COMMUNICATION
The alpha -Subunit of the Heterotrimeric G Protein G13 Activates a Phospholipase D Isozyme by a Pathway Requiring Rho Family GTPases*

Steven G. Plonk, Seung-Kiel Park, and John H. ExtonDagger

From the Department of Molecular Physiology and Biophysics and the Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

G13 belongs to the G12 family of heterotrimeric G proteins, whose effectors are poorly defined. The present study was designed to test if phospholipase D (PLD) is regulated by G13 and if Rho-type small GTPases are involved. Expression of the constitutively active Q226L mutant of the alpha -subunit of G13 in COS-7 cells stimulated the activity of a rat brain phospholipase D isozyme (rPLD1) co-expressed in the cells. Wild type Galpha 13 was ineffective unless the cells were incubated with AlF4-. rPLD1 was previously shown to be activated by constitutively active V14RhoA in COS-7 cells (Park, S. K., Provost, J. J., Bae, C. D., Ho, W. T., and Exton, J. H. (1997) J. Biol. Chem. 272, 29263-29272). When the endogenous Rho proteins of the cells were inactivated by treatment with C3 exoenzyme from Clostridium botulinum, the ability of Galpha 13Q226L to activate rPLD1 was greatly attenuated. Co-transfection of dominant negative N19RhoA and N17Rac-1, but not N17Cdc42Hs or N17Ras, also inhibited the activation. Expression of constitutively active Galpha q in COS-7 cells also activated rPLD1, but constitutively active forms of Galpha i2 and Galpha s were without effect. These findings support an effector role for PLD in G13 signaling and demonstrate a requirement for Rho GTPases in this response.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Phosphatidylcholine (PC)1 specific phospholipase D (PLD) hydrolyzes its substrate into phosphatidic acid (PA) and choline in response to growth factors and G protein-coupled receptor agonists (1). PA may act as a lipid messenger in the cell, inducing cytoskeletal rearrangements or growth-regulatory responses, and is implicated in the regulation of NADPH-oxidase and intracellular membrane trafficking and fusion events (1, 2). PA can also be converted to the protein kinase C (PKC) activator diacylglycerol by phosphatidic acid phosphohydrolase or to the G protein-coupled receptor agonist lysophosphatidic acid by a PA-specific phospholipase A2 (1).

Analysis of PLD activity from various tissues and subcellular fractions supports the existence of biochemically distinct isozymes differing in subcellular localization and responses to Ca2+, phosphatidylinositol 4,5-bisphosphate (PIP2), oleate, and detergents in vitro (1, 3). Studies of signal transduction pathways in intact cells support the involvement of PKC and Rho GTPases in the regulation of PLD (1, 2). In vitro experiments confirm a direct regulatory role for PKC, Rho-family, and ARF GTPases in PLD activation (1, 2). PIP2 also activates PLD in vitro and may be required for PLD regulation in vivo (1).

Mammalian PLD isoforms have been cloned including hPLD1a, hPLD1b, hPLD2, and rPLD2 (4-7). hPLD1a, a 1072-amino acid protein, is specific for PC and is regulated by PKC, ARF, RhoA, and PIP2 (4, 5). hPLD1b encodes a 1034-amino acid splice variant with properties similar to hPLD1a (5). hPLD2 is also PC-specific, has high basal activity both in vitro and in vivo, and is activated by PIP2, but not PKC, ARF, or RhoA (6).

rPLD1, cloned in our laboratory from a rat brain library using a fragment of hPLD1, is 91% identical to hPLD1b in amino acid sequence and is expressed in a wide variety of tissues (8). It is stimulated when co-expressed with constitutively active V14RhoA in COS-7 cells (8) and also by treatment of the cells with a PKC-activating phorbol ester (8) or lysophosphatidic acid.2 Like hPLD1, rPLD1 is activated directly by RhoA, Arf, and PKCalpha in vitro.2

The Rho-related G proteins Rho, Rac, and Cdc42 are members of the Ras superfamily of low molecular weight GTPases. They regulate multiple downstream effects, including the contractility and organization of the actin cytoskeleton (9-12). Rho is required for cytoskeletal, transcriptional, and PLD responses to some G protein-coupled receptor agonists (2, 12-14), although the pathways involved and the precise nature of the Rho-requirement remain to be defined.

