From the Centro de Biologia Molecular, Facultad de
Ciencias, Universidad Autonoma de Madrid, Canto Blanco,
28049 Madrid, Spain and the § Department of
Neurobiology, University of Alabama at Birmingham, Birmingham,
Alabama 35294-0021
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ABSTRACT |
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Recent evidence demonstrates that the Heterotrimeric guanine nucleotide-binding proteins (G
proteins)1 transduce signals
from cell-surface receptors for hormones, neurotransmitters, and growth
factors to intracellular second messengers and ion channels (1-5). One
subset of G proteins (the G The signals mediated through G proteins are subject to regulation by a
myriad of factors that can exert their effects throughout the signaling
pathway from surface seven-helix receptors (17) to intracellular
IP3 receptors (18-20). Some of these factors directly modify G proteins and alter G protein function. Pertussis toxin and
cholera toxin associate with certain G protein The evidence that signaling mediated through several different G
proteins can be regulated by PKC phosphorylation of G In Vitro Synthesis of RNA--
In vitro transcription
of sense RNA was performed as described previously (20). Recombinant
plasmids containing G Oocyte Expression and Electrophysiology--
These procedures
are described in detail elsewhere (35). Briefly, oocytes were
defolliculated and maintained at 18 °C in incubation medium
containing ND96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.4), 1.8 mM CaCl2, 50 µg/ml gentamycin, and 5% horse
serum. Whole cell currents were measured at room temperature using an
Axon Instruments GeneClamp in a standard two-microelectrode
voltage-clamp configuration. Current was measured on-line by chart
recorder and digitized using Axoscope software. Electrodes were filled
with 3 M KCl and had a resistance of 1-3 M GTP Measurements--
GTPase measurements were performed on cell
lysates prepared as described (37). Oocytes were homogenized in
ice-cold 10 mM Tris-Cl, pH 8.0, 0.32 M sucrose,
1 mM MgSO4, and the protease inhibitors
aprotinin (10 µg/ml), leupeptin (10 µg/ml), and
phenylmethylsulfonyl fluoride (200 µM). The homogenate
was pelleted twice at 1000 × g to remove cell debris,
and the supernatant fraction was centrifuged at 100,000 × g
for 30 min. GTPase measurements were made as described (38) and
performed for 15 min at room temperature in the presence of 100 µg of
membrane protein resuspended in 50 mM Tris-Cl and protease
inhibitors. Protein content was determined by the method of Bradford.
In Vivo Phosphorylation and Immunodetection--
Immunodetection
and phosphorylation experiments were performed using antibodies made
against peptides specific to G To examine the role of PKC in the functional regulation of
signaling mediated by the G subunits of some heterotrimeric GTP-binding proteins (G proteins) are
subject to modification by protein kinase C (PKC). For the family of G
proteins that activate the phospholipase C/inositol
trisphosphate/calcium/PKC pathway, such modification could result in G
protein autoregulation. To examine the potential regulation of members
of the G
q family by PKC phosphorylation, we
expressed the thyrotropin-releasing hormone (TRH) receptor in
combination with G
q, G
11,
G
14, G
15, or G
16 in
Xenopus oocytes and examined the regulation of signaling by
PKC activators and inhibitors. For G
16 and
G
15, the two family members of hematopoietic lineage,
PKC activators reduce both the magnitude and the time course of
TRH-mediated responses; PKC inhibitors have the opposite effect. The
PKC-mediated effects are evident in measurements of GTPase activity,
suggesting that the regulation is occurring early in the signaling
pathway. In vivo phosphorylation experiments demonstrate
that G
16 is a substrate for PKC modification. By
comparison, G
q is not phosphorylated by PKC in
vivo, and oocytes expressing G
q are not
functionally modulated by PKC. Repeated TRH stimulation of oocytes
expressing G
16 mimics the effects of PKC activators, and
this functional regulation is correlated with an increase in
G
16 phosphorylation. A mutant G
16 with
four consensus PKC phosphorylation sites removed is not phosphorylated in vivo, and TRH responses mediated through the mutant are
not regulated by PKC. These results demonstrate that signaling
involving hematopoietic G proteins is subject to PKC-mediated
autoregulation, at least in part, by phosphorylation of the G protein
subunit.
