From the Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612
Received for publication, September 5, 2002, and in revised form, January 31, 2003
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ABSTRACT |
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G2A, a G protein-coupled receptor for
which lysophosphatidylcholine (LPC) is a high affinity ligand, belongs
to a newly defined lysophospholipid receptor subfamily. Expression of
G2A is transcriptionally up-regulated by stress-inducing and
cell-damaging agents, and ectopic expression of G2A leads to growth
inhibition. However, the G proteins that functionally couple to G2A
have not been elucidated in detail. We report here that G2A ligand
independently stimulates the accumulation of both inositol
phosphates and cAMP. LPC does not further enhance inositol
phosphate accumulation but dose-dependently augments
intracellular cAMP concentration. Expression of
G G2A, named for its ability to cause accumulation of cells in
G2/M of the cell cycle, is a G protein-coupled receptor
(GPCR)1 with tumor
suppressor-like properties (1). Ectopic expression of G2A in
fibroblasts antagonizes BCR-ABL-mediated transformation. Unlike
most GPCRs, the expression of G2A is up-regulated by various DNA-damaging and stress-inducing stimuli (1). It was recently reported
that targeted deletion of the mouse G2A gene results in the development of a late-onset autoimmunity resembling systemic lupus erythematosus (2). T lymphocytes isolated from these mice display
enhanced sensitivity and proliferative responses to T cell receptor
stimulation, suggesting that G2A may negatively regulate peripheral
lymphocyte numbers or increase the threshold required for T cell
receptor activation (2). Because immune cells that recognize
self-antigens are destined for self-destruction and routinely undergo
apoptosis, disruption of this process can lead to autoimmunity (3).
Therefore, a possible explanation for the phenotype of
G2A Two receptors that share significant homology to G2A, OGR1 and TDAG8,
have been recently associated with potentially inhibitory functions.
TDAG8, or T cell death-associated gene 8, is an inducible gene that is up-regulated during T cell activation-induced apoptosis (4). OGR1, or ovarian cancer G protein-coupled receptor 1, has been
shown to cause pertussis toxin (PTX)-insensitive growth inhibition when
stimulated by its high affinity ligand, sphingosylphosphorylcholine (5). Furthermore, the respective genes for G2A, TDAG8, and OGR1 are
clustered in chromosome 14q31-32.1, a region in which mutations are
associated with T cell leukemias and lymphomas (1, 6). These properties
distinguish these 3 members of this GPCR subfamily from other
lysophospholipid receptors such as the Edg (endothelial
differentiation gene) family of receptors, many
of which have been shown to cause cellular proliferation by activating multiple G proteins (7). On the contrary, GPR4, a fourth
member of the G2A subfamily, is located on chromosome 19. Stimulation of GPR4-overexpressing cells with its high affinity ligand
sphingosylphosphorylcholine enhances DNA synthesis (8), indicating that
this receptor may share more functional properties with the Edg
subfamily of receptors than with the other receptors in the G2A subfamily.
The signaling properties of several lysophospholipid receptors, such as
platelet-activating factor receptor and Edg receptors, have been
extensively characterized. In comparison, much less is known about the
signaling properties of the G2A family of GPCRs. Studies conducted thus
far show that G2A couples to G The primary objectives of this study are to ascertain which G proteins
functionally couple to G2A and to determine the biological consequences
that result from activation of these G proteins. Using transfected
cells and primary lymphocytes, we found that G2A couples to multiple G
proteins that include G Materials--
PTX and 2',5'-dideoxyadenosine were obtained from
Calbiochem (San Diego, CA). The pCMV Cloning and Subcloning of Human G2A cDNA--
Wild type
human G2A was cloned as described in Ref. 1 from total RNA extracted
from a phorbol 12-myristate 13-acetate/ionomycin-stimulated (24 h)
Ramos RA1 B lymphoma cell line, using the forward primer XGR5
(5'-GTGAATGTGCCCAA TGCTACTG-3') and reverse primer XGR4
(5'-GTGGGCTCAGCAGGACTCCTC-3'). This PCR product was then amplified
using primers containing EcoRI and BamHI sites,
respectively: XGR7R1 (5'-CGGAATTCCCGCCATGTGCCCAATGCTACTG-3') and
XGR8Bam (5'-GCGGGATCCTCAGCAGGACTCCTCAAT-3'). The final PCR product was
subcloned into the pRK5 vector (BD Biosciences), and its full
sequence was confirmed by comparison with the GenBankTM
entry for human G2A. The N-terminal AU5 (TDFYLK)-tagged G2A construct (AU5-G2A) was created by PCR and subcloned into the pRK5 vector. Functionality of AU5-G2A was comparable with that of wild type G2A in
apoptosis assays, second messenger accumulation, and NF- Cell Culture, Transfection, and Luciferase Assay--
HeLa cells
were maintained in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 50 µg/ml streptomycin. Cells were transfected at 40-80% confluence in
6-well plates using LipofectAMINE Plus reagent (Invitrogen) as
previously described (12). Cells were transfected with pRK5, G2A,
and/or other constructs of interest and with 0.2 µg of the 3×
NF- RT-PCR and Detection of Cell Surface Expression--
To confirm
expression of G2A in transiently transfected HeLa cells or primary
lymphocytes by RT-PCR, first-strand cDNA was synthesized with 2 µg of total RNA isolated using the Trizol reagent and the Superscript
II preamplification system (Invitrogen). Fifteen percent of the first
strand cDNA synthesis product was then used for PCR with the
primers XGR3 (5'-CTCGTCGGGATCGTTCACTAC-3') and HG2AC1 as described
previously (1). GPR4 expression in primary cells was detected using
the primers FmidGPR4 (5'-CCGGGGCATCCTGCGGGCCG-3') and RevGPR4
(5'-GCTGGCGGCAGC ATCTTCAGC-3'). G2A receptor expression on the cell
surface was determined by using a 1:500 dilution of monoclonal anti-AU5
primary antibody (Covance, Denver, PA) and 1:200 fluorescein
isothiocyanate-conjugated goat anti-mouse secondary antibody. Stained
cells were analyzed on a Coulter Elite ESP flow cytometer with care to
exclude cellular debris and aggregates. Markers and statistics were
determined using WinMDI 2.8 software (facs.scripps.edu/software.html).
