Identification of G
13 as One of the G-proteins
That Couple to Human Platelet Thromboxane A2 Receptors*
Yasmine
Djellas
,
Jeanne M.
Manganello
,
Kostas
Antonakis§, and
Guy C.
Le Breton
¶
From the
Department of Pharmacology, University of
Illinois, Chicago, Illinois 60612 and the § Institut de
Recherches Scientifiques sur le Cancer, Chimie organique-biologique et
spectroscopique (UMR 133), CNRS B.P. 8, Villejuif 94802, France
 |
ABSTRACT |
Previous studies have shown that ligand or
immunoaffinity chromatography can be used to purify the human platelet
thromboxane A2 (TXA2)
receptor-G
q complex. The same principle of co-elution was used to identify another G-protein associated with platelet TXA2 receptors. It was found that in addition to
G
q, purification of TXA2 receptors by ligand
(SQ31,491)-affinity chromatography resulted in the co-purification of a
member of the G12 family. Using an antipeptide antibody
specific for the human G13
-subunit, this G-protein was
identified as G
13. In separate experiments, it was found
that the TXA2 receptor agonist U46619 stimulated [35S]guanosine 5'-O-(3-thiotriphosphate)
incorporation into G13
-subunit. Further evidence for
functional coupling of G13 to TXA2 receptors was provided in studies where solubilized platelet membranes were subjected to immunoaffinity chromatography using an antibody raised against native TXA2 receptor protein. It was found that
U46619 induced a significant decrease in G
q and
G
13 association with the receptor protein. These results
indicate that both G
q and G
13 are
functionally coupled to TXA2 receptors and dissociate upon
agonist activation. Furthermore, this agonist effect was specifically
blocked by pretreatment with the TXA2 receptor antagonist, BM13.505. Taken collectively, these data provide direct evidence that
endogenous G
13 is a TXA2 receptor-coupled
G-protein, as: 1) its
-subunit can be co-purified with the receptor
protein using both ligand and immunoaffinity chromatography, 2)
TXA2 receptor activation stimulates GTP
S binding to
G
13, and 3) G
13 affinity for the
TXA2 receptor can be modulated by agonist-receptor activation.
 |
INTRODUCTION |
Interaction of the prostaglandin endoperoxide analogue,
TXA2 1 (1, 2)
with platelet receptors (3-5) has been shown to modulate not only
hemostasis but also the development of thromboembolic diseases (6-9).
However, despite recent progress, the TXA2-mediated signal
transduction pathway is not completely understood. In this regard,
previous studies have shown that one mechanism by which TXA2 receptors act is through stimulation of phospholipase
C (PLC) leading to inositol 1,4,5-triphosphate (IP3)
production, and subsequent intracellular Ca2+ mobilization
(10-14). Furthermore, separate studies have linked this stimulation of
PLC activity to TXA2 receptor signal transduction through
the pertussis toxin-insensitive guanine nucleotide-binding protein
(G-protein) Gq (16, 17). On the other hand, experiments conducted in our laboratory provided evidence for the existence of
intraplatelet Ca2+ mobilization, which is independent of
IP3 production (15). This finding raised the possibility
that TXA2 receptors may also couple to a G-protein family
separate from Gq. Additional evidence in support of this
notion was provided by experiments showing that a C-terminal antibody
which recognizes the
-subunit of Gq and G11
was not able to completely inhibit U46619-stimulated GTPase activity
(16). Moreover, ligand and immunoaffinity chromatography purification
of the TXA2 receptor-G-protein complex allowed
co-purification of G-proteins distinct from Gq (17). Taken
together, these results led to the hypothesis that TXA2
receptors might couple to a G-protein(s) to stimulates platelet
aggregation independently of the Gq-PLC-IP3 pathway.