G13, a member of the G12 family of heterotrimeric G proteins, was first identified by the cDNA cloning of its alpha -subunit (15, 16). G13 is expressed in most cell lines and tissues and is especially abundant in human platelets (17). G13 is coupled to platelet thrombin and thromboxane A2 receptors (18, 19), suggesting a possible role in platelet activation. Immediate downstream effectors have not been identified for the G12 family, although downstream effects resulting from overexpression of constitutively active mutant forms of Galpha 12 and Galpha 13 have been described, including neoplastic transformation of cultured fibroblasts (20, 21), increased amiloride-sensitive sodium-proton exchange (22-24), induction of immediate early gene expression (13, 25), actin stress fiber formation and focal adhesion assembly (26), and activation of the c-Jun N-terminal kinase (JNK) cascade (27-29). A number of these effects can be blocked by dominant negative mutant forms of Rho-related G proteins, indicating a requirement at some level for these low molecular weight GTPases (13, 16, 23, 26-30).

The present study examines if PLD is a downstream effector of G13 and if Rho family GTPases are involved in the signal transduction pathway.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Plasmid Constructs-- A partial Galpha 13 cDNA, reverse transcription-polymerase chain reaction amplified from mouse brain total RNA, was used to clone a full-length cDNA from a mouse liver cDNA library (Stratagene) using standard methods. A Galpha 13Q226L mutant was made by overlap-extension polymerase chain reaction and confirmed by DNA sequencing. Wild type and Q226L were subcloned into the HindIII and EcoRI sites of the pcDNA3 expression vector (Invitrogen). rPLD1 in pcDNA3 (8), N19RhoA in pcDNA3, and Galpha qQ209L in pCMV4 were described previously (31). Galpha sQL and Galpha i2QL in pcDNA3 were provided by Dr. N. Dhanasekaran (Temple University, Philadelphia, PA), N17Cdc42 in pcDNA3 was provided by Dr. Shubha Bagrodia (Cornell University, Ithaca, NY), and N17Rac-1 in pCGT was provided by Dr. Linda Van Aelst (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Cell Culture and Transfection-- COS-7 cells (ATCC) were maintained in Dulbecco's modified Eagles's medium (Life Technologies, Inc.) plus 10% fetal bovine serum (Life Technologies, Inc.) under 5% CO2. Six-well plates were seeded with 2 × 105 cells/well and transfected with 2 µg of plasmid DNA and 6 µl of LipofectAMINE (Life Technologies, Inc.) per well according to the manufacturer's instructions. Opti-MEM (Life Technologies, Inc.) was substituted for Dulbecco's modified Eagles's medium during reduced serum incubations.

C3 Scrape Loading-- Cells were washed 24 h post-transfection, twice with phosphate-buffered saline (PBS) and once with scraping buffer (114 mM KCl, 15 mM NaCl, 5.5 mM MgCl2, 10 mM Tris-Cl pH 7.4) and then scraped off the plate in the absence or presence of 5 µg/ml Clostridium botulinum C3 exoenzyme (List Biologicals) as described (14). Cells were replated on polylysine and serum-starved 12 h later.

PLD Assay-- Cells were serum-starved (0.5% fetal bovine serum in Opti-MEM) at 24 h post-transfection for 16 h in the presence of 1 µCi/ml [3H]myristate (NEN Life Science Products), washed with PBS, and incubated in serum-free medium (Opti-MEM) for 50 min. PLD activity was then assayed as described previously (14). Cells were incubated in 0.3% 1-butanol for the indicated times. Cells were then washed with ice-cold PBS and stopped with methanol. Lipids were extracted, and the phosphatidylbutanol product was resolved by thin layer chromatography as described (14). Bands co-migrating with a phosphatidylbutanol standard were quantitated by scintillation counting.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