Assay for Apoptosis--
Tetramethylrhodamine ethyl ester (TMRE)
was used to measure changes in mitochondrial membrane potential.
Twenty-four hours post-transfection, cells were incubated at 37 °C
with 100 nM TMRE for 10 min and/or 1 µg/ml of Hoechst
33342 (Molecular Probes, Eugene, OR) for 2 min. Cells were then washed
and immediately visualized by Nikon Eclipse TE300 fluorescence
microscope and pictures captured using a Hamamatsu CCD camera and
SimplePCI software (C-Imaging Systems, Cranberry Township, PA).
Alternatively, after TMRE staining, both floating and attached cells
were collected by trypsinization, centrifuged at 1,200 rpm for 3 min,
washed twice with 1× phosphate-buffered saline, and then resuspended in 1× phosphate-buffered saline for analysis by flow cytometry. Samples were analyzed using the Coulter Elite ESP and percentages calculated using WinMDI 2.8 software. All samples were gated to exclude
cellular debris and aggregates and count a standard of 20,000 events.
Measurements for Second Messengers--
Inositol phosphate
accumulation was determined in transfected cells. Twenty-four hours
after transfection, the culture medium in each well was replaced with
inositol-free DMEM (ICN Biomedicals, Costa Mesa, CA) supplemented with
0.1% dialyzed fetal bovine serum and 3 µCi/ml myo-[3H]
inositol (PerkinElmer Life Sciences). Cells were then washed twice and
incubated in DMEM supplemented with 20 mM HEPES, 50 mM LiCl, pH 7.4, for 45 min, ± 1 µM LPC.
Accumulated InsP was measured using ion exchange chromatography and
scintillation counting. Cyclic-AMP was measured using a competitive
enzyme immunoassay (Biomol, Plymouth Meeting, PA). Twenty-four hours
after transfection, the medium was replaced with DMEM supplemented with
0.1% fetal bovine serum and cAMP production accumulated in the
presence of DMEM supplemented with 0.5 mM
isobutylmethylxanthine (IBMX), ± LPC for 45 min at 37 °C.
Alternatively, cells were pre-treated with PTX (500 ng/ml) or 100 µM 2',5'-dideoxyadenosine (DDA) for 4 h before
ligand stimulation. The medium was then removed and replaced with 0.1 M HCl. After the specified incubation times, p-nitrophenyl phosphate substrate was added, the reaction
was stopped, and OD was determined using a microplate reader at 405 nm. Standard cAMP curves and calculations were performed
according to the manufacturer's instructions. All cells were
co-transfected with pCMV Isolation of Primary Lymphocytes--
Anti-coagulated whole
blood from human donors was diluted 1:1 with Hanks Balanced Salt
Solution, and lymphocytes were separated using Ficoll-Paque PLUS
(Amersham Biosciences) gradient centrifugation according to the
manufacturer's instructions. The lymphocyte fraction was enriched for
CD4+ T cells using a CD4+ T cell isolation kit (Miltenyi Biotec,
Auburn, CA). The kit consists of a mixture of hapten-conjugated
specific cell surface marker antibodies (anti-CD8, CD11b, CD19, CD16,
CD36, and CD56) and an anti-hapten secondary antibody conjugated to
magnetic microbeads. Depletion of non-helper T cells is achieved by
incubation with the primary antibody mixture/secondary antibody and
subjecting the labeled cells to a magnetic column on an autoMACS cell
sorter (Miltenyi Biotec). The resulting population of cells were eluted
from the column, stained with an anti-CD4-fluorescein isothiocyanate
antibody (Dako, Carpinteria, CA) and subject to flow cytometric
analysis (85-90% CD4+ T cells, 10-15% red blood cells, as
determined by fluorescein isothiocyanate and forward scatter). Red
blood cells were lysed if necessary by quick (<10 s) resuspension of
cell pellet in a hypotonic solution followed by rapid addition of 2×
phosphate-buffered saline. CD4+ T cells were then stimulated with
vehicle or LPC for 90 min then lysed for cAMP analysis or incubated for
14-16 h, stained with TMRE, and analyzed by flow cytometry. In the
latter case, lymphocytes were kept in RPMI medium containing 5% of
donor serum, which was collected from the upper phase after gradient
centrifugation of diluted whole blood.
