dependent-TCR
CD3-mediated G protein activation, 4)
TCR
CD3-mediated PLC
activation and 5) G
11 modulation
of the tyrosine phosphorylation of the CD3
and
chains.
Figure 7:
Deficient tyrosine phosphorylation of CD3
proteins. A, 20 10
neo or G208A
transfectants were incubated with or without antibody OKT3. Cell
lysates prepared in RIPA buffer were used for immunoprecipitation with
an anti-
rabbit antiserum. The phosphotyrosine content of the
immunoprecipitated proteins was assessed by Western blotting with the
anti-phosphotyrosine antibody PY20 (upper panel). Loading and
specificity controls were evaluated with the anti-
antiserum (middle panel), or normal rabbit serum (lower panel),
respectively. The area of migration of phosphorylated
(
-PO
) or unphosphorylated (
)
chain, as well as the heavy (H) and light (L)
chains of the immunoprecipitating antibodies, are indicated. Results
are representative of four separate experiments using cells from three
independent transfections. B, the
chain was
immunoprecipitated from unstimulated or OKT3-stimulated neo or G208A
Jurkat cells as in A with the exception that Nonidet P-40
lysates were used instead. The immunoprecipitates were probed as in A with the PY20 mAb (upper panel), the anti-
antiserum (middle panel), or normal rabbit serum (lower
panel). The positions of a 70-kDa phosphoprotein,
chain, as
well as the immunoprecipitating antibody heavy (H) and light (L) chains are shown. Similar results were obtained from a
second experiment. C, ZAP-70 was immunoprecipitated from
Nonidet P-40 lysates (30
10
cell equivalents per
lane) prepared from OKT3-stimulated or unstimulated neo or G208A Jurkat
cells. The phosphotyrosine content of ZAP-70 (ZAP-70-PO
) was assessed with antibody
PY20 (upper panel) as described in A. The levels of
the precipitated ZAP-70 were determined with anti-ZAP-70 rabbit
polyclonal antiserum (middle panel). The lower panel represents an immunoblot of the same membrane with normal rabbit
serum. The results are representative of two independent experiments. D, the CD3
chain was immunoprecipitated from Nonidet
P-40 lysates (45
10
cell equivalents per lane)
prepared from OKT3- stimulated or unstimulated neo or G208A Jurkat
cells. The
chain from unstimulated cells was immunoprecipitated
by the post-lysis addition of OKT3. The phosphotyrosine content of the
chain (
-PO
) was assessed with
antibody PY20 (upper panel) as described in A. The
levels of the precipitated
chain were determined with the
anti-
rabbit polyclonal antiserum A 452 (middle panel).
The lower panel represents an immunoblot of the same membrane
with normal rabbit serum. A second experiment reproduced these results. E, the CD3
chain was precipitated from 20
10
unstimulated or OKT3-stimulated neo or G208A cells as in C. CD3
-associated phosphoproteins were detected with
antibody PY20 as described in A. The 70- and 80-kDa CD3
-associated phosphoproteins are indicated with arrowheads. This represents one experiment out of two with
similar results. In A and B, molecular mass markers
are shown to the right.
Figure 1:
Anti-CD3 antibodies stimulate
[
-
P]GTP exchange in membrane-associated G
proteins. A, purified Jurkat plasma membranes (Jurkat) or human peripheral blood T cell membranes (PBL) were incubated in photolabeling buffer in the absence
(-) or presence (+) of 10 µg/ml OKT3. Samples without
OKT3 received 10 µg/ml isotype control antibody, UPC-10. Incubation
was for 30 min on ice prior to [
-
P]GTP
labeling (see ``Materials and Methods''). For PBL membranes,
the dried gel was exposed for 4 days, Jurkat for 2 days. OKT3-enhanced
[
-
P]GTP binding in Jurkat membranes is
representative of 50 separate experiments, while PBL membrane labeling
is a single experiment. B, Jurkat membranes were treated with
(+) or without(-) OKT3 as described above. The resulting gel
was transferred to PVDF membrane, exposed to autoradiography (A) for 48 h, and then Western blotted with the indicated
antibodies (WB). Control Ab is an unrelated
anti-peptide antibody. Results represent two replicate experiments. C, Jurkat membranes were preincubated on ice for 30 min in
photolabeling buffer with or without the indicated amount of
anti-G
GTP binding motif antibody or antibody that had been
previously neutralized with specific peptide. The samples were then
incubated an additional 30 min on ice in the absence(-) or
presence (+) of OKT3 (samples without OKT3 received UPC-10) prior
to [
-
P]GTP labeling. Dried gels were
exposed for 48 h. Two independent experiments are
shown.