INTRODUCTION
Top
Abstract
Introduction
References
q family) is responsible for
coupling receptor-mediated signals to activation of PLC
;
in turn, PLC catalyzes the hydrolysis of phosphotidyl-4,5-bisphosphate to produce IP3 and diacylglycerol. IP3 causes
the release of Ca2+ from intracellular stores, and
diacylglycerol activates PKC (6-14). Five members comprise the
G
q family: G
q, G
11,
G
14, G
15, and G
16 (2). All
members are refractory for modification by pertussis toxin, although
they differ with respect to their potency for activation of PLC
and
their tissue distribution. For example, the human clone
G
16 and its murine homolog G
15 are
specifically expressed in cells of hematopoietic lineage (15, 16).
subunits to maintain
them in an inactive state or render them constitutively active,
respectively (21, 22). Post-translational events, such as
myristoylation and palmitoylation, also influence G protein action
(23). Recently, a family of proteins has been identified that act as
GTPase-activating proteins of heterotrimeric G protein
subunits.
Isoforms of these regulators of G protein signaling (RGS proteins) show
specificity toward particular G protein
subunits and exert their
effects by competing with downstream effectors for activated G
subunit binding (24, 25). Tyrosine kinases phosphorylate
G
q and the inhibition of tyrosine phosphorylation prevents accumulation of IP3 (26). Protein kinase C
directly phosphorylates members of other G protein families:
G
i, G
z, and G
12 (27-32).
Phosphorylation by PKC has been shown to regulate signaling through
G
z and G
12 by preventing the association
of the G
subunit with G
subunits (31, 32).
subunits is
particularly intriguing for members of the G
q family,
because such effects of PKC could represent a form of G protein
autoregulation. Although G
q does not appear to be
phosphorylated by PKC (32), the action of PKC on other members of the
G
q family is not known. Previously, we have
characterized the signaling properties of members of the
G
q family expressed in Xenopus oocytes (20, 33), exploiting the fact that the oocyte has an endogenous
Cl
channel that is normally activated by the
PLC/IP3/Ca2+ pathway and that can be used as a
sensitive tool for the electrophysiological characterization of G
protein activity (33, 34). In the present report, we used this system
to evaluate the regulation of G proteins of the G
q
family by PKC phosphorylation. We find that signaling mediated by
G
15 and G
16 is functionally regulated by
PKC and that this change in function is correlated with the level of
phosphorylation of the G
subunit. These results suggest an important
role for PKC in the regulation of G protein signaling in hematopoietic cells.
EXPERIMENTAL PROCEDURES
cDNA inserts were linearized by digestion
with appropriate restriction enzymes. The transcription of linearized
templates was performed in 7.6 mM Tris-HCl, pH 7.6, 6 mM MgCl2, 0.6 mM NaCl, and 10 mM dithiothreitol containing 0.5 mM each ATP,
CTP and UTP, 0.1 mM GTP, 0.5 mM
5'-(7-methyl)-GTP, and 180 units of the appropriate polymerase in a
total volume of 250 µl. The reaction mixture was incubated for 150 min at 37 °C. The DNA template was subsequently removed by treatment
with 5 units of RNase-free DNase I for 15 min at 37 °C. Free
nucleotides were removed using a Sephadex G-50 column. The mRNA was
phenol/chloroform-extracted and recovered by ethanol precipitation. The
RNA was dissolved in RNase-free water at the corresponding
concentration (see legends), divided into aliquots, and stored at
80 °C until used. A mutant G
16 was created in which
four consensus phosphorylation sites (Ser4,
Thr6, Ser53, and Ser336) were
simultaneously mutated to Ala (pALTER 1, Promega).
