 |
INTRODUCTION |
Thromboxane A2
(TxA2)1 is a
product of arachidonic acid metabolism and is synthesized upon
activation of a variety of cells, including platelets, vascular smooth
muscle cells, and macrophages. TxA2 exerts potent
biological activity, causing platelet aggregation and secretion,
vasoconstriction, and mitogenesis (1). These biological effects are the
consequence of the interaction of TxA2 with membrane
receptors. Although pharmacological studies suggested that different
TxA2 receptors (TPs) are expressed in different cell types
or even in a single cell (2), only one gene, encoding a heptahelical G
protein-coupled receptor, has been cloned (3). Deletion of this gene
renders mouse platelets unresponsive to thromboxane analogues and
abolishes the pressor response to infusion of TP agonists (4). Two
splice variants of the carboxyl-terminal tail of TP have been
identified. The first isoform, TP
, was cloned from a placental
library (5) and subsequently from megakaryocytic cell lines (6, 7). The
second, TP
, was cloned from an umbilical vein endothelial cell
library (8). Distinct functions for the two isoforms remain to be
established. Alternative splicing of the carboxyl-terminal tail may be
relevant to coupling of receptors with distinct G proteins. Examples
include the splice variants of the EP3 receptor for
prostaglandin E2 (9) and the angiotensin-II receptors
(10-13). Other regions of G protein-coupled receptors, including the
second and third intracellular loops, may also influence their
interaction with G proteins (14-16).
TPs have been shown to couple to members of the Gq (17-20)
and G12/G13 families (7, 21), whereas
conflicting data have been reported on their ability to couple with
Gi proteins (7, 17, 21-23). TPs do not appear to signal
via Gs. Although the domains of this receptor that regulate
interactions with G proteins remain to be defined, a naturally
occurring mutation in the first intracellular loop results in a mild
bleeding disorder and defective activation of phospholipase C (PLC)
(24).
Partial purification of the human platelet TP by ourselves and others
(18, 25) suggests its association with very high molecular weight G
protein(s). However, the identity of these protein(s) remains unknown.
Recently, a high molecular weight G protein, Gh, has been
identified and cloned (26-28). Gh may be activated via the
1B and
1D adrenoreceptor isoforms; this subtype specificity
involves determinants in their third intracellular loops (27, 29). In
turn, Gh activates a 69-kDa phosphatidylinositol PLC (28)
that has been identified as the PLC
1 subtype (30) through an 8-amino
acid portion near the carboxyl terminus (28). Gh may also
function as a tissue transglutaminase. The capacity of Gh
to function as a transglutaminase varies inversely with GTP binding
in vitro. However, the relevance of this "switch" function to the role of Gh in vivo is unknown.
Indeed, the role of Gh as a G protein has remained
controversial. This results in part from the restriction of studies of
its G protein function to a single receptor subfamily. Given our prior
copurification of the platelet TP with a high molecular weight G
protein, we have sought evidence for the association of this receptor
with Gh. We report that Gh is present in
cardiovascular cells that express both TP isoforms and that either may
be immunoprecipitated with Gh. However, whereas both
isoforms signal via Gq, only TP
signals via
Gh to activate PLC-dependent inositol phosphate formation.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Rat Gh cDNA was donated by Robert
M. Graham of The Victor Chang Cardiac Research Institute, New South
Wales, Australia. cDNAs for the human TP
and TP
isoforms were
contributed by Colin D. Funk of the University of Pennsylvania,
Philadelphia, and by J. Anthony Ware of the Beth Israel
Hospital, Boston, respectively. Gq
cDNA,
originally provided in pCMV5 vector, was donated by Gary L. Johnson,
National Jewish Center, Denver, CO. The cDNA for rat
Gs
was provided by David R. Manning of the University of Pennsylvania.
Oligonucleotides were synthesized by Genosys Biotechnologies Inc., The
Woodlands, TX. All the cell culture media were purchased from Life
Technologies Inc. Anion exchange resin AG 1-X8 and 30% acrylamide/bisacrylamide solution were purchased from Bio-Rad. Aprotinin, leupeptin, pefabloc, dithiothreitol, and restriction enzymes
were obtained from Roche Molecular Biochemicals, Mannheim, Germany. Phenylmethylsulfonyl fluoride (PMSF), CHAPS, and benzamidine were obtained from Sigma. 9,11-Dideoxy-9
,11
-methano-epoxy
prostaglandin F2
(U46619) and SQ29,548 were obtained
from Cayman Chemical Co., Ann Arbor, MI. Nonidet P-40 was obtained from
BDH, Poole, UK. [3H]SQ29,548 was obtained from NEN Life
Science Products.