Although this putative G-protein has not been identified, recent
reports have provided indirect evidence that it may belong to the
G12 family (18, 19). In one study, it was shown that activation of platelet TXA2 receptors led to increased
incorporation of the photo reactive GTP analogue
[
-32P]GTP azidoanilide into both G12 and
G13
-subunits, which may suggest coupling of
TXA2 receptors to these
-subunits (20). On the other
hand, as all the agonists tested (U46619, thrombin, ADP, and
vasopressin) produced [
-32P]GTP azidoanilide
incorporation, this labeling could also have been due to activation of
a downstream signaling event or to cross-talk between these separate
signal transduction pathways. In separate studies, it was shown that
the affinity state of TXA2 receptors transfected in COS-7
cells could be influenced by co-expression of G
13 (21).
Although this finding is consistent with the notion that
TXA2 receptors have the capacity to couple with
G13, it is not clear whether such coupling occurs at
physiological concentrations of receptor and/or G-protein.
Consequently, two independent reports have provided indirect evidence
that TXA2 receptors may couple to a G
subunit in the
G12/13 family. Based on these considerations, in the
present study we performed experiments to determine whether this
phenomenon occurs in a native platelet preparation using endogenous
concentrations of TXA2 receptor and G
subunits. To this
end, affinity purification of the receptor-G-protein complex was
employed to measure direct physical association of TXA2
receptors and G
12/13. This approach has been previously
applied in our laboratory to the identification of G
q as
one of the G-proteins associated with the platelet TXA2
receptors (17). It was found that, in addition to G
q,
purification of the TXA2 receptors resulted in co-elution
of G
13. Furthermore, agonist activation of
TXA2 receptors caused an increase in GTP
S binding to
G13
-subunit as well as dissociation of the
receptor-G
13 complex, providing evidence that
G
13 is indeed functionally coupled to platelet TXA2 receptors.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Outdated platelet concentrates were obtained from
Heartland Blood Services (Aurora, IL). SQ intermediate
(ethyl-[1S [1
,
2
-(Z),3
,4
]]-7-[[3-aminomethyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoate) for the synthesis of SQ31,491 was provided by Bristol-Myers Squibb Institute for Medical Research. BM13.177 and BM13.505 were generously supplied by Dr. K. Stegmeier, Roche Molecular Biochemicals
(Mannheim, Germany). [35S]GTP
S (1000-1300 Ci/mmol)
was purchased from Amersham Pharmacia Biotech. U44619 was purchased
from Cayman Chemicals; asolectin was from the American Lecithin Co.
(Atlanta, GA); CHAPS, protein A-Sepharose CL-4B, GTP
S,
o-phenylenediamine, and rabbit preimmune IgG were from
Sigma; Affi-Gel 102 and 4-chloro-1-naphthol (horseradish peroxidase
color development reagent) were from Bio-Rad; and horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L), biotinylated goat
anti-rabbit IgG (H+L), and the Vectastain ABC kit were purchased from
Vector Laboratories (Burlingame, CA).
Antibodies--
A 9-amino acid peptide corresponding to residues
40-48 of the human G
13 (P21, Table I) (22),
with a cysteine added at the N terminus to facilitate coupling to
carrier protein was synthesized by Chiron Mimotropes (Raleigh, NC). The
peptide was coupled to keyhole limpet hemocyanin using
m-maleimidobenzoic acid N-hydroxysuccinimide ester and injected into White New Zealand Pasteurella
multocida-free rabbits, according to previously described
procedures (23). Rabbit polyclonal antibodies against the C-terminal
region of G
q (G-QL, Table I) were produced as described
previously (16). Antibodies were purified from rabbit serum by
chromatography on protein A-Sepharose CL-4B, and the IgG fractions were
labeled with carrier-free Na125I (Amersham Pharmacia
Biotech) using the IODO-BEADS iodination reagent (Pierce). Rabbit
polyclonal IgG raised against residues 2-21 of G
12
(G-12) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Rabbit polyclonal IgG directed against the C-terminal region of
G
12 was a generous gift from J. Sylvio Gutkind (National Institutes of Health, Bethesda, MD) (24). Although this antibody has
been suggested to be specific for G
12, C-terminal
segments of G12 and G13 differ only by 4 amino
acids (18, 19). Consequently, this antibody may have limited
cross-reactivity with G13 and is named G-12/13.