. During an
experiment, the oocytes were clamped at
80 mV and superfused
continuously in ND96 medium; all drugs were applied in this solution.
For some drugs, Me2SO or ethanol was used as a solvent;
final concentrations of these chemicals were always less than 0.1%. To
minimize the contribution of the oocyte's endogenous G
o
to the measurements (G
o activates the PLC pathway in
oocytes; Ref. 36), all oocytes were treated for 24 h prior to
recording with 5 µg/ml pertussis toxin; this inhibits
G
o signaling but has no effect on signaling through the
pertussis toxin-insensitive G
q family (33).
16 (LARYLDEI) and
G
q (LNLKEYNL). Detection of G
protein was by Western
blot of oocyte membranes (39). Expressing oocytes were homogenized in
0.32 M sucrose in TE buffer (50 mM Tris-Cl, pH
7.5, 1 mM EDTA) with protease inhibitors. Crude membrane
pellets were obtained following 100,000 × g centrifugation
and resuspended in SDS sample buffer. Proteins were separated on 10%
SDS-polyacrylamide gel electrophoresis gels, transferred onto
polyvinylidene difluoride membranes (Pierce), immunoblotted with
antibody (1:800 dilution of primary; 1:1000 dilution of horseradish
peroxidase-conjugated secondary), and visualized using ECL reagents
(Amersham Pharmacia Bioetech). In vivo phosphorylation was
performed essentially as described (32). Oocytes expressing G
subunits were injected with 32Pi (0.5 mCi/ml)
3 h prior to antibody immunoprecipitation. For immunoprecipitation, the homogenization, centrifugation, and
resuspension steps were performed in RIPA buffer (20 mM
NaHEPES, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1%
sodium cholate, 1% Triton X-100, 0.5% SDS), 5 mM NaF, and
10 mM glycerophosphate. The resulting lysate was
precipitated with protein G-agarose and 0.8 mg of G
antibody overnight at 4 °C. The precipitate was centrifuged, resuspended, run
on a 6% acrylamide gel, and exposed to film. Where applicable, data
were analyzed by t test or one-way analysis of variance.
RESULTS
q family of heterotrimeric G
proteins, the TRH receptor was expressed in combination with various G
protein
subunits in Xenopus oocytes, and the resulting
TRH-mediated signal was measured (Fig.
1). The oocyte provides an excellent system for functional analysis of signals mediated by the
G
q family because it contains an endogenous
Ca2+-activated Cl
channel that acts as a
sensitive sensor for G
protein/PLC/IP3/Ca2+-mediated changes that
occur following receptor stimulation. We have demonstrated previously
that the ligand-induced TRH receptor couples to endogenous
G
q in oocytes (33). Furthermore, we have shown that
co-expression of the TRH receptor with different members of the
G
q family (G
q, G
11,
G
14, G
15, and G
16) results
in increases in TRH-induced Cl
currents; these results
indicate functional coupling of exogenously expressed G
subunits to
the TRH receptor in oocytes (20, 33).
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Fig. 1.
PMA-induced changes in the magnitude and
kinetics of TRH-induced Cl currents are specific to
G
16-injected and
G
15-injected oocytes.
A, oocytes were injected with 0.2 ng of TRH receptor cRNA
and 0.005 ng of G
16 cRNA 48 h prior to measurement.
TRH-induced Cl
currents in an individual oocyte were
measured before (control) and 20 min after (PMA)
injection of 1 µM (approximate final concentration) PMA.
The solid bars above each trace represent the application of
100 nM TRH. B, same as in A except
that oocytes were injected with 0.2 ng of TRH receptor cRNA and 0.5 ng
of G
q cRNA 48 h prior to measurement. C,
changes in the magnitude of peak Cl
currents of treated
oocytes expressing TRH receptor and the G protein
subunits of the
G
q family. The amount of cRNA injected was as follows:
0.2 ng of TRH receptor; 0.005 ng of G
15 and
G
16; 0.5 ng of G
q, G
11,
and G
14. The values in parentheses above the
bars denote the number of oocytes recorded first in control
conditions and then 20 min after PMA injection. A value of 100%
represents no change in responses between the two applications.