Cell Culture and Transfection--
All cDNAs used for COS-7
cell transfections, except for Gq
, were subcloned into
the mammalian expression vector pcDNA3 (Invitrogen, San Diego, CA).
COS-7 cells (ATCC, Rockville, MD) were cultured in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum, 2 mM
glutamine, 50 units /ml penicillin, and 100 µg/ml streptomycin under
5% CO2 at 37 °C. The cells were seeded at different densities, for transient transfection, depending on the experiment to
be performed. Cells were seeded in 24-well plates at a density of
4 × 104 cells/well for determination of inositol
phosphate formation, as described (19), grown overnight, and
transfected with a total amount of 0.4 µg of plasmid DNA, mixed with
2 µl of LipofectAMINE (Life Technologies, Inc.) in 250 µl of
serum-free medium (Opti-MEM). COS-7 cells were seeded in a two-well
Lab-Tek slide culture chamber (Nunc, Naperville, IL) for
immunofluorescence staining, at 1 × 105
cells/chamber, and transfected with a total amount of 0.8 µg of
plasmid DNA mixed with 5 µl of LipofectAMINE in 500 µl of Opti-MEM. The cells were plated in 60-mm dishes for immunoblotting, at 4 × 105 cells/dish, and transfected with a total of 4 µg of
plasmid DNA mixed with 20 µl of LipofectAMINE in 2 ml of Opti-MEM.
COS-7 cells were seeded in 100-mm culture dishes for
co-immunoprecipitation experiments, at 1.2 × 106
cells/dish and transfected with a total amount of 12 µg of plasmid DNA mixed with 50 µl of LipofectAMINE in 8 ml of OptiMEM. The cells
were seeded in 150-mm dishes at 3 × 106 cells/dish
and transfected with 20 µg of total plasmid DNA mixed with 140 µl
of LipofectAMINE in 14 ml of Opti-MEM for protein purification and
binding experiments. The total amount of DNA was kept constant in
control experiments by adding DNA from the vector pcDNA3.
HEL (ATCC) and CHRF-288 cells (donated by Lawrence F. Brass of the
University of Pennsylvania) were cultured in RPMI 1640 medium
containing glutamine, penicillin/streptomycin, and 20% fetal bovine
serum. Primary cultures of human aortic smooth muscle cells (HASMC)
were purchased from Clonetics (San Diego, CA) and used at passages
7-9. Human umbilical vein endothelial cells (HUVEC) were grown as
described (31); human umbilical vein smooth muscle cells (HUVSMC) were
donated by Elliot S. Barnathan and cultured as described (32). The
hepatoma cells HepG2 were donated by Rebecca A. Taub of the University
of Pennsylvania and cultured in Dulbecco's modified Eagle's medium
containing glutamine, penicillin/streptomycin, and 10% fetal bovine serum.
Extraction and Amplification of RNA--
Total RNA was extracted
from HepG2, HEL, CHRF-288, HUVEC, HUVSMC, and HASMC by the acid
guanidinium/phenol/chloroform method using the Trizol reagent (Life
Technologies Inc.). Fifty ml of human blood was collected from healthy
volunteers in 9 ml of a sterile solution containing 1.5% citric acid
and 2.5% sodium citrate, pH 6.5. Platelet-rich plasma was obtained by
centrifugation and filtered through a PXLTM8 leukocyte
removal filter (Pall Biomedical Corp., Fajardo, Puerto Rico), as
described (33). Total RNA was extracted as described above and
resuspended in 100 µl of diethylpropylcarbonate-treated water.
One µg of total RNA or, in the case of platelets, 8 µl of the RNA
solution, was reverse-transcribed (RT) in a 20-µl volume using the
1st strand cDNA synthesis kit from Roche Molecular Biochemicals. Five µl of the RT mixture were amplified by polymerase chain reaction (PCR) in a volume of 50 µl using the ExpandTM High
Fidelity PCR System (Roche Molecular Biochemicals) and 0.5 µg of each primer.
Primers used for Gh amplification were designed on the
basis of human tissue transglutaminase (5'-ATCTACCAGGGCTCGGCCAA-3' and
5-ACTCCACCCAGCAGTGGAAG-3') (34) corresponding to nucleotides (nt)
634-653 and nt 1134-1153 (28, 34); when using the cDNA of rat
Gh, primers were designed on the basis of the corresponding sequences of rat Gh corresponding to nt 504-523 and
1004-1023, respectively (5'-ATCTACCAGGGCTCTGTCAA-3' and
5'-ACTCCACCCAGCAGTGGAAA-3') (26).