Ligand-independent Coupling of G2A to G
It was recently reported that certain G
We also employed a loss-of-function approach that utilizes RGS
constructs to block specific G protein pathways (17, 18). P115RhoGEF,
an effector that couples G Ligand-independent and LPC-induced Elevation of
cAMP--
GPCR-mediated activation of adenylate cyclase and the
subsequent production of cAMP involve the activation of
G
To examine the dose effect of LPC on G2A-mediated cAMP elevation, we
treated both mock-transfected and G2A-transfected cells with increasing
concentrations of LPC (Fig. 3B). Indeed, there exists a
correlation between LPC dose and G2A-mediated cAMP elevation, with a
maximum response at 0.5 µM LPC. At Expression of G2A Causes Apoptosis--
Evidence exists that
expression of G2A is induced by various DNA-damaging agents such as
chemotherapeutics, UV, and x-ray. Furthermore, it is known that
expression of G2A causes NIH 3T3 cells to accumulate at G2/M in the
cell cycle and that it inhibits transformation of cells by the oncogene
BCR-ABL (1). These findings suggest a potential link between G2A
expression and suppression of cell growth, and prompted us to examine
whether G2A also induces apoptosis. HeLa cells were transfected to
express G2A as described above. 24-48 h after transfection,
G2A-transfected cells, but not mock (vector)-transfected cells, began
to display significant cytoplasmic shrinkage, cell rounding, and
membrane blebbing (Fig. 4A).
Expression of G2A also caused a decrease in mitochondrial membrane
potential as determined by negative staining with TMRE. TMRE is a
cationic, lipophilic dye that is dissipated when there is a large
reduction in mitochondrial membrane potential, an event that occurs in
most cases of both receptor-mediated and stress-induced apoptosis (21).
As a result, apoptotic cells show much less staining with this
rhodamine-based dye, whereas live cells stain positively with TMRE
(Fig. 4B and also Fig.
5A). Hoechst 33342 was used to
stain condensed nuclear chromatin in apoptotic cells. As shown in Fig.
4B, expression of G2A produced approximately twice as many
apoptotic cells, which stained positively with Hoechst and negatively
with TMRE (white arrows). Regardless of the presence of
serum, there was a 2-3-fold increase in apoptosis in G2A-transfected cells compared with mock-transfected cells (data not shown).
Furthermore, several other cell lines, including NIH 3T3, COS-7, Saos2,
and U20S, were also susceptible to G2A-mediated apoptosis (data not shown), suggesting that this effect is not restricted to one cell type.
To confirm that the apoptotic cells were those that expressed G2A, we
used a G2A-GFP fusion construct (1) for transfecting HeLa cells. We
found that the cells that underwent apoptosis were indeed GFP-positive
(data not shown). In control experiments, expression of the empty
vector, pEGFP, did not increase the number of apoptotic cells.
To examine whether G2A-mediated apoptosis requires caspase activation,
cells were either co-transfected with the viral serpin caspase-8
inhibitor, CrmA, or treated with 100 µM of the
pan-caspase inhibitor, z-vad-fmk. Quantitation of apoptosis was
determined by flow cytometric analysis of TMRE-stained cells. In Fig.
5A, density plots showing TMRE fluorescence intensity are
plotted against side scatter (90LS). The R3 region designates the
percentage of events that no longer stain with TMRE and are therefore
indicative of apoptotic cells. In the first row, G2A-transfected cells
(right-hand column) display over a 2-fold increase in the
percentage of apoptotic cells compared with the mock-transfected cells
(left-hand column). The two caspase inhibitors, compared
with Bcl-2, had a greater effect in rescuing cells from G2A-mediated
apoptosis, as implicated by the fold change in apoptosis (see Fig.
5B). The anti-apoptotic protein Bcl-2 decreases the basal
level of apoptosis, but G2A co-transfection with Bcl-2 still results in
more than a 2-fold increase in apoptosis over the control (Bcl-2
alone). On the other hand, G2A co-transfection with CrmA (or zvad
treatment) results in only a 1.2-1.3-fold increase in apoptosis over
control (CrmA or zvad alone), suggesting that caspase inhibitors have a
greater effect in protecting cells from G2A-mediated apoptosis than
Bcl-2.
G2A-mediated Apoptosis Involves Multiple G Proteins--
Given
that other GPCRs that couple to G
Given the ability of LPC to induce apoptosis in G2A-expressing HeLa
cells, we next examined the effect of LPC stimulation in cells that
normally express endogenous G2A receptor, i.e. lymphocytes. By RT-PCR, we confirmed expression of G2A in primary T lymphocytes isolated from the blood of healthy human donors (Fig. 6B).