The identity of the proteins displaying GTP binding upon
TCRCD3 engagement is unknown. However, the protein(s) migrating
at 42/43 kDa have a molecular mass consistent with that of
subunits belonging to the Gq family(23, 24) . In view
of the fact that these G proteins activate PLC-
(6) , we
decided to focus our attention on the further analysis of the 42/43-kDa
band. Jurkat cell membranes were stimulated with antibody OKT3 or
control antibody, followed by photolabeling with
[
-
P]GTP. The samples were resolved by
SDS-PAGE and transferred to a PVDF membrane. As seen in Fig. 1B, incubation with OKT3 induced enhanced
[
-
P]GTP binding. Western blotting of that
membrane with anti-G
q/11 or G
16 anti-peptide antibodies,
reveals the presence of these G
subunits and their comigration
with the [
-
P]GTP-labeled proteins
comprising the 42/43-kDa band (Fig. 1B). To determine
the nucleotide specificity of this band, we performed competition
assays utilizing excess cold adenine or guanine triphosphate. We
stimulated Jurkat cell membranes with OKT3 in the presence of
increasing concentrations of cold ATP or GTP that were in excess of the
amounts present in the photolabeling buffer (2.5 µMversus 0.05 µM, respectively). While
OKT3-induced GTP labeling of the 42/43-kDa band remains strong, even in
the presence of a 1000-fold molar excess of ATP, labeling in the
presence of a 100-fold excess of cold GTP dramatically inhibits the
signal, and a 250-fold excess nearly abolishes it completely (Fig. 2, upper panel). Densitometric analysis of the
resulting autoradiogram is shown in Fig. 2, lower
panel.
Figure 2:
Specificity of guanine nucleotide binding. A, Jurkat plasma membranes were incubated in photolabeling
buffer for 30 min on ice in the absence(-) or presence (+)
of 10 µg/ml OKT3 (samples without OKT3 received 10 µg/ml
isotype control antibody, UPC-10) prior to the addition of increasing
quantities of cold nucleotides as indicated. The cold ATP and GTP added
was in addition to that already present in the photolabeling buffer
(2.5 µMversus 0.05 µM,
respectively). The [-
P]GTP-labeling assay
was performed, and dried gels were exposed for 48 h. The data are
representative of three independent experiments. B,
densitometric analysis of the autoradiogram shown in A.
Although the pattern of OKT3-induced GTP labeling was
reproducible within more than 50 individual experiments, variability
was noted with regard to the intensity of the signals. This
variability, however, allowed the identification of two distinct bands
comprising the 42/43-kDa signal (Fig. 1C). In an
attempt to further characterize these proteins as heterotrimeric G
protein subunits, we performed OKT3-induced GTP-labeling
experiments utilizing Jurkat membranes that had been preincubated with
an anti-peptide antibody raised against the sequence GAGESGKSTIVK, one
of the conserved GTP-binding motifs present in all known G protein
subunits(25) . Such pretreatment significantly inhibited
subsequent OKT3-induced GTP labeling of the 42/43-kDa protein doublet
in a dose dependent manner (Fig. 1C). Densitometric
analysis of the resulting autoradiogram for experiment I reveals a
signal reduction of 40 and 47% with 1 and 2 µl of antibody
preincubation, respectively. Reductions of 40 and 57% were observed in
experiment II. To assess the specificity of the antibody inhibition, we
neutralized the anti-GTP binding motif antibody with the specific
GAGESGKSTIVK peptide prior to preincubation with Jurkat membranes. Such
pretreatment resulted in a 73% reversal of the antibody inhibitory
effect in experiment I and a 100% reversal in experiment II (Fig. 1C). It is unlikely that these proteins represent
other non-
subunit GTP-binding proteins that may cross-react with
this antibody since the closely related Ras GTP-binding protein,
containing the GTP-binding sequence GAGGVGKSALTI, is not recognized. (
)It should be noted that stimulation in the presence of
GDP
S resulted in a similar pattern of inhibition (data not shown).