PLD Activation by Galpha 13-- To study the effect of G13 signaling on PLD, we expressed wild type Galpha 13 and GTPase-deficient, constitutively active Galpha 13Q226L together with a Rho-responsive PLD isoform, rPLD1, by liposome-mediated transient transfection of COS-7 cells. High level expression of the proteins and similar levels of rPLD1 expression between different co-transfections were confirmed by Western analysis (not shown). Expression of wild type Galpha 13 had little or no effect on either endogenous PLD activity or that of rPLD1. Galpha 13Q226L also had no effect on the activity of the endogenous PLD, but markedly stimulated rPLD1 activity (Fig. 1A). This activation of rPLD1 by Galpha 13Q226L was consistent with an effector role for PLD in G13 signaling. However, since Galpha 13 signaling was chronically activated by the prolonged expression of the Q226L mutant in this experiment, it was not clear how direct or indirect the activation of rPLD1 might be.


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Fig. 1.   Effect of mutational and aluminum fluoride-stimulated activation of Galpha 13 on rPLD1 activity. A, PLD activity was measured in COS-7 cells transfected with 0.6 µg/well of pcDNA3 vector containing no insert, Galpha 13, or Galpha 13Q226L, with and without 0.6 µg of co-transfected rPLD1 cDNA. Vector was added when needed to adjust the total amount of DNA to 2 µg/well. Cells were serum-starved and labeled with [3H]myristate as described under "Experimental Procedures" and incubated with 0.3% 1-butanol for 1 h. Radioactivity incorporated into phosphatidylbutanol was quantitated as described under "Experimental Procedures." The results are plotted as the means of at least four independent experiments ± S.E. B, COS-7 cells were transfected as above with the indicated combinations of Galpha 13 and rPLD1. PLD activity was measured in cells preincubated with 0.3% 1-butanol for 10 min followed by 10 µM AlCl3 + 10 mM NaF for 30 min. The results are the means of three independent experiments ± S.E.

To rule out mechanisms arising from long term activation of the pathway or consequently altered expression of PLD regulators, we examined Galpha 13 signaling in an acutely regulated system. Aluminum fluoride activates heterotrimeric G proteins due to its ability to mimic the gamma -phosphoryl group of GTP when complexed with the GDP-bound alpha -subunit (32). We expressed wild type Galpha 13 with and without rPLD1 and then treated the cells with aluminum fluoride for 30 min (Fig. 1B). Fluoride treatment of the cells yielded a small stimulation of the endogenous PLD activity, which was not affected by overexpression of wild type Galpha 13. rPLD1 activity was potently stimulated by fluoride treatment when co-expressed with Galpha 13, but not when expressed alone. This Galpha 13-dependent acute stimulation of rPLD1 by aluminum fluoride effectively rules out mechanisms requiring long term activation of Galpha 13 and is consistent with an effector role for PLD in G13 signaling.

Requirement for Rho Family GTPases-- To investigate the potential role of Rho in the regulation of rPLD1 by Galpha 13, we inhibited its function by treating the cells with C3 exoenzyme from C. botulinum, an ADP-ribosyltransferase that inactivates Rho (33). Efficient ADP-ribosylation was confirmed by in vitro assays with C3 (33), which showed that cellular Rho from the C3-scraped cells was efficiently modified as indicated by a subsequent loss of ADP-ribosylation in vitro (Fig. 2A). We then looked at the effect of C3 toxin on the activation of rPLD1 by Galpha 13Q226L. C3 had little or no effect on the endogenous PLD activity or that of the expressed rPLD1. It did, however, significantly diminish the effect of Galpha 13Q226L on rPLD1 activity, consistent with a requirement for Rho in the activation of rPLD1 by active Galpha 13 (Fig. 2B).


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Fig. 2.   Effect of loading COS-7 cells with C3 exoenzyme of C. botulinum on rPLD1 activation by Galpha 13Q226L. A, transfected COS-7 cells were scrape-loaded with bovine serum albumin or C3 toxin and replated as described under "Experimental Procedures." Twenty-eight h later, cellular extracts were prepared and treated in vitro with C3 toxin in the presence of [32P]NAD as described (33) to measure the extent of ADP-ribosylation of Rho proteins. The extracts were then resolved by SDS-polyacrylamide gel electrophoresis and subjected to autoradiography. B, COS-7 cells transfected with the indicated plasmid constructs were scrape-loaded with bovine serum albumin or C3 toxin and replated as described under "Experimental Procedures." Cells were starved and labeled as described and then incubated with 0.3% 1-butanol for 1 h. Radioactivity incorporated into phosphatidylbutanol was quantitated as described under "Experimental Procedures." The results are plotted as the means of three independent experiments ± S.E.

