The Lysophospholipid Receptor G2A Activates a Specific Combination of G Proteins and Promotes Apoptosis*

Phoebe LinDagger and Richard D. Ye§

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Galpha q and Galpha 13 with G2A potentiates G2A-mediated activation of a NF-kappa B-luciferase reporter. These results demonstrate that G2A differentially couples to multiple G proteins including Galpha s, Galpha q, and Galpha 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 Galpha 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 Galpha 13- and Galpha s-mediated pathways.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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-/- mice is that G2A plays a role in programmed death of these self-recognizing T cells.

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 Galpha 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 Galpha i (10). However, other GPCRs that couple to Galpha 13 and Galpha 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.

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 Galpha s, Galpha q, and Galpha 13. Exogenous expression of G2A results in constitutive activation of these G proteins, whereas LPC can further stimulate Galpha s activation. Activation of this specific group of G proteins by G2A contributes to caspase-mediated apoptosis.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- PTX and 2',5'-dideoxyadenosine were obtained from Calbiochem (San Diego, CA). The pCMVbeta (beta -galactosidase) vector and the caspase inhibitor z-vad-fmk were purchased from Promega (Madison, WI). The 3× NF-kappa B luciferase reporter plasmid was constructed as described previously (12). beta -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) Galpha subunits were gifts from Drs. Cindy Knall and Gary Johnson (University of Colorado, Denver, CO). The expression vector containing the beta gamma scavenger, myc-tagged beta -adrenergic receptor kinase (beta ARK) carboxyl terminus fragment linked to transmembrane domain of CD8, was kindly provided by Dr. Silvio Gutkind (National Institutes of Health). Anti-Galpha q and anti-Galpha 13 polyclonal antibodies were acquired from Santa Cruz Biotechnologies (Santa Cruz, CA).

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-kappa B activation. The G2A-green fluorescent protein (GFP) fusion construct was prepared as described in Weng et al. (1).

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-kappa B-luciferase reporter construct and 0.02 µg of pCMVbeta (beta -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 beta -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 beta -galactosidase. Normalized data for all samples were plotted using Prism software (Version 3.0, GraphPad, San Diego, CA).

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 pCMVbeta , and all data were normalized against beta -galactosidase to account for variances in transfection efficiency.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Ligand-independent Coupling of G2A to Galpha q-- G2A is a transcriptionally regulated, stress-inducible GPCR that has been shown to ligand-independently couple to Galpha 13 in the activation of RhoA (6, 9). We examined whether G2A also activates other Galpha proteins because Galpha 13-coupling GPCRs are often found to interact with more than one G protein. Upon GPCR activation, certain Galpha subunits (e.g. Galpha q) and beta gamma subunits activate phospholipase-Cbeta (PLCbeta ), 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 PLCbeta 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 Galpha i/Galpha o and prevents functional coupling to their respective GPCRs, did not inhibit G2A-mediated increase in InsP accumulation. To determine the role of Gbeta gamma in this response, HeLa cells were transfected with an expression construct containing the carboxyl terminus fragment of beta ARK (beta ARKct), a known beta gamma scavenger (13). It was found that beta ARKct did not inhibit G2A-mediated InsP accumulation (Fig. 1B). These results suggest that G2A activates a Galpha protein, most likely Galpha q, regardless of LPC stimulation. As a positive control, expression of a constitutively active Galpha 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 beta gamma subunit scavenger, beta ARKct. Equivalent protein expression of the myc-tagged beta ARKct was shown by immunoblotting with 1:1000 anti-myc monoclonal antibody. All samples were co-transfected with beta -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.

It was recently reported that certain Galpha proteins, including Galpha q and Galpha 13, are involved in GPCR-mediated activation of NF-kappa 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-kappa B luciferase reporter and G2A, and in some samples, one of the Galpha proteins. As shown in Fig. 2A, expression of G2A resulted in a potent induction of NF-kappa B-mediated transcription characterized by an increase in luciferase activity. Co-expression of Galpha q was found to augment G2A-induced luciferase activity by ~90%, whereas co-expression of Galpha 13, known to couple to G2A (6, 9), also potentiated luciferase reporter activity. Neither of these Galpha proteins was able to induce the expression of NF-kappa 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-kappa B-luciferase activity (data not shown).