G
common, a rabbit polyclonal IgG recognizing G-protein
-subunits of the Gs and Gi families, was
purchased from Calbiochem.
Membrane Preparation and Solubilization--
Human platelet
membranes were prepared from platelet concentrates and solubilized
using 10 mM CHAPS as described previously (5). Typically,
this method resulted in a 60-70% solubilization of platelet membrane
protein, yielding a final protein concentration of 2-3 mg/ml.
Ligand Affinity Chromatography Purification of the Thromboxane
A2 Receptor-G-protein Complex--
TXA2
receptors were purified as described previously (5). Briefly, the
TXA2 receptor antagonist SQ31,491 was immobilized to
Affi-Gel 102, and CHAPS-solubilized membranes (4 mg of protein) in
buffer A (20% glycerol, 500 mM KCl, 0.2 mM
EGTA, and 0.5 mg/ml asolectin) were incubated with the matrix
overnight. Unbound proteins were eluted as flow-through, and the column
was washed with buffer D (20 mM Tris-HCl, 10 mM
CHAPS, 20% glycerol, 500 mM KCl, 0.2 mM EGTA,
0.5 mg/ml asolectin, pH 7.4). TXA2 receptors and
receptor-associated proteins were then eluted with buffer D containing
50 mM TXA2 receptor antagonist, BM13.177 (25,
26). After elution of the first 1-ml fraction, the flow was stopped for
30 min and restarted to elute the subsequent 1-ml fractions.
TXA2 receptor binding activity as well as G
q
immunoreactivity were found to be concentrated in the first fraction
following the 30-min incubation (17). A modification of this method was
used in order to allow further identification of the TXA2
receptor-associated G-proteins (27). Specifically, after unbound
proteins were washed with buffer D, 3 µg/ml 125I-G-QL
IgG, 125I-G-12 IgG, or 125I-G-13 IgG (or the
same protein concentration of 125I-labeled preimmune IgG
(PI IgG)) was added, and the reaction mixture was allowed to incubate
for 1 h at 20 °C. Unbound antibodies were washed with buffer D,
and elution of TXA2 receptors and receptor-associated proteins was performed as described above. The elution fractions were
counted for 125I activity and specific binding attributable
to G
q, G
12, or G
13 was
defined as the difference between the counts eluted from the
125I-antibody columns minus the counts eluted from the
125I-PI IgG column.
Immunoaffinity Chromatography Purification of the Thromboxane
A2 Receptor-G-protein Complex--
Solubilized platelet
membranes were prepared as described (5), and the CHAPS concentration
was adjusted to 2 mM. The preparation (4 mg of protein) was
then incubated with an immunoaffinity matrix coupled to an
anti-TXA2 receptor antibody (TxAb) for 1 h at 20 °C
(28). 125I-Labeled G-QL IgG, G-13 IgG, or PI IgG was added
(final concentration 150 µg/ml), and the reaction mixture was allowed
to incubate for 5 min. The preparation was then incubated with vehicle
or the TXA2 agonist U46619 (100 nM) (29) for an
additional 5 min. The matrix was loaded on a column and washed with
buffer D to elute unbound proteins. The column was eluted with 100 mM glycine (pH 2.5), and the 3-ml elution fraction was
counted for 125I activity. Specific binding attributable to
G
q or G
13 was defined as the difference
between the counts eluted from the 125I-G-QL or
125I-G-13 columns, respectively, minus the counts eluted
from the 125I-PI IgG column. Eluted counts were normalized
to the amount of purified TXA2 receptor protein, as
measured by densitometric analysis of the immunoaffinity column elution
fractions immunoblotted with TxAb. In experiments where the
TXA2 receptor antagonist BM13.505 (30) was used to block
U46619 effects, BM13.505 (10 µM) was incubated for 30 min
before addition of U46619.