Inasmuch as the data argue strongly in favor of G protein
subunit activation via TCR
CD3 engagement, we sought further proof
that the 42/43-kDa protein indeed represents a G
q/11 subunit. To
investigate this, we performed a set of GTP exchange experiments
similar to those described above, except that, instead of photoaffinity
labeling and SDS-PAGE analysis, we solubilized Jurkat membranes that
had been treated with control or OKT3 antibody and immunoprecipitated
with specific G protein
subunit antibodies, normal rabbit serum,
or OKT3. Cherenkov counting of the resulting immune complexes reveals a
7.3-fold increase in specific [
-
P]GTP
binding to G
q/11 G proteins upon OKT3 stimulation as compared to
the control antibody (Fig. 3). In contrast,
[
-
P]GTP binding to G
s and
G
i
was not increased significantly over the control
stimulation. Interestingly, a smaller, but significant enhancement in
[
-
P]GTP binding could also be detected in
immunoprecipitates of OKT3-treated membranes (Fig. 3).
Figure 3:
Specific
[-
P]GTP exchange in G
q/11 following
anti- CD3
stimulation. Purified Jurkat plasma membranes were
stimulated with 10 µg/ml anti-CD3
antibody or isotype control (UPC-10) as follows. Membranes were placed in photolabeling
buffer on ice for 30 min in the presence of the antibodies followed by
a 3-min, 30 °C incubation. The samples were then incubated with
[
-
P]GTP for an additional 3 min at 30
°C. Samples were solubilized in Nonidet P-40 lysis buffer,
precleared, then immunoprecipitated with the indicated antibodies or
NRS. Radioactivity associated with the resulting protein A-Sepharose
pellet was determined by Cerenkov counting. The data represent the mean
(± S.E.) of two independent
experiments.
Figure 4:
Genistein and tyrphostin inhibit anti-CD3
-induced [
-
P]GTP exchange. Jurkat
plasma membranes were preincubated on ice for 30 min in photolabeling
buffer with either 2% Me
SO, genistein, or tyrphostin at the
indicated concentrations followed by an additional 30-min incubation on
ice in the absence(-) or presence (+) of 10 µg/ml OKT3.
Samples without OKT3 received 10 µg/ml isotype control antibody,
UPC-10. The [
-
P]GTP labeling assay was
performed, and dried gels were exposed for 48 h followed by
densitometric analysis of the resulting autoradiogram. UPC-10
stimulation was assigned a value of zero. Genistein and tyrphostin
inhibition is representative of five and three independent experiments,
respectively.
Figure 5:
Characterization of G11
transfectants. A, 1 µg of poly(A)
mRNA
was isolated from neo, G208A, and wt Jurkat transfectants and analyzed
by Northern blotting using a G
11-specific probe. The neo lane was exposed for longer time in order to produce a visible
autoradiography signal. B, 75 µg of plasma membrane from
the same cell types were utilized for the Western blot analysis of
G
11 and CD3
expression. C, 5
10
neo, G208A, or wt Jurkat cells were stained with antibody OKT3 (solid line histograms) or isotype control antibody RPC5 (dotted line histograms) and analyzed by flow cytometry.
Results are shown as cell number (linear scale) versus fluorescence intensity (log scale). Similar results were
produced from two independent experiments.
Figure 6:
Physical association of CD3 with
G
11. A, 1
10
neo or G208A Jurkat
cells were lysed in Nonidet P-40, and immunoprecipitations performed
using antibodies OKT3 (anti-CD3
) or WT31 (anti-TCR
).