The products of the RT were also amplified, using primers based on the
sequence of the human TP, upstream of the splicing site and, thus, we
were able to amplify both TP
and TP
. The following primers were
used: 5'-CCTTCCTGCTGAACACGGTCA-3' (nt 572-592) and
5'-GATATACACCCAGGGGTCCAG-3' (nt 847-867).
The absence of leukocyte contamination in the platelet preparations was
confirmed by using the platelet cDNA in PCR reactions with primers
based on the sequence of the leukocyte marker HLA-DQb as described
previously (33). The HLA-DQb primers used were 5'-GTCTCAATTATGTCTTGGAA-3' and 5'-TGCCACTCAGCATCTTGCT-3' corresponding to nt 37-56 and 730-748, respectively.
Control experiments were carried out using human genomic DNA (0.5 µg/reaction) or platelet RNA (not reverse-transcribed, 2 µl/reaction) as templates in PCR reactions with human Gh primers.
cDNA was denatured at 94 °C for 3 min, and then 30 amplification
cycles were performed. The denaturation step was at 94 °C for 1 min
and the elongation step at 72 °C for 1 min. The annealing conditions
were 60 °C for 1 min when using human Gh or TP primers and 55 °C for 1 min when using HLA-DQb or rat Gh
primers. PCR products were electrophoresed, and the gel was blotted on
nylon transfer membranes (HybondTM-N+, Amersham Pharmacia Biotech).
Hybridization was carried out at 45 °C from 3 h to overnight
using 32P-labeled oligonucleotide probes. The
Gh oligonucleotide probe was 5'-GTGGCATGGTCAACTGCAACGAT-3'
corresponding to nt 809-831. The HLA-DQb oligonucleotide probe was
5'-TCGGTGGACACCGTATGCAGACAC-3' corresponding to nt 361-384. After
hybridization, the membranes were washed and autoradiographed.
Immunoblotting--
Immunoblotting was performed under reducing
conditions as described previously (35) using an anti-Gh Ab
(NeoMarkers, Fremont, CA) (1 µg/ml) followed by a
peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch)
diluted 1:5000. Anti-Gq
, anti-Gq/11
, and
anti-Gs
Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and were used at 0.5 µg/ml; a peroxidase-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch) was used as secondary Ab diluted 1:5000.
Partial Protein Purification--
We carried out a partial
protein purification of the plasma membrane proteins to detect
Gh in platelets by immunoblotting. Outdated human platelets
obtained from the Philadelphia American Red Cross were filtered through
a PXLTM8 filter to remove contaminating leukocytes. Low speed
centrifugation was carried out to remove red blood cells. Platelets
were resuspended in 5 mM Tris-HCl, pH 7.4, containing 0.1 µg/ml prostaglandin E1 and sonicated 3 times for 30 s on ice. After a centrifugation at 1,000 × g to remove unbroken cells, the platelet membranes were centrifuged at
100,000 × g for 40 min. The pellet was resuspended by
sonication in a buffer containing 20% glycerol, 5 mM EDTA,
pH 7.4, 0.5 mM dithiothreitol, 0.5 mM
4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (pefabloc), 1 µg/ml leupeptin, and 10 µg/ml aprotinin. CHAPS was then added at a
final concentration of 10 mM.
Membranes were prepared for partial protein purification from COS-7
cells transfected with rat Gh. Cells were washed twice with
phosphate-buffered saline and scraped in 25 mM HEPES, pH 6.5, 150 mM NaCl, 10 mM MgCl2, 5 mM KCl containing 1 mM benzamidine and 128 µg/ml pefabloc. Cells were sonicated, and membranes were prepared as
described previously for platelets.
Partial protein purification was performed using pre-swollen DE52
(diethylaminoethyl cellulose) ion exchanger (Whatman) washed with 200 mM Tris-HCl, pH 8, and 1 mM EDTA. The columns
were equilibrated with 2 volumes (~3 ml) of column buffer (20 mM Tris-HCl, pH 8, 0.1 mM EDTA, 5 mM CHAPS) and finally washed with the same buffer that
contained 0.5 mM dithiothreitol, 1 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.5 mM pefabloc. Two mg of platelet
membrane or 0.5 mg of membrane from COS-7 cells were loaded on the
columns. The columns were washed 3 times with 0.5 ml of column buffer, and the proteins were eluted three times with 0.5 ml of the same buffer
containing increasing concentrations of NaCl (0.1-0.5 M). The proteins eluted at each step were trichloroacetic
acid-precipitated, washed with ice-cold acetone, and dissolved in
Laemmli sample buffer. Immunoblotting for Gh was performed
as described previously.