Assay of [35S]GTP
S Binding--
Solubilized
platelet membranes (4 mg of protein) were prepared as described (5) and
incubated with 10 µM GTP
S (5 × 106
cpm [35S]GTP
S) for 5 min at room temperature in the
presence of 1 µM GDP. The incubation was then allowed to
proceed for an additional 15 min at room temperature in the presence or
absence of 10 nM U46619. The preparation was added to 20 µl of G-13 IgG, which had been preincubated with 55 µl of a 10%
(w/v) suspension of protein A-Sepharose. The immunoprecipitates were
collected, washed to remove nonspecifically bound proteins and
incubated with 1 mM G-13 peptide for 1 h at room
temperature to specifically elute G13
-subunits. The
fractions were then added to 5 ml of EconosafeTM (Research
Product International, IL) and analyzed by scintillation spectrometry.
Eluted counts were normalized to the amount of immunoprecipitated G13
-subunit, as measured by densitometric analysis of
the elution fractions immunoblotted with G-13 IgG.
ELISA--
Immulon 2 microtiter plates were coated with either
12.5 µg of synthetic peptide or 125 µg of solubilized platelet
membranes. Following incubation for 1 h at room temperature, the
plates were washed three times with modified Tyrode's buffer
containing 0.1% bovine serum albumin, 5 mM dextrose, 1 mM CaCl2, 5 mM HEPES, pH 7.4, and
then blocked by incubation for 1 h with 5% bovine serum albumin
in the same buffer. Serial dilutions of antisera were applied to the
wells and incubated for an additional 1 h at room temperature. The
wells were washed three times with the modified Tyrode's buffer, and
bound antibodies were detected by incubation for 1 h with goat
anti-rabbit IgG (H+L) conjugated to horseradish peroxidase. After
extensive washing, the color reaction was developed by addition of 50 µl of 0.4 mg/ml o-phenylenediamine, 0.012%
H2O2 in 80 mM citrate phosphate, pH
5. An equal volume of 2 N H2SO4 was
then added, and the presence of specific antibodies was measured by
absorbance at 490 nm.
Polyacrylamide Gel Electrophoresis and Immunoblot Assay--
The
affinity column eluates were first concentrated using Millipore
Ultrafree-MC filters. 20-40 µl of sample (30-40 µg of protein)
was then subjected to SDS-PAGE according to the method of Laemmli (31)
using 10% minigels, under nonreducing conditions, and the proteins
were electrophoretically transferred onto nitrocellulose membranes
according to the method of Towbin et al. (32). After transfer, the nitrocellulose membranes were blocked with 3% gelatin in
Tris-buffered saline and incubated overnight at room temperature with
the indicated dilution of G-QL, G-12/13, or G
common IgG. The blots were washed and treated with biotinylated goat anti-rabbit IgG (H+L) as the secondary antibody. The immunoreactive proteins were
detected with avidin and horseradish peroxidase, followed by 0.5 mg/ml
4-chloro-1-naphthol.
Statistical Analysis--
Data were analyzed according to
Student's paired t test (*, p < 0.05; **,
p < 0.005).
 |
RESULTS |
In order to purify TXA2 receptor-associated
G-proteins, solubilized platelet membranes were subjected to ligand
affinity chromatography, and the column was eluted with the
TXA2 receptor antagonist BM13.177 (5, 25, 26). Elution
fractions were then immunoblotted with G-protein
-subunit specific
antibodies. As illustrated in Fig. 1,
G-QL IgG, which recognizes Gq and G11
-subunits, blotted a major protein band in the molecular mass region
of 42 kDa and two minor bands at approximately 38 kDa in solubilized
platelet membranes and the ligand column elution fraction. This pattern of primary labeling at 42 kDa and secondary labeling at lower molecular
masses has been previously described in human platelets and other
tissues and has been attributed to proteolytic fragments of
G
q (16). Furthermore, blotting with G-12/13 IgG also
revealed immunoreactivity for the
-subunit of G12/13.
Thus, a single band in the molecular mass range of 43-44 kDa was
observed both in solubilized platelet membranes and the ligand affinity
column eluate (Fig. 1). On the other hand, an antibody against
G
common revealed that members of the G
s
and G
i families were only present in solubilized
platelet membranes but not in the affinity column eluate (data not
shown). These results indicate that the ligand chromatography-purified
TXA2 receptor-G-protein complex is selectively enriched in
both G
q and a member(s) of the G
12
family.