The immunoprecipitates were then analyzed by Western blotting with
anti-G
q/11 (upper panel), or normal rabbit serum (lower panel). The OKT3 immunoprecipitations represent one of
three replicate experiments with similar results; the WT31
immunoprecipitation is from a single experiment. B, the same
membrane shown in A was stripped and reprobed with anti-G
(upper panel) or normal rabbit serum (lower panel). C, 3
10
human thymocytes were lysed in
Nonidet P-40 and immunoprecipitated with antibody OKT3 or RPC5 isotype
control. The samples were then analyzed by Western blotting with the
anti-G
q/11 antiserum (upper panel) or normal rabbit serum (lower panel). The results represent a single experiment.
Molecular weight markers are shown to the right. The positions
of migration of the G
11 and G
proteins as well as the heavy
chain (H) of the immunoprecipitating antibodies are shown to
the left. The shadows seen in the lower panel of part A represent the leading edges of the H chain
bands.
The physical association of G proteins
with CD3 , but not with TCR
/
is further confirmed by
the detection of G protein
subunits in the same
immunoprecipitated samples that are described above (Fig. 6B, upper panel, lanes
1-4). Here, as in Panel A, no signals were observed
in a NRS control immunoblot of the same membrane (Fig. 6B, lower panel, lanes
1-4).
In like experiments, immunoprecipitation of human
thymocyte lysates with antibody OKT3, but not with RPC5 (IgG2a)
antibody control, reveals the coprecipitation of Gq/11 protein (Fig. 6C, upper panel, lanes 1 and 2). The specificity of the G
q/11 antiserum is confirmed
once more by the absence of any detectable signal with NRS blotting (Fig. 6C, lower panel, lanes 1 and 2).
Immunoprecipitation of the chain from OKT3-stimulated neo
Jurkat cells coprecipitated a 70-kDa phosphoprotein (Fig. 7B, upper panel) that we suspected to be
ZAP-70(29) . To confirm this, we specifically
immunoprecipitated ZAP-70 from control or OKT3-stimulated neo or G208A
Jurkat and found that OKT3-stimulated G208A cells exhibit decreased
tyrosine phosphorylation of ZAP-70 as compared to that from neo Jurkat (Fig. 7C, upper panel). The quantity of ZAP-70
protein immunoprecipitated from both cell types is equivalent as
demonstrated by ZAP-70 Western blots (Fig. 7C, middle panel). The fact that ZAP-70 is not seen in Fig. 7A is due to the treatment of the
immunoprecipitates with RIPA buffer that tends to disrupt physical
associations.
The chain of the CD3 complex is also
phosphorylated on tyrosine residues following TCR
CD3
engagement(29) . We find that CD3
immunoprecipitated from
OKT3-stimulated G208A cells display deficient phosphotyrosine content
as well (Fig. 7D, upper panel). In contrast,
neo Jurkat cells exhibit significant levels of
chain
phosphorylation (Fig. 7D, upper panel). To
control for the amounts of CD3
loaded in each lane, we probed the
same membrane with an anti-
rabbit polyclonal antibody. Clearly
the differences in CD3
phosphorylation cannot be attributable to
differences in the amount of protein loaded (Fig. 7D,
compare upper and middle panels).
Among the
tyrosine phosphorylated proteins that can be coprecipitated with
anti-CD3 antibodies upon TCR
CD3 triggering, is an unknown
protein of 80-kDa whose phosphorylation in G208A cells appears to be
unaffected (Fig. 7E). Thus, the deficiency in tyrosine
phosphorylation seen in the G208A transfectants is selective, and not a
general defect in tyrosine kinase activity. This is also confirmed by
comparing total tyrosine kinase activity between anti-CD3-stimulated
neo and G208A cells following immunoprecipitation and Western blotting
with anti-phosphotyrosine antibodies (data not shown).