D, changes in the time course of responses of oocytes
treated as in C. Data are plotted as the time for the peak
response to decay to one-half its maximal value. To control for changes
in time course due to differences in peak Cl
currents,
oocytes were selected post hoc such that peak
Cl
currents for each group were within 10% of each
other. The values in parentheses above the bars denote the
number of oocytes recorded in each condition.
Fig. 1, A and B, shows results from individual
oocytes recorded first during control conditions and then 20 min after
injection of the PKC-activating phorbol ester PMA. Under control
conditions, application of 100 nM TRH to oocytes expressing
the TRH receptor and either G16 (Fig. 1A) or
G
q (Fig. 1B) resulted in characteristic inward Cl
currents. Following PMA treatment, oocytes
expressing G
16 showed a reduction in both the peak
amplitude and the time course of these TRH-mediated Cl
currents (Fig. 1A); oocytes expressing G
q
were unaffected by PMA treatment (Fig. 1B).
Summary data for oocytes expressing TRH receptor and each of the
PLC-activating G proteins are shown in Fig. 1, C and
D. PMA significantly reduced peak TRH-mediated
Cl currents in oocytes expressing G
16
(p < 0.05) and G
15 (p < 0.05); PMA had no effect on oocytes expressing G
q,
G
11, and G
14 (Fig. 1C). In
addition to examining the effect on peak responses, we also
investigated the effect of PMA on the response time course. Because the
magnitude of the peak response could potentially influence the time
course of the response, the post-PMA peak response data from Fig.
1C were examined; only peak Cl
currents that
were of comparable size were included in the time course analyses. For
oocytes showing comparable peak TRH-mediated Cl
currents
following PMA treatment, only oocytes expressing G
16 or
G
15 showed PMA-mediated decreases in response time
course (Fig. 1D). These results suggest that PKC regulates
the cascade induced by TRH receptor activation only in the presence of
G
16 or G
15 subunits.
To elucidate further the PKC-dependent modulation of
G16 signaling, TRH-induced currents were measured in the
presence of PMA, staurosporine, or the inactive phorbol ester 4
PDD
(Fig. 2). Fig. 2A shows data
from a single oocyte recorded first during control conditions and then
20 min after injection of PMA. These results, from a separate oocyte
batch, reiterate the PMA effect shown in Fig. 1; TRH-mediated
Cl
currents in oocytes injected with PMA exhibit a
reduction in both the peak amplitude and the time course of the
response. Treatment with staurosporine, a PKC inhibitor, had the
opposite effect; both the peak amplitude and the response time course
of TRH-mediated currents were increased (Fig. 2B). To rule
out the possibility that the reduction in G
16-mediated
signaling seen with PMA treatment was due to desensitization of the TRH
receptor (because of repeated applications of TRH), a subset of
G
16-expressing oocytes was recorded first during control
conditions and then 20 min following injection of water. Neither peak
currents nor the time course of the response were altered in these
oocytes (Fig. 2C).
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Data summarizing the modulation of the magnitude of the peak response
are shown in Fig. 2D. PMA injection caused a 56% reduction (p < 0.01) of peak Cl currents in
oocytes expressing TRH receptors and G
16; staurosporine caused a 39% increase (p < 0.01). Neither injection
of vehicle solution nor 4
PDD altered the TRH-mediated currents,
which further supports the role of PKC in mediating this effect.
Concentration-response data for both staurosporine and PMA are shown in
Fig. 2E and demonstrate that the modulation by PKC
activators and inhibitors is saturable.