Binding Assays--
Binding assays were performed in COS-7 cells
transfected with either TP
or TP
, in the presence or in the
absence of Gh. Membranes were prepared as described
previously (7). Briefly, the cells were washed twice in buffer A (25 mM HEPES, pH 6.5, 125 mM NaCl, 10 mM MgCl2, 5 mM KCl) and scraped in
buffer B (as buffer A, plus 1 mM benzamidine, 128 µg/ml
pefabloc, and 10 µM indomethacin). Cells were homogenized
with a glass-glass homogenizer and unbroken cells removed by
centrifugation. Membranes were centrifuged at 100,000 × g for 45 min, resuspended in buffer B at a concentration of
300 µg/ml, and stored at
80 °C until use.
Radioligand binding was performed, incubating the thromboxane receptor
antagonist [3H]SQ29,548 (1-40 nM) with 50 µg of membrane protein in a total volume of 200 µl for 2 h at
4 °C (36).
We tested for the significance of differences using Student's
two-tailed t test.
Immunofluorescence Staining--
Immunofluorescence staining was
performed as we have described previously (36), using anti-TP
Ab
diluted 1:1000 or anti-TP
Ab diluted 1:500 plus or minus
anti-Gh Ab diluted at 2 µg/ml. Fluorescein
isothiocyanate-conjugated donkey anti-rabbit IgG and tetramethylrhodamine-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch) diluted 1:1000 were used as secondary Abs.
Co-immunoprecipitation--
Transfected COS-7 cells, HEL cells,
or HASMC were washed twice in PBS and lysed with 10 mM
Tris-HCl, pH 8, 140 mM NaCl, 0.5% Triton X-100, 1 mM PMSF, 10 µg/ml aprotinin, and 1 µg/ml leupeptin. When indicated, cells were stimulated with the TP agonist, 300 nM U46619, for 5 min at 37 °C before lysis.
The DNA was shorn by passing the cell lysate through a 21-gauge needle.
The lysate was then centrifuged at 4 °C at 3,000 rpm for 10 min and
then at 13,000 rpm for 30 min at 4 °C. Sodium deoxycholate and
sodium dodecyl sulfate (SDS) were added to the supernatant at a final
concentration of 1% and 0.1%, respectively. After a further
centrifugation at 13,000 rpm at 4 °C for 10 min, the clear supernatant was used for immunoprecipitation experiments which were
performed in Eppendorf tubes precoated for 10 min with lysis buffer (10 mM Tris-HCl, pH 8, 140 mM NaCl, 0.5% Triton
X-100).
Five hundred µg of HASMC lysate, 400-600 µg of HEL cell lysate, or
300-400 µg of transfected COS-7 cell lysate were used for immunoprecipitation experiments, and the volume was adjusted to 1 ml
with lysis buffer. Samples were precleared for 30 min at 4 °C with
normal rabbit serum covalently coupled to Sepharose CL-4B and
immunoprecipitated with immunoaffinity Sepharose for either anti-TP
or anti-TP
Abs, as described (36). The beads were washed twice with
1 ml of ice-cold radioimmune precipitation buffer (50 mM
Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS), once with 1 ml of
ice-cold Tris/saline/azide (10 mM Tris-HCl, pH 7.5, 140 mM NaCl), and once with 1 ml of ice-cold 0.05 M
Tris-HCl, pH 6.8. The beads were resuspended in 80 µl of Laemmli
sample buffer, boiled, and loaded onto 10% SDS-polyacrylamide gels.
The proteins were transferred onto nitrocellulose membranes (Schleicher
& Schuell, Göttingen, Germany) in 25 mM Tris-HCl, 192 mM glycine buffer, pH 8.3, containing 0.01% SDS and 20%
methanol. Immunoblotting with anti-Gh antibody was
performed as described above.
Control co-immunoprecipitation experiments were carried out using an
anti-rhodopsin antibody obtained from Biodesign International, Kennebunkport, ME. Coupling of the antibodies to protein G-Sepharose beads was then performed. Briefly, 500 µl of a 20% slurry of protein G-Sepharose 4B (Amersham Pharmacia Biotech, Uppsala, Sweden) in 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2,
150 mM NaCl, 0.5% Nonidet P-40, 0.01 units/ml aprotinin
were incubated with 2.5 µg of the anti-rhodopsin Ab or 10 µl of
normal rabbit serum for 5 h at 4 °C under constant agitation.