In the present investigation, we provide evidence that the G
protein family q/11, a known activator of PLC , is physically
associated with the CD3 complex of the T cell antigen receptor and
becomes activated upon TCR
CD3 engagement; this activation being
dependent on the participation of tyrosine kinases. Furthermore, by
utilizing function-deficient mutants of G
11, we have been able to
demonstrate that Gq/11 proteins are involved in pathways that mediate
both tyrosine phosphorylation of CD3 proteins and IP
generation upon TCR engagement. We provide the first
demonstration of physical and functional association between the PLC
-activating G protein G
q/11 (6) and the TCR
CD3
molecular complex. The fact that the TCR
CD3 complex does not
belong to the family of classic seven-transmembrane receptors suggests
the possibility that G proteins couple to the CD3 complex via a
mechanism similar to that employed by other non seven-transmembrane G
protein-coupled
receptors(30, 31, 32, 33) .
Therefore, the topology of seven-membrane spanning domains may not be
an absolute requirement for receptor/G protein coupling.
It is
interesting that immunoprecipitation with anti-TCR antibody does not
reveal physical interaction with Gq/11. This could be due to the
fact that, under the experimental conditions employed here, not all
associations between G
q/11 and TCR
CD3 proteins may have been
preserved. Alternatively, G
q/11 may simply not interact physically
with TCR
/
.
The physical association between TCRCD3
and G protein strongly suggests receptor-mediated G protein activation.
We reveal for the first time that in isolated plasma membranes of
Jurkat T cells, as well as peripheral blood T cells, antibody-mediated
perturbation of the TCR
CD3 complex results in significantly
enhanced binding of [
-
P]GTP among several
membrane-associated proteins. Of these, the 42/43-kDa species display
characteristics of bona fide G
subunits.
Immunoprecipitation of specific G
subunits from OKT3 activated
Jurkat plasma membranes reveals a 7.3-fold increase in
[
-
P]GTP binding to G
q/11 G proteins.
The enhancement in nucleotide exchange seems to be specific for this
family of G proteins, as G proteins representing two other abundantly
expressed families, G
s and G
i
, do not appear to
undergo any significant GTP exchange upon CD3 perturbation.
Interestingly, a significant enhancement in
[
-
P]GTP binding could also be detected in
complexes immunoprecipitated with the anti-CD3
antibody, OKT3.
This likely reflects [
-
P]GTP binding to
G
q/11 proteins associated with CD3
.
Anti-CD3 engagement
indicates IP production in membrane preparations of control
Jurkat T cells that is ablated in membranes isolated from cells that
had been transfected with a ``function-deficient'' form of
G
11. Similar results are obtained when membranes from these two
cell types are incubated with GTP
S. GTP
S, a nonhydrolyzable
guanine nucleotide analog, activates G proteins by circumventing the
need for receptor engagement and irreversibly dissociating the
subunit from
/
. The fact that GTP
S does not induce
IP
production in membranes isolated from G208A
transfectants suggests that this mutant may function in a
``dominant-negative'' manner. The failure of G208A membranes
to generate IP
upon antibody engagement cannot be due to
deficient CD3 expression as they express normal levels of the receptor.
We believe that in isolated cell membranes, the contribution of PLC
1 to IP
generation is negligible, if any, since it has
been convincingly demonstrated that this isoform resides in the
cytoplasm at the resting state and is recruited to the membrane only
upon activation(34) . Although we cannot exclude completely the
possibility that some small fraction of PLC
1 remains at the
membrane, this could not account for the majority of IP
generated. The reasons being that first, OKT3-induced IP
production is inhibited in the presence of GDP
S (data not
shown) and second, IP
generation is ablated in membranes
prepared from G208A transfectants. Therefore, the Gq family of G
proteins must be responsible for the CD3-mediated IP
production observed in isolated membranes. In contrast, when whole
cells of either the G208A or control transfectants were stimulated with
anti-CD3
antibody, no significant differences in IP
generation were observed (data not shown). This suggests that PLC
1 might be able to compensate for the defect in G
protein-mediated, PLC
-specific IP
production. This
interpretation is corroborated by additional experiments, not presented
here, in which we were unable to detect deficiencies in intracellular
Ca
mobilization, following OKT3 antibody stimulation,
within G208A cells. However, when the human muscarinic type 1 receptor,
a known Gq-coupled receptor not normally expressed in Jurkat
cells(35) , was transiently transfected into G208A or neo
Jurkat, Ca
mobilization upon carbachol stimulation
was observed only in neo Jurkat.