Data summarizing the modulation of the response time course are shown
in Fig. 2F. Only peak Cl currents presented in
Fig. 2D that were of comparable size, 490 nA ± 10%
(across all conditions), were included in the time course analyses. In
vehicle-injected or 4
PDD-injected oocytes expressing TRH receptors
and G
16, the length of time required for the TRH-induced response to decline to one-half its peak amplitude was approximately 13 s. In PMA-injected oocytes, this decline was significantly faster (4.5 s, p < 0.01). In staurosporine-injected
oocytes, the decline was slowed to 24 s (p < 0.01). These results demonstrate that agents which either activate or
inhibit PKC regulate ligand-induced receptor-mediated
G
16 signaling by altering both the amplitude and the
time course of the receptor response.
The evidence that a PKC activator could inhibit signaling through
G16, coupled with the fact that G
16
stimulation results in activation of PKC, suggested the hypothesis that
G
16 activation could autoregulate further
G
16 signaling via PKC activation. To test this
possibility, we designed experiments in which Cl
currents
were measured during repeated TRH stimulation in oocytes expressing TRH
receptor and G
16. Conditions were chosen to minimize homologous desensitization of the receptor upon repeated TRH
applications. The results of one such experiment are shown in Fig.
3A. First, base-line
TRH-mediated peak responses were obtained for oocytes in all
conditions. Next, oocytes were injected with PMA, staurosporine, or
vehicle. Twenty minutes later, oocytes were repeatedly challenged with
30-s applications of TRH at 4-min intervals. Repeated TRH stimulation
of oocytes expressing G
16 resulted in a
time-dependent decrease in the peak response that
approached the levels obtained in oocytes treated with PMA. Oocytes
treated with PMA showed the characteristic reduction in peak currents
that were insensitive to further reduction following repeated TRH
stimulation. Oocytes treated with staurosporine showed the
characteristic increase in peak currents but failed to show a decrease
in peak response following repeated TRH application. Oocytes injected
with G
q, which is insensitive to PKC regulation, did not
show a decrease in peak Cl
currents during repeated TRH
stimulation. The evidence that repetitive stimulation caused a
reduction in the TRH-induced response (i) only in the presence of
G
16, (ii) similar to that seen with PMA treatment, and
(iii) under conditions in which receptor homologous desensitization was
negligible suggests the possibility that the G
16 cascade
can be feedback-regulated.
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To rule out further that desensitization of the receptor was
contributing to the reduction in G16-mediated signaling
during repetitive stimulation, we performed heterologous
desensitization experiments using repetitive stimulation of multiple
receptors that couple to G
16 in oocytes (20).
Experiments were performed similar to those discussed in Fig.
3A using oocytes expressing G
16 and both TRH
and 5HT2c receptors. These data are shown in Fig. 3B. Once
again, base-line TRH-mediated peak responses were obtained for oocytes
in all conditions. Next, oocytes were injected with PMA or vehicle.
Twenty minutes later, oocytes were re-tested with TRH and then
repeatedly stimulated with 30-s applications of 5HT at 4-min intervals.
After three applications of 5HT, the oocytes were re-tested with TRH.
Oocytes expressing G
16 showed decreases in TRH-mediated
Cl
currents following repeated 5HT stimulation that were
comparable to the decreases obtained in PMA-treated oocytes. Oocytes
expressing G
q did not show stimulation-mediated
decreases in Cl
currents. Thus, repeated
G
16 stimulation, even through different receptors, is
capable of down-regulating the G
16-mediated signal. These data not only reinforce the possibility of
G
16-mediated autoregulation but suggest that this
signaling cascade can be regulated by any pathway that activates PKC.