Interestingly, we found that the transcript for GPR4, the low affinity
receptor for LPC, is barely detectable in primary T lymphocytes. As
depicted in Fig. 6, T lymphocytes that were stimulated with nanomolar
concentrations of LPC (0.01 or 0.5 µM) showed a dramatic
increase in apoptosis as shown by an increase in the number of cells
that had reduced TMRE staining (from 27 to 87%). Furthermore, this
increase in apoptosis correlated with a dose-dependent
increase in cAMP elevation (Fig. 6C).
A number of G proteins, including G
To determine whether G2A utilizes G Our results reveal primarily two novel findings. The first
involves the differential activation of G proteins by G2A depending on
whether or not it is stimulated by LPC. According to our second messenger accumulation measurements and NF- The second new finding is that expression of G2A causes
caspase-dependent apoptosis via the activation of a
specific combination of G proteins and that LPC stimulation of G2A
enhances apoptosis. The fact that G2A expression results in apoptosis
is consistent with the implications derived from G2A-deficient mice,
which develop autoimmune disease, as well as the growth inhibitory
properties displayed by G2A-expressing Rat-1 fibroblasts and mouse bone
marrow cells (1, 2). Recent evidence also shows that G2A-deficient mice
have a faster disease progression in BCR-ABL-driven leukemia than wild
type mice, thus supporting the notion that G2A plays a negative role in
regulating peripheral lymphocyte numbers by causing apoptosis (28).
Furthermore, we demonstrate here that the viral serpin caspase-8
inhibitor, CrmA (29), is just as effective in rescuing cells from
G2A-mediated apoptosis as the pan-caspase inhibitor z-vad-fmk, whereas
Bcl-2 did not have as great an effect (Fig. 5). Because caspase-8 is
thought to be a target of receptor-mediated apoptotic pathways such as
those involving tumor necrosis factor- We have attempted to identify the proximal signaling mechanisms for
G2A-mediated apoptosis. Our results confirm studies conducted by two
separate groups (6, 9) describing the ability of G2A to couple
functionally and ligand-independently to G An important issue remains, concerning the ability of T lymphocytes to
express G2A and survive, whereas expression of G2A in HeLa cells causes
cell death even without ligand. This may be because T cells normally
express low amounts of G2A and have achieved an equilibrium that allows
them to survive until increased circulating concentrations of LPC are
made available to the receptor, thereby causing the cells to undergo
apoptosis. On the other hand, when up-regulation of the receptor occurs
(e.g. when prompted by stress-inducing stimuli, or when
overexpressed in HeLa cells), then cells may undergo apoptosis with or
without the presence of LPC.
G2A has been implicated in the control of cellular proliferation and
negative regulation of self-recognizing lymphocytes. Its ligand, LPC,
has been implied in a variety of cellular processes such as chemotaxis
of monocytes and T cells and growth inhibition of smooth muscle cells
and endothelial cells (32, 33), although these phenomena have yet to be
correlated with a specific cell surface receptor. As previous studies
(7) have described, other lysophospholipids (e.g.
lysophosphatidic acid or sphingosine 1-phosphate) in mediating cell
proliferation and angiogenesis through their respective GPCRs, our
finding that LPC promotes apoptosis through G2A provides the first
account of a lysophospholipid (LPC) causing apoptosis through a
distinct GPCR-mediated mechanism. We are currently investigating
whether other receptors of this subfamily also possess growth
inhibitory functions.
q and G
13 with G2A potentiates
G2A-mediated activation of a NF-
B-luciferase reporter. These results
demonstrate that G2A differentially couples to multiple G proteins
including G
s, G
q, and G
13,
depending on whether it is bound to ligand. G2A-transfected HeLa cells
display apoptotic signs including membrane blebbing, nuclear
condensation, and reduction of mitochondrial membrane potential.
Furthermore, G2A-induced apoptosis can be rescued by the caspase
inhibitors, z-vad-fmk and CrmA. Although apoptosis occurs
without LPC stimulation, LPC further enhances G2A-mediated apoptosis
and correlates with its ability to induce cAMP elevation in both HeLa
cells and primary lymphocytes. Rescue from G2A-induced apoptosis
was achieved by co-expression of a G
12/13-specific
inhibitor, p115RGS (regulator of G protein
signaling), in combination with
2',5'-dideoxyadenosine treatment. These results
demonstrate the ability of G2A to activate a specific combination of G
proteins, and that G2A/LPC-induced apoptosis involves both
G
13- and G
s-mediated pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice is that G2A plays a role in programmed death
of these self-recognizing T cells.
13 and activates RhoA,
leading to actin stress fiber formation and serum-response factor-mediated transcription. These observations were made while G2A
was still considered an orphan receptor (6, 9). LPC was subsequently
discovered to be the high affinity ligand for G2A and could stimulate
PTX-sensitive intracellular Ca2+ transients and
extracellular signal-related kinase (ERK) phosphorylation in
transfected cells, suggesting the involvement of G
i
(10). However, other GPCRs that couple to G
13 and
G
i (e.g. protease-activated receptor-1
and certain Edg receptors) do not display functional properties similar
to that of G2A (7, 11). Moreover, although G2A has been previously
characterized as having growth inhibitory effects, at least one
contradictory study exists that depicts G2A as capable of inducing
morphological changes resembling oncogenic transformation (9).