The G208A mutant of G11 has a
substitution at amino acid position 208 where glycine has been replaced
with alanine. The region where this glycine residue is found is highly
conserved among all known G
subunits and is believed to be
involved in the interaction with the guanine
nucleotide(36, 37, 38) . Similar mutations,
previously described for the
subunits of G
s and
G
i(36, 37, 38) , behave as
``dominant-negative'' mutants, as their lack of function is
due to the inability of the GTP-containing
subunit to dissociate
from its
/
partner, a prerequisite step for signal
transduction (36, 37, 38) .
The Gq family
of G proteins is intimately involved in the mechanism of CD3 and
chain tyrosine phosphorylation and the subsequent tyrosine
phosphorylation of ZAP-70, upon T cell receptor engagement, as G208A
Jurkat cells are deficient in these events. The diminution in
phosphorylation is due to the mutation and not the mere overexpression
of the G
11 protein, as overexpression of the wild type, nonmutated
G
11 gene at levels equivalent to that seen in G208A transfectants
has no effect on the OKT3-induced tyrosine phosphorylation of these
proteins. Previous data by Cenciarelli et al.(39) support the involvement of GTP-binding proteins in
the tyrosine phosphorylation of CD3
chain. These investigators
found that tyrosine phosphorylation of the
chain was synergized
by addition of GTP
S to permeabilized, antigen-specific, T cell
hybridomas that were cross-linked with an anti-CD3
antibody(39) . Interestingly, GTP
S alone, without CD3
engagement, did not induce
chain phosphorylation, while
stimulation in the presence of GDP
S, an inhibitor of G protein
activation, ablated
chain phosphorylation (39) .
The G
protein-mediated regulation of CD3/ZAP-70 phosphorylation and
subsequent signal transduction may have profound effects on the T cell
activation process. This is evident by several reports where both
humans with genetic defects in ZAP-70 expression and mice lacking
ZAP-70 all together, are deficient not only in T cell development, but
activation as well(40, 41, 42, 43) .
The mechanism by which G11 regulates CD3/ZAP-70 phosphorylation is
not understood, but it may be related to the observations recently
reported by Lev et al.(44) and van Biesen et
al.(45) . These investigators demonstrated that
cytoplasmic tyrosine kinases, one of which was identified as PYK2 (44) , are responsible for the interaction between the
signaling pathway regulated by G proteins and the one regulated by
tyrosine kinases. Previous observations have also suggested a
cross-talk among G proteins and tyrosine kinases. Examples include the
activation of a 50-kDa GTP-binding protein by the v-Fps protein
tyrosine kinase, which in turn leads to gene expression(46) ,
epidermal growth factor receptor activation of PLC
by way of a
G
i-coupled mechanism (31) and vasopressin, bombesin, and
bradykinin receptor-mediated tyrosine
phosphorylation(47, 48) . It is also of interest to
note that Hausdorff et al. (49) have shown that purified
G
s can be phosphorylated in vitro by the tyrosine kinase
pp60
, although the physiological relevance
of this observation remains unclear.
In conclusion, our data suggest
that trimeric GTP-binding proteins of the Gq family are activated upon
TCRCD3 engagement; that this activation is dependent upon
tyrosine kinases, and once activated, G proteins have a modulatory role
in certain early, important tyrosine phosphorylation events during T
cell signaling, and through their stimulation of PLC
, enhance the
pool of second messengers. Alterations in phosphorylation patterns of
relevant substrates may be critical to the interpretation of antigenic
signals by T cells as immunogenic or tolerogenic, as recently suggested
by Sloan-Lancaster et al.(50) and Madrenas et
al.(51) .