Assuming that all of the components of the
PLC/IP3/Ca2+ pathway are the same following
activation by different members of the Gq family, the
data showing that only some members are modulated by PKC suggest that
the action of PKC occurs at the level of G protein activation. To
support this hypothesis further, two additional experiments were
performed. First, to confirm that the regulation by PKC was not
occurring at the level of the IP3 receptor or at the level
of the Ca2+-activated Cl
channel,
TRH-mediated G protein activation was bypassed by direct injection of
IP3 into the oocyte. The results of this experiment are
shown in Fig. 4. Fig. 4A shows
the responses of an oocyte-expressing TRH receptor and
G
16 before and after treatment with PMA. One minute
after the PMA measurement, the oocyte was injected with IP3. The PMA-induced decrease in peak response following
TRH stimulation was reversed by IP3 (Fig. 4B,
p < 0.05), suggesting that components of the signaling
cascade downstream of IP3 are not the site of the
regulation.
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To confirm that the modulation of function by PKC was occurring at the
level of the G protein, GTPase measurements were performed on oocytes
expressing TRH receptors and either G16 or
G
q. These results are shown in Fig.
5A. Activation with TRH
resulted in a greater than 20-fold increase in GTPase activity in
oocytes expressing either G
16 or G
q over
no-agonist control conditions. There was an approximately 4-fold
increase in TRH-mediated GTPase activity in oocytes expressing the TRH
receptor alone (data not shown), suggesting that the majority of the
GTPase activity in the oocytes expressing either G
16 or
G
q was due to overexpression of these G proteins.
Pretreatment with PMA caused a reduction in GTPase activity in
G
16-expressing oocytes (p < 0.05) but
not in G
q-expressing oocytes. These results suggest that
the action of PMA occurs at a step in the cascade that can affect GTP
hydrolysis, either at the level of the G protein or at the level of G
protein/receptor coupling.
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In theory, a reduction in GTPase activity due to PMA treatment could
result either from a decrease in intrinsic GTPase activity (either
directly or through a change in GTPase-activating protein function) or
from a decrease in the number of activated G protein molecules
(e.g. through an inhibition of G protein/receptor coupling). In the former case, a decrease in GTPase activity would be correlated with an increase in Cl channel activation; in the latter
case, TRH-mediated Cl
channel activation would be
decreased. Since oocytes expressing G
16 show a decrease
in both GTPase activity and Cl
currents, the data are
consistent with a model in which PMA interferes with receptor-mediated
activation of the G protein. To address this hypothesis further, we
repeated the GTPase measurements in the presence of increasing TRH
concentrations (Fig. 5B), and we compared these results to
peak TRH-mediated Cl
currents (Fig. 5C).
Treatment of oocytes with both PMA and staurosporine resulted in a
change in the maximal amount of GTPase activity that could be elicited;
this effect was mirrored by functional changes as assessed by
Cl
current activation. These data suggest that PKC
activators and inhibitors act to alter the fraction of activable
G
16 molecules.
If the fraction of activable G16 molecules is reduced, perhaps by
interfering with the coupling of the TRH receptor to
G
16, then the time course to peak Cl
channel activation should be altered. Changes in the latency to peak
activation following TRH application in oocytes treated with either PMA
or staurosporine are shown in Fig. 5D. PMA treatment resulted in a 3-fold slowing in response latency; staurosporine treatment produced a small decrease in the latency. The much smaller change in latency in the presence of staurosporine may reflect a
smaller amount of endogenous PKC phosphorylation in control oocytes in
that oocyte batch.
Since the above data were consistent with the hypothesis that PKC is
acting on the G protein, experiments were performed to determine if
G16 is phosphorylated by PKC and if the level of phosphorylation correlated with the functional effects of PKC activation. These results are shown in Fig.
6. We first examined the effect of PMA
treatment on oocytes expressing either G
16 or
G
q. For this experiment, oocytes were preincubated with
labeled phosphate in the absence or presence of PMA, homogenized, and immunoprecipitated using subtype-specific antibodies. The specificity of the antibodies was confirmed in a Western blot analysis of oocyte
membranes expressing either G
16 or G
q
(Fig. 6A). Autoradiographs of extracts immunoprecipitated
with the G
16 antibody revealed a band only in oocytes
expressing G
16 and treated with PMA (Fig. 6B). This band was absent or near absent in oocytes not
treated with PMA or in oocytes expressing G
q and treated
with PMA. These results indicate that phosphorylation of
G
16 is specific and observed upon activation of PKC.