The protein G-Sepharose was washed three times with radioimmune
precipitation buffer and used for the immunoprecipitation. Three to
five hundred micrograms of HASMC lysate, 500-700 µg of HEL cell
lysate, or 300 µg of the lysate of COS-7 cells transfected with
Gh were precleared for 30 min and immunoprecipitated
overnight. After washing as described above, samples were analyzed by
electrophoresis and immunoblotted. The same immunoprecipitation
procedure was performed using a bovine retina preparation, which was
kindly provided by Dr. John H. Parks, University of Pennsylvania.
Proteins (~500 µg at a concentration of 3 mg/ml) were solubilized
by adding 1/10 volume of 10% sodium deoxycholate and radioimmune
precipitation buffer (containing 1 mM PMSF, 10 µg/ml
aprotinin, and 1 µg/ml leupeptin) up to 1 ml and by shaking at
4 °C for 5 h. Proteins were precleared for 30 min and
immunoprecipitated overnight with an anti-rhodopsin antibody coupled to
protein G-Sepharose, as described above. Immunoblotting was performed
using the anti-rhodopsin Ab diluted at 2.5 µg/ml, followed by a
peroxidase-conjugated donkey anti-mouse IgG diluted 1:5000.
Inositol Phosphate Formation--
Inositol phosphate formation
was measured in COS-7 cells stimulated with U46619 for 30 min at
37 °C. Reactions were terminated by aspiration of the medium and by
the addition of 750 µl of ice-cold 10 mM formic acid
(36). Agonist-stimulated inositol phosphate formation is expressed as a
percentage of the non-stimulated sample. We tested for significant
differences using analysis of variance followed by Bonferroni's
multiple comparison test between all pairs.
 |
RESULTS |
Identification of Gh Message--
Total RNA was
extracted from human platelets, HUVEC, HUVSMC, and HASMC, that are
known to express TPs. We also sought Gh expression in
megakaryocytic cell lines (HEL and CHRF-288) that have several platelet
characteristics and have been used as models to study platelet
structure and function (37, 38). We used the HepG2 hepatoma cell line
as a positive control for Gh expression, since it is known
that Gh is highly expressed in liver (26). Using primers
specific for human Gh, a fragment of 520 bp was amplified as demonstrated in Fig. 1A. A
fragment of 520 bp was also amplified from the cDNA of rat
Gh, cloned into pcDNA3. The specificity of the PCR
products was confirmed by hybridization with a 32P-labeled
internal oligonucleotide (Fig. 1B). Sequence analysis of the
PCR product obtained with HepG2 cDNA confirmed that the 520-bp
fragment corresponds to human Gh (data not shown). When platelet cDNA was used, no bands were evident by staining of the gel, although a positive band was detected after hybridization, demonstrating that platelets also express Gh mRNA (Fig.
1B).

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Fig. 1.
Identification of Gh message by
RT-PCR. RNA from HepG2, HEL, CHRF-288, HUVEC, HUVSMC, HASMC, and
platelets was reverse-transcribed and amplified by PCR. Rat
Gh cDNA was used as positive control. A,
agarose gel stained with ethidium bromide; B, Southern blot
of the gel followed by hybridization with a specific internal
oligonucleotide.
|
|
No fragments were obtained when using human genomic DNA as template,
indicating that the PCR products were derived from the respective
cDNAs. Amplification of the Gh fragment from platelets depended strictly on cDNA synthesis, since no products were formed when using platelet RNA in the PCR reaction.
The purity of our platelet preparation was tested by performing RT-PCR,
followed by Southern blotting and hybridization, with primers specific
for the leukocyte marker HLA-DQb. However, a product was not formed,
indicating that contaminating leukocytes were absent from the platelet preparation.
The expression of TPs in the cell types used in these experiments was
confirmed by RT-PCR using oligonucleotides common to TP
and TP
: a
positive band of 296 bp was obtained with all the cDNA preparations
tested (data not shown).
Identification of Platelet Gh by
Immunoblotting--
The results obtained by RT-PCR demonstrated the
presence of Gh message in platelets, but the level of
expression appears to be low, since a positive band was observed only
after Southern blotting and hybridization. This is consistent with our
failure to detect clear bands corresponding to Gh by
Western blotting, using a whole cell lysate or a platelet membrane
preparation. To increase the concentration of Gh to a level
detectable by immunoblotting, we carried out a partial purification of
the membrane proteins by ion-exchange chromatography. As a control, the
same purification steps were performed using membranes from COS-7 cells
transfected with rat Gh. Immunoblotting revealed a band
corresponding to Gh in the 0.1 M NaCl fraction
from human platelets and in the 0.3 M NaCl fraction from
COS-7 cells transfected with rat Gh (Fig. 2). The differential retention of rat
versus human Gh is attributable to species
divergence (39).