Therefore, an essential question that remains unanswered is whether G2A
has a direct effect on cell fate.
s, G
q, and G
13. Exogenous expression of G2A results in constitutive
activation of these G proteins, whereas LPC can further stimulate
G
s activation. Activation of this specific group of G
proteins by G2A contributes to caspase-mediated apoptosis.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(
-galactosidase) vector and
the caspase inhibitor z-vad-fmk were purchased from Promega (Madison,
WI). The 3× NF-
B luciferase reporter plasmid was constructed as
described previously (12).
-galactosidase reaction buffer and
substrate were obtained from Clontech (Palo Alto,
CA). LPC, 16:0, was purchased from Avanti Polar Lipids (Alabaster, AL).
Myc-tagged GRK2 RGS and p115RGS were generous gifts from Dr.
Tohru Kozasa (University of Illinois at Chicago). Expression vectors
for Bcl-2 and CrmA were kindly provided by Dr. Elena Efimova
(University of Illinois at Chicago). Expression vectors containing wild
type and constitutively active (QL) G
subunits were gifts from Drs.
Cindy Knall and Gary Johnson (University of Colorado, Denver, CO). The
expression vector containing the
scavenger, myc-tagged
-adrenergic receptor kinase (
ARK) carboxyl terminus fragment
linked to transmembrane domain of CD8, was kindly provided by Dr.
Silvio Gutkind (National Institutes of Health). Anti-G
q
and anti-G
13 polyclonal antibodies were acquired from
Santa Cruz Biotechnologies (Santa Cruz, CA).
B activation. The G2A-green fluorescent protein (GFP) fusion construct was prepared as described in Weng et al. (1).
B-luciferase reporter construct and 0.02 µg of pCMV
(
-galactosidase expression vector) for normalization of transfection
efficiency. Total DNA was made equivalent between samples by adding
empty vector (pRK5). Twenty-four hours post-transfection, cells were
starved for 16-18 h in serum-free DMEM and treated with ligand for
4 h if necessary. Cells were then washed twice with 1×
phosphate-buffered saline, lysed with 1× Reporter Lysis Buffer
(Promega), and supernatant was collected. Luciferase substrate
(Promega) and
-galactosidase substrate were added to two different
aliquots of supernatant and luminescence measured with a Femtomaster
FB12 luminometer (Berthold Detection Systems, Pforzheim, Germany).
Luciferase activities were normalized against
-galactosidase.
Normalized data for all samples were plotted using Prism software
(Version 3.0, GraphPad, San Diego, CA).
, and all data were normalized against
-galactosidase to account for variances in transfection efficiency.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q--
G2A
is a transcriptionally regulated, stress-inducible GPCR that has been
shown to ligand-independently couple to G
13 in the
activation of RhoA (6, 9). We examined whether G2A also activates other
G
proteins because G
13-coupling GPCRs are often found
to interact with more than one G protein. Upon GPCR activation, certain
G
subunits (e.g. G
q) and
subunits
activate phospholipase-C
(PLC
), which cleaves phosphoinositol
diphosphate into two products, diacylglycerol and inositol
triphosphate. Thus, the accumulation of InsP is a useful and effective
indication of GPCR-mediated PLC
activation. G2A was exogenously
expressed in HeLa cells, a human carcinoma cell line devoid of
endogenous G2A. Expression of G2A was confirmed by RT-PCR, and cell
surface expression of the receptor was observed by flow cytometric
analysis using a monoclonal antibody against an AU5 tag that was fused
to the N terminus of G2A. Approximately 30% of transfected cells
showed a high level of cell surface receptor expression 24 h after
transfection (data not shown). Expression of G2A resulted in a 3.5-fold
increase of InsP accumulation, as compared with mock-transfected cells (Fig. 1A). Stimulation of
transfected cells with the G2A agonist LPC did not further increase
InsP accumulation. PTX, which ADP-ribosylates G
i/G
o and prevents functional coupling to
their respective GPCRs, did not inhibit G2A-mediated increase in InsP
accumulation. To determine the role of G
in this response, HeLa
cells were transfected with an expression construct containing the
carboxyl terminus fragment of
ARK (
ARKct), a known
scavenger (13). It was found that
ARKct did not inhibit G2A-mediated
InsP accumulation (Fig. 1B). These results suggest that G2A
activates a G
protein, most likely G
q, regardless of
LPC stimulation. As a positive control, expression of a constitutively
active G
q (Q209L) led to a ~3-fold induction of InsP
accumulation (data not shown).
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Fig. 1.
G2A causes ligand-independent,
PTX-insensitive InsP accumulation. A, transfected
cells were labeled with [3H]myoinositol in inositol-free,
serum-free DMEM in the presence of 50 mM LiCl. InsP levels
were then measured in pRK5 versus G2A-transfected cells ± PTX 500 ng/ml for 4 h and/or 1 µM 16:0 LPC for 45 min. B, pRK5 or G2A-transfected cells were
co-transfected with 200 ng of the subunit scavenger,
ARKct.