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To determine if phosphorylation of G16 is correlated
with receptor activation, oocytes expressing TRH receptor and
G
16 were preincubated with labeled phosphate,
unstimulated or repetitively stimulated with various applications of
TRH, homogenized, and immunoprecipitated. Repetitive TRH stimulation
resulted in an increase in the state of G
16
phosphorylation (Fig. 6C). The amount of G
16
phosphorylation following repetitive TRH application was correlated
with the reduction in peak TRH-mediated Cl
currents as
assessed by voltage clamp (Fig. 6D). These results demonstrate that the phosphorylation of G
16 is
influenced by receptor stimulation and that the degree of
G
16 phosphorylation correlates with the amount of
receptor-mediated activation. The evidence that both repetitive
stimulation of the receptor and PMA treatment induce G
16
phosphorylation supports the hypothesis that G
16 can be
regulated by feedback phosphorylation.
If phosphorylation of G16 by PKC is responsible for the
functional regulation of TRH-mediated responses, then oocytes
expressing a mutant G
16 protein that is not
phosphorylated by PKC should not mediate modulation of TRH-induced
responses. To test this hypothesis, we simultaneously mutated four
sites on G
16 (Ser4, Thr6,
Ser53, and Ser336) that are consensus sequences
for PKC phosphorylation which are not present in G
q. We
expressed this mutant along with TRH receptors in oocytes. The results
of this experiment are shown in Fig. 7. Unlike wild-type G
16, the mutant G
16
protein was not phosphorylated in vivo by PMA (Fig.
7A). In addition, TRH stimulation of oocytes expressing the
mutant G
16 resulted in inward Cl
currents
that appeared comparable to wild-type G
16; however, these currents could not be regulated by PMA (Fig. 7B). In
the same batch of oocytes, whereas oocytes expressing wild-type
G
16 showed the characteristic regulation of peak
Cl
currents in the presence of PMA and staurosporine,
oocytes expressing the mutant G
16 were not regulated
(Fig. 7C). The response time course in the mutant-expressing
oocytes was also not regulated (Fig. 7D). These data
strongly support the hypothesis that PKC phosphorylation of
G
16 is responsible for the functional regulation of
TRH-mediated signaling.
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DISCUSSION |
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G proteins serve as both the upstream initiators and the
downstream targets of a variety of intracellular second messengers. An
important aspect of these cascades must be their ability to prevent
uncontrolled cell stimulation. Such regulation of G protein signaling
could occur through second messenger pathways that are part of an
autoregulatory feedback loop or that are initiated by cross-talk
between second messenger systems. Several types of kinases are known to
modify different proteins of the G protein cascade. These include G
protein-coupled receptor kinases (40, 41), tyrosine kinases (26), and
second messenger kinases (42). In the present report, we show that
signaling mediated by two members of the Gq family that
are endogenously expressed in hematopoietic cells (G
16
and G
15) is regulated by activators and inhibitors of
PKC. A decrease in G
16 signaling is concomitant with
in vivo G
subunit phosphorylation by PKC. We show that
repeated receptor/G
16-coupled activation of the PLC
pathway is sufficient to inhibit downstream signaling and to induce
G
subunit phosphorylation; this result associates PKC
phosphorylation with regulation of the receptor-mediated response.
Additionally, these results demonstrate that stimulation of one
receptor results in desensitization of a second receptor signal when
both receptors are coupled to G
16. These data suggest (i) a mechanism for autoregulation of the receptor-activated
G
16/G
15 transduction pathway and (ii) a
mechanism for regulatory cross-talk by any transduction pathway that
activates PKC.
We believe that the phosphorylation of G16 by PKC is
responsible for the functional regulation of TRH-mediated signaling. In
fact, we show that a mutant G
16 protein that is not
phosphorylated by PKC does not mediate modulation of TRH-induced
responses, suggesting that PKC phosphorylation of G
16 is
responsible for the functional regulation of TRH-mediated signaling.