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Fig. 2.
Identification of Gh protein by
immunoblotting. Membrane proteins from human platelets and from
COS-7 cells transfected with rat Gh cDNA were partially
purified by chromatography through a DE52 anion exchanger and eluted
with different concentrations of NaCl (0-0.5 M). Crude
cell lysate of Gh-transfected COS-7 cells (C)
was also loaded on the gel, as a control. Molecular mass markers (kDa)
are indicated in the left margin of the figure.
|
|
Overexpression of TPs and G Proteins in COS-7 Cells--
To
address the interaction of TPs with Gh, we used a
cotransfection approach in COS cells that has been described previously (14, 40-42). Binding of [3H]SQ29,548 to both TP
and
TP
was saturable, and receptor density in both TP
- and
TP
-transfected cells was similar (Fig.
3). In addition, neither density nor
affinity of either TP isoform, as detected by binding of the antagonist
[3H]SQ29,548, was altered by cotransfection with
Gh (Fig. 3). COS-7 cells that were not transfected failed
to bind [3H]SQ29,548 detectably, although TP message
could be amplified from these cells (data not shown).

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Fig. 3.
Binding of [3H]SQ29,548 to
membranes of transfected COS-7 cells. Membranes from COS-7 cells
transfected with TP + vector (+vec), TP + Gh, TP + vector, or TP + Gh ( 50 µg)
were incubated with increasing concentrations of
[3H]SQ29,548 for 2 h at 4 °C. Nonspecific binding
was measured in the presence of 25 µM unlabeled ligand.
Results represent the mean ± S.E. of 6-7 experiments.
|
|
The expression of G proteins in non-transfected COS-7 cells and in
cells transfected with TP
or TP
plus Gh,
Gq
, or Gs
was confirmed by
immunoblotting. A strong signal corresponding to Gh was
observed when Gh was overexpressed together with either TP
or TP
. A band of slightly higher molecular weight, usually also observed in non-transfected cells, may represent monkey
Gh, probably expressed constitutively in COS-7 cells. Both
Gq
and Gs
were detected when they were
overexpressed with either TP
or TP
, whereas no signal was
detected in non-transfected cells. When non-transfected cells were
probed with an anti-Gq/11
Ab, a band of the expected
molecular weight was observed. Detection of a positive band with the
anti-Gq/11
Ab, but not with the anti Gq
Ab, may reflect higher expression of G11
than
Gq
in COS-7 cells (Fig.
4). Immunofluorescence staining was
performed to assess the coexpression of the TP isoforms with
Gh in these transiently transfected cells. The majority of
the transfected cells stained positive both for the respective TPs and
Gh (data not shown).

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Fig. 4.
Identification of G proteins in transfected
COS-7 cells. Lysates from mock-transfected (M) COS-7
cells or from cells transfected with TP ( ) or TP ( )
together with Gh (lanes 2 and 3),
Gq (lanes 5 and 6), or Gs
(lanes 8 and 9) were analyzed by SDS-PAGE and
immunoblotting. Anti-Gh (lanes 1-3),
anti-Gq (lanes 4-6), anti-Gs
(lanes 7-9), or anti-Gq/11 (lane 10)
antibodies were used for immunoblotting. Molecular mass markers (kDa)
are indicated in the left margin of the figure.
|
|
Physical Coupling of TPs with Gh--
TPs were
immunoprecipitated using specific Abs which we previously raised
against TP
and TP
(36). These experiments were carried out in
COS-7 cells transfected with TP
+ Gh, TP
+ Gh, or only with Gh, as well as in control
cells, such as HEL and HASMC.
TP
+ Gh and TP
+ Gh-transfected COS-7
cells were stimulated or not with the TP agonist, U46619.
Gh was detected in TP
and TP
immunoprecipitates,
irrespective of whether the cells had been previously stimulated with
the agonist (Fig. 5A). COS-7
cells transfected with Gh only express very low levels of
their endogenous TPs, detectable by RT-PCR but not by
[3H]SQ29,548 binding.

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Fig. 5.