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Fig. 1.
Immunoblot of solubilized platelet membranes
and the ligand affinity column eluate against G-QL and G-12/13
antisera. Proteins were separated by SDS-PAGE and transferred to
nitrocellulose membranes as described under "Experimental
Procedures." The membranes were incubated overnight with 1:300
dilution of G-QL IgG or 1:200 dilution of G-12/13 IgG. Lanes
1 and 3, solubilized platelet membranes; lanes
2 and 4, ligand affinity column eluate.
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|
In order to determine whether TXA2 receptors purified in
complex with either G
12 or G
13, the
ligand affinity eluate was first probed using a new antibody
specifically directed against G
13. This polyclonal
antibody was generated by immunizing rabbits with a peptide sequence
unique to an internal segment of the human G13
-subunit
(G-13, Table I) (22). Evaluation of the
sera and IgG revealed that specific antibodies against
G
13 were successfully raised. Specifically, the
anti-G-13 serum was shown by ELISA to react in a
concentration-dependent manner with its cognate peptide (Fig. 2A). Moreover,
evaluation of solubilized platelet membranes by ELISA revealed positive
immunoreactivity against an endogenous platelet protein (Fig.
2B). In addition, immunoblotting of solubilized platelet
membranes with G-13 serum and IgG demonstrated a predominant 43-44 kDa
protein (Fig. 3, lanes
1 and 2, respectively), consistent with the
molecular mass previously described for G13
-subunit (18, 19, 22). The blotting of this band could be prevented by
preincubation of G-13 IgG with its cognate peptide (Fig. 3, lane 3). To utilize this antibody for
quantitative evaluation of G
13, G-13 IgG was iodinated
by standard procedures.

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Fig. 2.
Reactivity of G-13 antiserum against its
cognate peptide and solubilized platelet membranes. Microtiter
wells were coated with 12.5 µg of G-13 peptide (panel A)
or 125 µg of solubilized platelet membrane proteins (panel
B). Various dilutions of G-13 antiserum ( ) or preimmune
antiserum ( ) were added to the wells. Immunoreactivities were
detected by ELISA, as described under "Experimental Procedures."
Each point is the mean of triplicate values obtained from two separate
experiments. Standard error of the mean was typically less than
10%.
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Fig. 3.
Immunoblot of solubilized platelet membranes
against G-13 antiserum and G-13 IgG. Proteins were separated by
SDS-PAGE and transferred to nitrocellulose membranes as described under
"Experimental Procedures." The membranes were incubated overnight
with 1:50 dilution of G-13 antiserum (lane 1), 1:50 dilution
of G-13 IgG (lane 2), or 1:50 dilution of G-13 IgG
preincubated with 14.4 µg/ml (100 µM) G-13 peptide
(lane 3).
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In the next experiments, solubilized platelet membranes were incubated
with the ligand affinity matrix (5). The column was washed with buffer
and then equilibrated with 125I-G-13 IgG. The
TXA2 receptor-G-protein complex was next eluted by BM13.177
(25, 26), and the elution fractions were quantitated for
125I. As G
q is known to couple to
TXA2 receptors (16, 17), a positive control experiment was
conducted using 125I-G-QL IgG. Control experiments were
also performed using 125I-labeled preimmune IgG to
determine nonspecific binding of both G-QL and G-13 IgG. Reported
specific binding represents the difference between the counts eluted
from the 125I-anti-
-subunit IgG column and the counts
eluted from the 125I-labeled preimmune IgG column. Using
this procedure, it was found that TXA2 receptors
co-purified not only with G
q but also with G
13. Thus, Fig. 4
illustrates that the affinity column eluate contained 53 ± 5%
and 24 ± 5% specific binding for G
q
(solid bar) and G
13
(open bar), respectively. These findings,
therefore, provide evidence that both G
q and
G
13 are in direct physical association with endogenous
platelet TXA2 receptors.