Equivalent protein expression of the myc-tagged
ARKct was shown by
immunoblotting with 1:1000 anti-myc monoclonal antibody. All samples
were co-transfected with
-galactosidase (20 ng) to account for
differing transfection efficiencies, and the data represent the fold
increase of normalized cpm readings. Data were collected in duplicate
and expressed as mean ± S.D.
proteins, including
G
q and G
13, are involved in GPCR-mediated
activation of NF-
B (12, 14-16). We used this property of G proteins
to further confirm functional coupling of G2A to these two G proteins.
HeLa cells were co-transfected with expression constructs for a NF-
B
luciferase reporter and G2A, and in some samples, one of the G
proteins. As shown in Fig. 2A,
expression of G2A resulted in a potent induction of NF-
B-mediated
transcription characterized by an increase in luciferase activity.
Co-expression of G
q was found to augment G2A-induced
luciferase activity by ~90%, whereas co-expression of
G
13, known to couple to G2A (6, 9), also potentiated luciferase reporter activity. Neither of these G
proteins was able
to induce the expression of NF-
B-driven luciferase activity when
expressed alone, indicating that the observed enhancement results from
specific functional coupling with G2A. Consistent with the
ligand-independent nature of these coupling events, LPC stimulation did
not further increase G2A-induced NF-
B-luciferase activity (data not
shown).
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[in a new window]
Fig. 2.
Functional coupling of G2A to
G q and
G
13 in
NF-
B luciferase reporter expression.
A, cDNA (200 ng) encoding either wild type
G
q or G
13 were co-transfected with pRK5
or G2A along with the NF-
B-luciferase reporter and pCMV
.
Twenty-four hours post-transfection cells were lysed and luciferase or
-galactosidase substrate were added. Luminescence readings
for luciferase were normalized against those for
-galactosidase.
Equivalent protein expression of wild type G
q or
G
13 was determined in pRK5 versus
G2A-transfected samples by immunoblotting with anti-G
q
and anti-G
13 polyclonal antibody (1:1000).
B, a schematic of the truncated RGS constructs.
C, co-expression of p115RGS or GRK2RGS with G2A
(or pRK5), NF-
B-luciferase, and
-galactosidase causes inhibition
of G2A-mediated NF-
B activity. Equivalent protein expression of
myc-tagged p115RGS and GRK2RGS is shown by immunoblotting with an
anti-myc monoclonal antibody. Data represent the mean ± S.D. of
duplicate determinations in one of three separate experiments.
13 to the activation of RhoA,
contains an N-terminal RGS domain that serves to down-regulate the
G
13 signal (19). GRK2 contains an N-terminal RGS domain that binds to and specifically inhibits G
q signaling
(20). The two RGS truncation constructs shown in Fig. 2B
were used to down-regulate signaling by G
13 and
G
q, respectively. Our results show that either GRK2RGS
or p115RGS alone inhibited G2A-induced NF-
B activation by 50 and
36%, respectively (Fig. 2C). When both RGS constructs were
used together, a more complete inhibition of G2A-mediated NF-
B
activation was observed. These data provide additional support for the
functional coupling of G2A to G
q and G
13.
s in most cases. On the other hand, if there is an
inhibition of stimulated adenylate cyclase and/or sensitivity to PTX,
activation of the G
i/o family is inferred. To test these
possibilities in G2A-transfected cells, we measured cAMP production
with and without LPC stimulation, in the presence or absence of PTX
pre-treatment. Without LPC stimulation, expression of G2A induces a
~3-fold elevation in intracellular cAMP (Fig.
3A). This cAMP elevation is
partially inhibited by the cell-permeable adenylate cyclase inhibitor,
DDA. LPC stimulation further increases cAMP elevation by an additional
60%, from 3-fold to ~5-fold. This effect was also inhibited by DDA.
PTX pre-treatment enhances G2A-mediated cAMP elevation by ~30%.
Based on these observations, it appears that G2A couples to
G
s, and possibly G
i, in the absence of
LPC stimulation, in that the ligand-independent increase in cAMP is
slightly enhanced by abrogating the inhibitory effect of
G
i with PTX pre-treatment. The inhibitory activity by
G
i appears to be relatively weak, as LPC stimulation
further increased cAMP levels, indicating that G2A-mediated activation
of G
s offsets the inhibition by
G
i.
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Fig. 3.
G2A-mediated cAMP elevation.
A, accumulated cAMP levels were measured in control
versus G2A-transfected cells, ± PTX (500 ng/ml)
pre-incubation for 4 h, ± pre-incubation with 100 µM DDA for 4 h, or ± 1 µM LPC
for 1 h. Cells were stimulated with LPC in DMEM supplemented with
0.5 mM IBMX. Cyclic-AMP levels were measured using
competitive enzyme immunoassay. B, increasing doses of
LPC (0.05-25 µM) were used to stimulate control and
G2A-transfected cells for 1 h, 37 °C in the presence of 0.5 mM IBMX. All samples were transfected with and normalized
against -galactosidase and represent the mean ± S.D. of
duplicate determinations in one of three separate
experiments.