Immunoprecipitation of Gh with
TP and TP in the
lysate of COS-7 cells transfected with TP + Gh (A, left panel), TP + Gh (A, right panel), or
Gh alone (B). Cells transfected with
TP + Gh or TP + Gh were either stimulated
with U46619 before lysis (stim.) or not stimulated
(unstim.). Immunoprecipitations were carried out using
anti-TP (TP Ab), anti-TP (TP
Ab), or anti-rhodopsin (rhod Ab) antibodies, and the
immunoprecipitated samples (ip) were analyzed by
SDS-polyacrylamide gel electrophoresis. Normal rabbit serum
(nrs) samples used for the preclearing, an aliquot of the
supernatant (s) of the immunoprecipitations, and a crude
cell lysate sample (C) were also run in the gel.
Molecular mass markers (kDa) are indicated in the left
margin of the figure.
|
|
Even under these circumstances, Gh was readily detectable
in immunoprecipitates of either isoform (Fig. 5B). Thus,
Gh appears to possess high affinity for TPs, as it can be
immunoprecipitated even when TP expression is low. It is unlikely that
this reflects nonspecific immunoprecipitation of Gh, as
Gh was not detected in the normal rabbit serum sample that
we used for the preclearing step (Fig. 5B) and was not
immunoprecipitated by a control anti-rhodopsin Ab (Fig.
5A).
Rhodopsin is specifically expressed in retina and is absent in COS-7
cells. In contrast to our findings in COS-7 cells, we could
immunoprecipitate rhodopsin from a retinal preparation with the
anti-rhodopsin Ab (data not shown). Consistent with our observations in
COS-7 cells, Gh was again detected in samples
immunoprecipitated with either TP
or TP
Abs from both HEL (Fig.
6A) and HASM cells (Fig.
6B). Again, Gh was not detected either in the
normal rabbit serum samples used for preclearing or when the
anti-rhodopsin Ab was used for immunoprecipitation (Fig. 6,
A and B). Thus, Gh appears to favor
immunoprecipitation with either TP
or TP
.

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Fig. 6.
Immunoprecipitation of Gh with
TP and TP in the
lysate of HEL (A) and of HASMC
(B). Cell lysates were immunoprecipitated with
anti-TP (TP Ab), anti-TP (TP Ab), or
anti-rhodopsin (rhod Ab) antibodies. Normal rabbit serum
(nrs) samples used for preclearing, an aliquot of the
supernatant (s) of the immunoprecipitations, and a crude
cell lysate sample (C) were also run in the gel.
Molecular mass markers (kDa) are indicated in the left
margin of the figure.
|
|
Functional Coupling of TPs with Gh--
COS-7 cells
were transfected with either TP
or TP
with or without
Gh, Gq
, or Gs
.
Gq
and Gs
were used as positive and negative controls for TP-mediated signaling events, respectively (2).
U46619 dose-dependently increased inositol phosphate
production in cells transfected with TP
alone, with an
EC50 of 3.48 ± 0.34 nM (n = 6). When cells had been transfected with both TP
and Gh, a further increase of inositol phosphate was observed
(Fig. 7A); however, the
EC50 (4.27 ± 0.81 nM) was not
significantly altered. The agonist-stimulated increase of inositol
phosphate formation was higher in TP
+ Gq
-transfected
cells than in TP
+ vector-transfected cells. Consequently, the
EC50 (0.83 ± 0.08 nM) was significantly
(p < 0.05) lower than in cells transfected with TP
+ vector. As expected, TP
did not activate PLC via Gs. Agonist-stimulated TP
+ Gs
-transfected cells did not
produce more inositol phosphate than cells transfected with TP
+ vector. The EC50 (6.87 ± 2.48 nM) for
agonist was also similar (p = not significant) to that
observed in TP
+ vector-transfected cells.

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|
Fig. 7.
U46619-stimulated production of IP. IP
production in COS-7 cells transfected with TP (A) or
TP (B) along with the vector pcDNA3
(+vec), as a control, Gh
(+Gh), Gq (+Gq),
or Gs (+Gs) was measured after 30 min
incubation with different concentrations of U46619. Results are
expressed as percentage of the inosital phosohate (IPs)
production measured in the non-stimulated sample (control) and
represent the mean ± S.E. of six experiments performed in
duplicate. * p < 0.05; ** p < 0.01, compared with control.
|
|
Similar experiments were carried out in cells transfected with TP
.