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Fig. 4.
Ligand affinity chromatography purification
of TXA2 receptor-G-protein complex. Solubilized
platelet membranes were subjected to ligand affinity chromatography as
described under "Experimental Procedures," and the purified
TXA2 receptor-G-protein complex was probed using
125I-labeled G-QL, G-13 and G-12. Results are expressed in
counts per minute (cpm) of specifically eluted 125I-labeled
IgG and represent the mean ± S.E. of four separate experiments.
Statistical significance was evaluated using a two-sample Student's
t test (*, p < 0.05; **, p < 0.005).
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The next series of experiments was performed to determine whether
G
12 also copurified as part of the TXA2
receptor-G-protein complex. These studies employed a specific antibody
directed at a unique N-terminal segment of G
12 (G-12,
Table I), which was also iodinated for quantitative purposes. Again,
the TXA2 receptor-G-protein complex was purified by ligand
affinity chromatography and the amount of G
12 present in
the eluate was determined using 125I-G-12 IgG. As above,
specific binding was determined by parallel experiments using
125I-labeled preimmune IgG. It was found that, in contrast
to G
13, G
12 did not appear to co-purify
with TXA2 receptors (Fig. 4, hatched
bar). In these experiments, it can be seen that the counts attributable to G
12 are less than the counts
representing nonspecific binding by preimmune IgG. Although this
decrease is not significant, it can be explained on the basis that G-12
IgG is enriched in immunoglobulins against G
12 and
consequently contains a lesser percentage of nonspecific proteins than
preimmune IgG. As nonspecific protein is presumably responsible for
nonspecific binding observed with preimmune IgG, the difference between
125I-G-12 IgG counts and 125I-labeled preimmune
IgG counts yields a negative number. The same phenomenon would also
suggest that the specific binding observed for both G-QL and G-13 IgG
(Fig. 4) is probably underestimated, as each of these IgG fractions
contain less nonspecific proteins than their preimmune IgG controls.
Furthermore, this consideration would indicate that the relative
percentage of specific binding with G-QL and G-13 IgG may not
necessarily represent the actual distribution of G
q and
G
13 within the TXA2 receptor-G-protein complex. Taken together, the above results provide evidence that in
addition to G
q, platelet TXA2 receptors are
coupled to endogenous G
13.
In the next series of experiments, the agonist U46619 was used to
determine whether G
13 is functionally coupled to
TXA2 receptors. In these studies, solubilized platelet
membranes were incubated with [35S]GTP
S in the
presence and absence of U46619 (10 nM) and subjected to
immunoprecipitation with G-13 IgG. Immunoprecipitated
G
13 was eluted using 1 mM G-13 peptide, the
elution fractions were counted for 35S and the counts were
normalized for the amount of purified G13
-subunit. It
can be seen in Fig. 5A that
TXA2 receptor activation led to a 44 ± 18%
(p = 0.05) increase in [35S]GTP
S
binding to G
13.

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Fig. 5.
A, effect of U46619 treatment on
[35S]GTP S binding to G 13. Solubilized
platelet membranes were incubated with [35S]GTP S in
the presence and absence of U46619 (10 nM) and subjected to
immunoprecipitation using G-13 IgG as described under "Experimental
Procedures." Results are expressed as percentage of increase in
[35S]GTP S binding to immunoprecipitated
G 13 compared with vehicle and represent the mean ± S.E. of six separate experiments. Statistical analysis measuring the
effect of U46619 on [35S]GTP S binding to
G 13 was performed using a two-sample Student's
t test. B, effect of U46619 treatment on
G q and G 13 association with
TXA2 receptors. Solubilized platelet membranes were
subjected to immunoaffinity chromatography purification as described
under "Experimental Procedures," and the purified TXA2
receptor-G-protein complex was probed with 125I-labeled
G-QL and G-13 IgG in the presence and absence of U46619 (100 nM). Results are expressed in counts per minute (cpm)
normalized for the amount of purified TXA2 receptor protein
and represent the mean ± S.E. of five separate experiments.