10 µM LPC, there was no further increase in cAMP. This may
be due to nonspecific effects or decreasing activity of LPC at
concentrations
10 µM (5).
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Fig. 4.
Exogenous G2A expression causes
apoptosis. A, HeLa cells were transfected with 200 ng of empty vector, pRK5, or G2A DNA per well in a 6-well plate and
visualized with light microscopy. B, 24 h after
transfection, cells were stained with 100 nM TMRE for 8 min
and 1 µg/ml Hoechst 33342 for an additional 2 min and
visualized with fluorescence microscopy. White
arrows, Hoechst (blue) positive/TMRE negative cells.
The pictures shown are representative of 5-6 similar images taken in 3 repeated experiments.
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Fig. 5.
Effects of caspase inhibitors and
Bcl-2 on G2A-mediated apoptosis. A, HeLa cells
were transfected with 200 ng of either pRK5 (vector) or an
expression construct of G2A (G2A). In some samples, 200 ng
of expression vector for either CrmA or Bcl-2 were co-transfected;
alternatively, cells were incubated with 100 µM of the
pan-caspase inhibitor, z-vad-fmk. Twenty-four hours post-transfection
cells were harvested, stained with 100 nM TMRE for 10 min,
and analyzed by flow cytometry. R3 region depicts cells that show
reduced staining with TMRE. B, relative increases in % of apoptotic cells as depicted by the R3 region in A. A
representative set of data was shown in this figure. The same results
were obtained in two other experiments.
13 and
G
q (e.g. PAR-1 and the GPCR encoded by open
reading frame 74 of Kaposi's sarcoma-associated herpesvirus) (11, 16,
22) do not cause apoptosis, we speculated that G2A may activate a
distinct combination of G proteins that leads to apoptosis. To
ascertain which G
proteins G2A utilizes to induce apoptosis, we
first sought to investigate the role of LPC-stimulated cAMP elevation
on G2A-mediated apoptosis. LPC stimulation enhanced G2A-mediated
apoptosis in a dose-dependent manner that correlated with
its effect on cAMP elevation (Fig.
6A), i.e. the concentration of LPC that induced maximal apoptosis (0.5 µM) coincided with the dose that stimulated peak
elevation of cAMP (Fig. 3B). This result indicates that
G
s activation, either alone or in combination with other
signals, may be critical in G2A-mediated apoptosis.
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Fig. 6.
Dose effect of LPC on G2A-mediated
apoptosis. A, G2A or pRK5-transfected HeLa cells
were treated with increasing doses of LPC (0.05-25 µM)
for 12 h, harvested, stained with 100 nM TMRE for 10 min, and analyzed by flow cytometry. B, RT-PCR showing
the expression of G2A and GPR4 transcripts in primary T lymphocytes
(upper left). Primary lymphocytes were stimulated overnight
with vehicle, LPC at 0.01 or 0.5 µM and then stained for
TMRE. M1 marker designates the percentage of cells that show
reduced TMRE staining (apoptotic cells). Actual percentages are shown
in parentheses. C, primary lymphocytes were
stimulated with LPC for 90 min in the presence of 0.5 mM
IBMX before lysis for cAMP measurement (open bars).
Alternatively, lymphocytes were pre-treated with 500 ng/ml PTX for 30 min before LPC stimulation (solid bars).
13,
G
q, and G
s, have been implicated in
GPCR-mediated apoptosis (23-26). We therefore examined the ability of
constitutively activated G
subunits (with Q
L mutation) to induce
apoptosis in our system. The results indicate that expression of
G
sQL alone did not induce apoptosis in HeLa cells,
whereas expression of either G
13QL or
G
qQL induced apoptosis to a small extent (Fig.
7A). Interestingly, when both
G
13QL and G
sQL were transfected together,
a synergism was achieved, which mimics the level of apoptosis of that
seen in G2A-transfected cells. In contrast, when G
qQL
was expressed in combination with G
sQL or
G
13QL, there was no additive or synergistic effect. All
G
(QL) constructs have been tested for constitutive activity by
either second messenger accumulation assays (InsP for
G
qQL and cAMP for G
sQL) or by the
NF-
B- and serum response element-luciferase reporter assays
(for G
13QL) (data not shown).
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Fig. 7.
G s and
G
13 are critical to
G2A-dependent apoptosis. A, HeLa cells
were transfected with 200 ng of the expression constructs for active
mutants (QL) of G
q, G
s, or
G
13, either alone or in combination. B,
either GRK2RGS or p115RGS were co-transfected with pRK5 or G2A.