Furthermore, the evidence that signaling mediated by G
q,
a protein that is highly related to G
16 and which
activates a comparable signaling pathway in oocytes, is not affected by
PKC supports the conclusion that the PKC effect on G
16
is direct. We cannot rule out that some other protein in the oocyte may
be responsible for some of the PKC-mediated effects in the
G
16 pathway, and the likelihood that the functional effects of PKC are solely due to its action on G
16 is
remote given the multiple mechanisms through which G protein-mediated signals have been shown to be modified. For instance, PKC can have
functional effects by acting on various seven-helix receptors and on
downstream signaling components. Furthermore, PKC may not be the only
mechanism acting to control G
16 activity. We and others
(19, 20) have demonstrated that chronic activation of the PLC pathway
by high expression of wild-type or GTPase-deficient G
16
induces signal adaptation through down-regulation of IP3 receptors. Thus, G
16-mediated signaling cascades
initiated by different receptors in hematopoietic cell types is subject
to regulatory control involving multiple mechanisms, including PKC autoregulation and chronic desensitization of IP3 receptors.
We are presently determining the site(s) necessary for this effect and
the mechanism(s) by which such biochemical modification causes
functional inhibition of G16 signaling. For
G
z and G
12, PKC phosphorylation occurs in
the amino-terminal
binding region of the G
subunit and
inhibits signaling by preventing trimer association (31, 32). Two
serine residues and one threonine residue are present in this region of
G
16 and G
15 that could serve as the sites
of PKC phosphorylation. The G
z and G
12
studies also suggest that the active phosphorylation sites are
surrounded by arginine residues (31, 32). One arginine-rich serine site is present in G
16 and G
15; it is found
near the carboxyl terminus in a putative receptor-binding region. Our
GTPase data suggest that PKC phosphorylation of G
16
reduces the number of activable G protein molecules; a mechanism in
which phosphorylation of a site interferes with receptor/G protein
coupling is consistent with these data.
Our data reveal that the only members of the Gq family
that are functionally regulated by PKC phosphorylation are the
G
15 and G
16 subunits. These two G
proteins have a number of interesting features. They can be activated
by a wide variety of functionally different receptors (43); the
potential for these G proteins to couple to a broad spectrum of
receptors makes them a good target for feedback regulation in order to
prevent aberrant signaling. Regulation of the expression and activity
of G
16 may be critical for controlling cellular
proliferation rates. For instance, GTPase-deficient G
16
signaling inhibits growth rates of small cell lung carcinoma (44).
Also, these G protein subunits can be distinguished from other G
proteins by their specific and tightly regulated expression; they are
restricted to hematopoietic cell types (15, 16), and the expression
pattern is subject to change during cellular differentiation (45-49).
In this regard it is intriguing to note that disruption of
G
16 expression in Jurkat T-cells inhibits activation of
T-cell responses and that G
16 proteins may be involved in the negative regulation of TCR signaling (50).
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FOOTNOTES |
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* This work was supported in part by W. M. Keck Foundation Grant 931360 and National Institutes of Health Grant CA13148 (to M. W. Q.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Neurobiology, CIRC 446, University of Alabama at Birmingham, 1719 Sixth Ave. South, Birmingham, AL 35294-0021. Tel.: 205-975-5098; Fax: 205-975-5097; E-mail: quick{at}nrc.uab.edu.
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ABBREVIATIONS |
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The abbreviations used are:
G proteins, heterotrimeric guanine nucleotide-binding proteins;
IP3, inositol trisphosphate;
PKC, protein kinase C;
PLC, phospholipase C;
PMA, phorbol 12-myristate 13-acetate;
TRH, thyrotropin-releasing
hormone;
4-PDD, 4-
-phorbol 12,13-didecanoate;
5HT, serotonin.
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REFERENCES |
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