Statistical analysis measuring the effect of U46619 on
G q and G 13 association with
TXA2 receptors was performed using a two-sample Student's
t test (*, p < 0.05; **, p < 0.005).
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|
In separate experiments, an affinity column matrix coupled to an
antibody raised against native TXA2 receptor protein (TxAb) was used to purify the receptor-G-protein complex (16, 28). Briefly,
solubilized platelet membranes were incubated with the affinity matrix
and the coupling of G-protein
-subunits to TXA2 receptors was evaluated in the presence and absence of U46619 (29).
Evidence has been previously provided that agonist activation of a
seven-transmembrane receptor results in the dissociation of its coupled
G-protein
-subunits (33). Based on these considerations, excess
125I-labeled anti-G-protein antibody was added to the
column matrix prior to U46619 treatment in order to trap
-subunits
that may be released upon receptor activation. The excess
125I-labeled anti-
-subunit antibody was then washed from
the column matrix and the TXA2 receptor-G-protein complex
was eluted with glycine 100 mM, pH 2.5. Elution fractions
were then counted for 125I activity and normalized to the
amounts of purified TXA2 receptor protein. Again, specific
binding was determined in parallel experiments using
125I-labeled preimmune IgG. Consistent with the results
obtained with ligand affinity chromatography, both G
q
and G
13 co-purified with platelet TXA2
receptors. Specific binding attributable to G-QL IgG and G-13 IgG was
42 ± 11% and 54 ± 10%, respectively (Fig. 5B,
open bars). Furthermore, treatment with 100 nM U46619 (Fig. 5B, solid
bars) caused a significant decrease in the amount of
specifically eluted 125I-labeled G-QL and G-13 IgG. The
magnitude of the agonist-induced decrease in G-protein-receptor
association was 44 ± 15% and 32 ± 1% for
G
q and G
13, respectively. Finally,
pretreatment with a TXA2 receptor antagonist, BM13.505 (10 µM) (30), completely inhibited U46619-induced
receptor-G-protein complex dissociation (Fig. 5B,
hatched bars). Taken together, these results
demonstrate that in addition to G
q, human platelet
TXA2 receptors are functionally coupled to
G
13.
 |
DISCUSSION |
The present study employed ligand affinity (5) and immunoaffinity
chromatography techniques (28) to purify and identify G-proteins
associated with human platelet TXA2 receptors. These techniques have previously been used for the purification of
TXA2 receptor-G-protein complexes from solubilized platelet
membranes (17). Using both ligand and immunoaffinity chromatography, it was found that in addition to G
q, a member of the
G
12 family of G-proteins co-purifies with platelet
TXA2 receptors. This G-protein was identified as
G
13 using an antibody raised against a unique internal
sequence of human G13
-subunit (22). Additional studies demonstrated that this G13
-subunit was functionally
coupled to TXA2 receptors. Specifically, the
TXA2 receptor agonist U46619 stimulated
[35S]GTP
S incorporation into G13
-subunit as well as caused dissociation of this subunit from
TXA2 receptors.