Agonist-stimulated inositol phosphate production only exceeded that in
cells transfected TP
+ vector in those cotransfected with TP
+ Gq
(Fig. 7B). Although Gq
caused a shift of the U46619 dose-response curve to the left, the
EC50 calculated in TP
+ Gq
-transfected
cells was not significantly different from that found in TP
+ vector-transfected cells (TP
+ vector: EC50 = 5.36 ± 1.89 nM; TP
+ Gq
: EC50 = 1.97 ± 0.29 nM, p = not significant). No significant increase in agonist-induced inositol phosphate production was observed in either TP
+ Gh or TP
+ Gs
-transfected cells versus TP
+ vector-transfected cells. Similarly, no differences were found in the
EC50 (TP
+ Gh: EC50 = 5.75 ± 0.75 nM; TP
+ Gs
EC50 = 5.10 ± 2.08 nM, p = not significant).
 |
DISCUSSION |
Gh is a newly characterized high molecular weight G
protein that can be activated via the
1 adrenoreceptor (27, 29). We
have previously found that human platelet TPs, purified roughly 2,000-fold, were associated with an uncharacterized G protein of a
similar, high (~70 kDa) molecular mass (25). Given these observations, we decided to address the possibility that TPs might actually transmit intracellular signals via Gh.
Furthermore, given the uncertain role of the TP isoforms, we sought to
investigate whether they might couple with differential affinity to
either Gh or Gq
. Gh is expressed
in cells of the cardiovascular system which express TPs. Thus,
platelets express message for both isoforms, although they appear to
translate only TP
(43). Vascular smooth muscle cells express both
isoforms, whereas endothelial cells appear to express only TP
(8).
All of these cell types, as well as megakaryocytic cell lines, express
Gh.
Both TP isoforms physically associate with Gh, as reflected by studies
involving co-immunoprecipitation. Thus, immunoblotting of samples from
lysates of COS-7 cells transfected with rat Gh, together
with TP
or TP
, demonstrated that Gh
immunoprecipitates with either isoform. Similar results were obtained
in HEL and HASMC, cells in which the proteins were not overexpressed.
These results are consistent with our earlier finding that a high
molecular weight G protein copurifies with platelet TPs (presumably
TP
). Stimulation of the cells with TP agonists, which promote
receptor-G protein coupling, was not required for this association.
However, this is unsurprising. Precedent for co-immunoprecipitation of receptors and G proteins in non-stimulated cells has already been established (44, 45). Indeed, the affinity of TPs for Gh
seems to be high. For example, immunoprecipitation of Gh
with TPs occurs even when using a lysate of COS-7 cells overexpressing
Gh but not the receptors. COS-7 cells express low levels of
endogenous TPs, which can be detected by RT-PCR, but not by binding of
the TP antagonist [3H]SQ29,548. To address the
specificity of the co-immunoprecipitation of Gh with TPs,
we sought to replicate these findings using an antibody directed
against rhodopsin. Rhodopsin is expressed specifically in retina and is
absent from COS-7, HEL, or HASM cells. Gh did not
immunoprecipitate with either the anti-rhodopsin Ab or with the IgG
fraction of the normal rabbit serum used for the preclearing step.
Thus, immunoprecipitation of Gh with TP
and TP
appears to reflect a specific interaction.
To address the possibility that the TP-Gh association is of
functional relevance, we utilized transfected COS-7 cells (14, 40-42)
measuring inositol phosphate production as an index of G protein-dependent PLC activation.
Dose-dependent stimulation of inositol phosphate production
was observed in response to agonist in cells transfected with either
isoform alone, whereas cotransfection of the cells with
Gq
amplified the response to agonist. By contrast, cotransfection with Gs
had no effect, as expected.
Similar to our observation with Gq
, cotransfection of
Gh with TP
enhanced the agonist-dependent
increase in inositol phosphate formation over that found in cells
transfected with TP
alone. By contrast, a similar effect was not
observed in cells transfected with TP
.
The role of Gh as a conventional G protein has remained
controversial, in part reflecting the restriction of evidence to a single subfamily of G protein-coupled receptors, the
adrenoreceptors. We now provide evidence for association of this
protein with a distinct receptor, the TP, a member of the eicosanoid
receptor subfamily. Gh also functions as a tissue
transglutaminase, and it is now recognized that it binds and
cross-links only specific substrates that may be relevant to cell death
and survival (46). Interestingly, its transglutaminase function varies
inversely with its binding of GTP (26), raising the possibility that
the switch between these functions might be relevant to cellular
survival. We have shown that TP isoforms are expressed with
Gh in cardiovascular cells and associate with this protein.
However, their differential ability to signal via Gh
provides the first suggestion of distinct roles for these receptor
isoforms in vivo.