Alternatively, transfected cells were treated with 100 µM
of DDA. Twenty-four hours after transfection/treatment, cells were
harvested, incubated with 100 nM TMRE for 10 min, and
analyzed by flow cytometry. Relative decrease in the percentage of
apoptotic cells (TMRE negative) as compared with G2A alone are shown as
mean ± S.D. from duplicate samples.
s- and/or
G
13-mediated pathways to mediate apoptosis, we used
various inhibitors and RGS constructs to attempt pathway-specific
rescue from G2A-induced apoptosis. Both the p115RGS construct and the
adenylate cyclase inhibitor DDA partially rescued cells from
G2A-mediated apoptosis (Fig. 7B). In comparison, GRK2RGS was
much less effective. When DDA and p115RGS were used simultaneously,
G2A-induced apoptosis was inhibited to a greater extent. The various
constructs and inhibitors did not have a great effect on the basal
level of vector-transfected cells that show reduced TMRE staining (data
not shown). These results indicate that G
s and
G
13 play critical roles in G2A-mediated apoptosis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B reporter data, G2A may
ligand-independently couple to a unique combination of G proteins: G
q, G
s, and G
13
(G
i involvement appears to be minimal at most). LPC
stimulation of G2A-transfected cells enhances activation of G
s, resulting in a greater elevation of cAMP (Fig. 3,
A and B) while failing to alter inositol
phosphate levels (Fig. 1A). On the other hand, previous
reports (10) describe PTX-sensitive LPC signaling through G2A causing
ERK phosphorylation and an increase in intracellular
[Ca2+], implying the involvement of G
i.
Although this discrepancy may be due to differences in the cell lines
utilized, it is also noted that LPC stimulation of G
s
was not specifically investigated in that study. Our finding that LPC
induces G2A-mediated G
s activation is consistent with a
previous report demonstrating the ability of LPC to stimulate G
protein-dependent elevation of cAMP in platelets (27),
indicating the importance of investigating the LPC effects in cells
that endogenously express G2A.
/FasL, the ability of
CrmA but not Bcl-2 to inhibit G2A-mediated apoptosis suggests that G2A
may share more similarities to known receptor-mediated pathways than it does to stress-induced pathways. Indeed, there exists an autoimmune lymphoproliferative syndrome similar to systemic lupus
erythematosus, which causes defective lymphocyte apoptosis due
to mutations of the Fas receptor (30). Whether G2A recruits any of the
direct upstream components of the death receptor pathways such as
Fas-associated death domain, Tumor necrosis factor receptor
1-associated death domain, or the Ser/Thr kinase RIP, or whether
G2A enhances tumor necrosis factor/Fas-mediated apoptosis may be an
interesting direction to pursue in future studies.
13. As of
late, there has been a revision in the conventional model of ligand-receptor interactions to incorporate the finding that several native GPCRs (not virally encoded), such as certain serotonin receptors
(31), exhibit a significant level of basal activity in the absence of
ligand. Virally encoded GPCRs (e.g. US28 and KSHV-GPCR) have
also been shown to activate multiple G proteins ligand-independently,
the result being cellular transformation rather than apoptosis (14,
22). In fact, recent evidence indicates that similar to G2A, KSHV-GPCR
ligand-independently activates G
13 in the same cell line
tested in this report (16). The dramatic difference in phenotype
(transformation versus apoptosis) may be due to the
combination of G proteins activated specifically by G2A. Activation of
G
13, in combination with G
s-mediated
pathways may be required, in our cell system, to mediate apoptosis
instead of transformation. In fact, in an additional experiment, we
have seen that co-expression of KSHV-GPCR with constitutively active G
sQL successfully mimics the level of apoptosis seen
with G2A, whereas expression of either alone has no effect on
apoptosis.2 Therefore, it
appears that a complex web of events in which certain G protein
pathways work in the context of other activated pathways must occur to
achieve the ultimate result, in this case, apoptosis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Tohru Kozasa for generously providing p115RGS and GRK2RGS, Dr. Bellur S. Prabhakar and Dr. Prasad Kanteti for scientific discussions, Hairong Sang for cDNA cloning, Dr. Karen Hagen and Virginia Mezo for flow cytometry analysis, and Sharon Chou for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant AI40176.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.
Recipient of a predoctoral fellowship from the American Heart
Association, Midwest Affiliate.
§ To whom correspondence should be addressed. Tel.: 312-996-5087; Fax: 312-996-7857; E-mail: yer@uic.edu.
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M209101200
2 P. Lin and R. D. Ye, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G
protein-coupled receptor;
LPC, lysophosphatidylcholine;
InsP, inositol
phosphate;
PTX, pertussis toxin;
ERK, extracellular signal-related
kinase;
DDA, dideoxyadenosine;
RGS, regulator of G protein signaling;
ARK,
-adrenergic receptor kinase;
z, benzyloxycarbonyl;
fmk, fluoromethyl ketone;
TMRE, tetramethylrhodamine ethyl ester;
IBMX, isobutylmethylxanthine;
NF-
B, nuclear factor
B;
GFP, green
fluorescent protein;
KSHV, Kaposi's Sarcoma-associated Herpes virus;
Edg, endothelial differentiation gene;
DMEM, Dulbecco's modified
Eagle's medium;
RT, reverse transcription;
ct, carboxyl terminus;
GRK, G protein-coupled receptor kinase.
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