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Fig. 2.   Functional coupling of G2A to Galpha q and Galpha 13 in NF-kappa B luciferase reporter expression. A, cDNA (200 ng) encoding either wild type Galpha q or Galpha 13 were co-transfected with pRK5 or G2A along with the NF-kappa B-luciferase reporter and pCMVbeta . Twenty-four hours post-transfection cells were lysed and luciferase or beta -galactosidase substrate were added. Luminescence readings for luciferase were normalized against those for beta -galactosidase. Equivalent protein expression of wild type Galpha q or Galpha 13 was determined in pRK5 versus G2A-transfected samples by immunoblotting with anti-Galpha q and anti-Galpha 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-kappa B-luciferase, and beta -galactosidase causes inhibition of G2A-mediated NF-kappa 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.

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 Galpha 13 to the activation of RhoA, contains an N-terminal RGS domain that serves to down-regulate the Galpha 13 signal (19). GRK2 contains an N-terminal RGS domain that binds to and specifically inhibits Galpha q signaling (20). The two RGS truncation constructs shown in Fig. 2B were used to down-regulate signaling by Galpha 13 and Galpha q, respectively. Our results show that either GRK2RGS or p115RGS alone inhibited G2A-induced NF-kappa B activation by 50 and 36%, respectively (Fig. 2C). When both RGS constructs were used together, a more complete inhibition of G2A-mediated NF-kappa B activation was observed. These data provide additional support for the functional coupling of G2A to Galpha q and Galpha 13.

Ligand-independent and LPC-induced Elevation of cAMP-- GPCR-mediated activation of adenylate cyclase and the subsequent production of cAMP involve the activation of Galpha 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 Galpha 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 Galpha s, and possibly Galpha i, in the absence of LPC stimulation, in that the ligand-independent increase in cAMP is slightly enhanced by abrogating the inhibitory effect of Galpha i with PTX pre-treatment. The inhibitory activity by Galpha i appears to be relatively weak, as LPC stimulation further increased cAMP levels, indicating that G2A-mediated activation of Galpha s offsets the inhibition by Galpha 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 beta -galactosidase and represent the mean ± S.D. of duplicate determinations in one of three separate experiments.

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 >=  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).

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.


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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.

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 Galpha 13 and Galpha 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 Galpha 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 Galpha 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).

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 Galpha 13, Galpha q, and Galpha s, have been implicated in GPCR-mediated apoptosis (23-26). We therefore examined the ability of constitutively activated Galpha subunits (with Qright-arrowL mutation) to induce apoptosis in our system. The results indicate that expression of Galpha sQL alone did not induce apoptosis in HeLa cells, whereas expression of either Galpha 13QL or Galpha qQL induced apoptosis to a small extent (Fig. 7A). Interestingly, when both Galpha 13QL and Galpha sQL were transfected together, a synergism was achieved, which mimics the level of apoptosis of that seen in G2A-transfected cells. In contrast, when Galpha qQL was expressed in combination with Galpha sQL or Galpha 13QL, there was no additive or synergistic effect. All Galpha (QL) constructs have been tested for constitutive activity by either second messenger accumulation assays (InsP for Galpha qQL and cAMP for Galpha sQL) or by the NF-kappa B- and serum response element-luciferase reporter assays (for Galpha 13QL) (data not shown).


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Fig. 7.   Galpha s and Galpha 13 are critical to G2A-dependent apoptosis. A, HeLa cells were transfected with 200 ng of the expression constructs for active mutants (QL) of Galpha q, Galpha s, or Galpha 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.

To determine whether G2A utilizes Galpha s- and/or Galpha 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 Galpha s and Galpha 13 play critical roles in G2A-mediated apoptosis.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-kappa B reporter data, G2A may ligand-independently couple to a unique combination of G proteins: Galpha q, Galpha s, and Galpha 13 (Galpha i involvement appears to be minimal at most). LPC stimulation of G2A-transfected cells enhances activation of Galpha 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 Galpha i. Although this discrepancy may be due to differences in the cell lines utilized, it is also noted that LPC stimulation of Galpha s was not specifically investigated in that study. Our finding that LPC induces G2A-mediated Galpha 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.

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-alpha /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.

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 Galpha 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 Galpha 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 Galpha 13, in combination with Galpha 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 Galpha 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.

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.

    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.

Dagger 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; beta ARK, beta -adrenergic receptor kinase; z, benzyloxycarbonyl; fmk, fluoromethyl ketone; TMRE, tetramethylrhodamine ethyl ester; IBMX, isobutylmethylxanthine; NF-kappa B, nuclear factor kappa  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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ABSTRACT
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RESULTS
DISCUSSION
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