The G
12 family of G-proteins defines the fourth and the
most recently discovered class of
-subunits (18, 19). The members of
this family share high sequence homology and are ubiquitous and
immunodetectable in most membranes of various mammalian cells and
tissues (22, 34, 35). However, despite intensive research in the past
years, no definitive effector(s) has been assigned to either
G
12 or G
13 (36, 37). Both
G
12 and G
13 are oncogenic, and expression
of their mutationally activated forms stimulates cell proliferation and
induces neoplastic transformation in NIH3T3 and Rat1 cells (24,
38-40). Furthermore, GTPase-deficient mutants of G
12
and G
13 have been shown to stimulate Jun
kinase/stress-activated protein kinase (JNK/SAPK) in NIH3T3, HEK293,
and COS-1 cells (41, 42). In addition, both activated
-subunits have
been shown to stimulate stress fiber formation/focal adhesion assembly
in Swiss 3T3 cells (43) and induce apoptosis when transfected in Chines
hamster ovary or COS-7 cells (44, 45). Finally, signal transduction
through G
12 and G
13 appears to involve
small molecular weight GTP-binding proteins such as RhoA, cdc42, and
Ras (46-48). However, even though there is similarity between
G
12 and G
13-associated pathways, evidence
has been provided that both subunits seem to fulfill distinct cellular
and biological functions. Specifically, G
12 but not
G
13 has been shown to be involved in the transcriptional activation of the serum response element (47). On the other hand,
G
13 but not G
12 is involved in the
induction of inducible nitric-oxide synthase in MCT cells (49) and in
lysophosphatidic acid-induced activation of Rho (50). Other studies
showed that, whereas the guanine nucleotide exchange factor (GEF) for
Rho, p155RhoGEF, was able to act as a GTPase-activating protein toward both G
12 and G
13, only G
13
bound to p155RhoGEF and stimulated its capacity to catalyze nucleotide
exchange on Rho (51, 52). In addition, disruption of the gene encoding
G13
-subunit in mice impaired the ability of endothelial
cells to develop into organized vascular system, resulting in
intrauterine death and demonstrating a role for G
13 in
the regulation of cell movement and developmental angiogenesis
(53).
Two potential effectors for G
13 have been proposed that
would be of interest in the signal transduction pathways associated with TXA2 receptors in platelets. In this connection,
G
13 has been shown to stimulate the ubiquitously
distributed Na/H exchanger isoform, NHE1 (46, 53-55). Moreover,
substitution of C-terminal residues from
z conferred on
13 the ability to respond to stimulation by the
D2-dopamine receptor and to activate NHE1 in an
agonist-dependent manner (54).
In platelets, regulation of Na/H exchange has been shown to modulate
receptor-mediated phospholipase A2 and phospholipase C
activation as well as intracellular Ca2+ mobilization (57,
58). In regard to platelet TXA2 receptors, it was found
that U46619 caused an increase in intracellular pH, which was required
for full U46619-induced Ca2+ mobilization (59). Thus,
coupling of TXA2 receptors to NHE1 activity stimulation
could be a possible mechanism by which G
13 is involved
in TXA2-mediated signal transduction in platelets.
In addition to its indirect effects on intracellular Ca2+
via Na/H exchanger activity stimulation, G
13 has also
been implicated in the activation of L-type
Ca2+ channels (60, 61). Specifically, in rat portal vein
myocytes, the heterotrimer
13
1
3 couples to the
angiotensin AT1A receptors to increase cytoplasmic
Ca2+ concentration (60). Furthermore, it was found that the

dimer released from
13 upon angiotensin
AT1A receptor activation was responsible for the activation
of L-type Ca2+ channels (61). Although
extracellular Ca2+ influx through L-type and
non-L-type Ca2+ channels has been associated
with TXA2 receptor-mediated contraction in rat aorta (62),
no such channels have been identified on the platelet surface thus far.
In summary, the present data demonstrate that platelet TXA2
receptors are functionally coupled to G
13. The
physiological significance of the signal transduction pathway
associated with such coupling requires further investigation.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grant HL-24530, NATO Grant CRG-940595, and Training Grant "Cellular Signaling in the Cardiovascular System" T32 HL07692, and
was conducted under the auspices of the Association for U.S.-French Biomedical Cooperation. This work was presented in part at the annual
meeting of the American Society of Hematology, Miami Beach, FL,
December, 1998.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
Pharmacology, College of Medicine, University of Illinois, 835 S. Wolcott Ave. (M/C 868), Chicago, IL 60612. Tel.: 312-996-4929; Fax:
312-996-1225; E-mail: gcl{at}tigger.uic.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
TXA2, thromboxane A2;
PLC, phospholipase C;
IP3, inositol 1,4,5-triphosphate;
GTP
S, guanosine
5'-O-(3-thiotriphosphate);
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PAGE, polyacrylamide gel electrophoresis;
TxAb, TXA2 antibody;
ELISA, enzyme-linked immunosorbent assay;
PI, preimmune;
GEF, guanine nucleotide exchange factor.
 |
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