To further address the requirement for Rho and related GTPases, we expressed dominant negative mutant forms of these G proteins together with Galpha 13Q226L and rPLD1 and measured PLD activity. These dominant negative mutants are thought to inhibit signaling through endogenous Rho-related small G proteins by forming stable, inactive complexes with guanine nucleotide exchange factors required for their activation (34). N17Ras, N19RhoA, N17Rac, and N17Cdc42 alone had no effect on rPLD1 activity (not shown). When co-expressed with both Galpha 13Q226L and rPLD1, N19RhoA and N17Rac partially blocked the activation of rPLD1 while N17Ras and N17Cdc42 did not (Fig. 3). Combination of N19RhoA and N17Rac completely blocked the activation. These results are consistent with a requirement for exchange factor-mediated regulation of Rho family GTPases such as Rho and Rac in the activation of rPLD1 by constitutively active Galpha 13.


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Fig. 3.   Effect of dominant negative RhoA, Rac, Cdc42, and Ras mutants on rPLD1 activation by Galpha 13Q226L. PLD activity was measured in COS-7 cells transfected with 0.5 µg/well each of plasmid constructs encoding Galpha 13Q226L and rPLD1 and 1 µg/well of the indicated dominant negative mutant GTPases (0.5 µg + 0.5 µg when N19RhoA and N17Rac were combined). Vector was added to adjust the total amount of DNA per well to 2 µg. Cells were starved and labeled as described under "Experimental Procedures" and incubated with 0.3% butanol for 1 h. Radioactivity incorporated into phosphatidylbutanol was quantitated as described, and results were expressed as a percentage of the maximal response. The results plotted are the means of at least three independent experiments ± S.E.

rPLD1 Is Activated by Mutationally Active alpha -Subunits of the G12 and Gq Families-- To determine the selectivity of G12 family proteins in the activation of rPLD1, we examined its regulation by other heterotrimeric G protein alpha -subunits. Accordingly, we expressed mutationally active alpha -subunits of the G12, Gq, Gi, and Gs families with and without rPLD1 and then measured PLD activity (Fig. 4). We found that the activity of rPLD1 was potently stimulated by constitutively active Galpha 13Q226L and Galpha qQ209L, but not by Galpha i2Q205L or Galpha sQ227L.


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Fig. 4.   Effect of expression of mutationally active G protein alpha -subunits of the G12, Gq, Gi, and Gs families on rPLD1 activity. PLD activity was measured in COS-7 cells transfected with 0.6 µg/well of each of the indicated cDNAs encoding mutationally active G protein alpha -subunits both with and without rPLD1 co-transfection (0.6 µg/well). Vector was added to adjust the total amount of DNA to 2 µg/well. Cells were serum-starved and labeled as described under "Experimental Procedures" and incubated with 0.3% 1-butanol for 1 h. Radioactivity incorporated into phosphatidylbutanol was quantitated. The results are plotted as the means of four independent experiments ± S.E.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

This report supports the hypothesis that rPLD1, a widely expressed rat hPLD1b homolog, serves as a downstream effector in G13 signaling. Both mutational activation and fluoride stimulation of expressed Galpha 13 led to potent stimulation of co-expressed rPLD1 activity (Fig. 1).3 The acute regulation by fluoride-stimulated Galpha 13 rules out mechanisms requiring long term activation of the pathway and is consistent with an effector role for rPLD1.4

Mutational and fluoride-stimulated activation of Galpha 13 had no effect on endogenous PLD (Fig. 1). This is probably due to differences between rPLD1 and the endogenous PLD in COS cells. Previous work in this laboratory has indicated the absence of Rho-reponsive PLD activity in COS cells (8). This has been observed both in cells transfected with V14RhoA and also with in vitro assays performed by incubation of COS cell extracts with RhoA and GTPS (8). In both cases, the Rho response could be reconstituted by expression of rPLD1 (8).

We found that C3 toxin, which is selective for Rho (33), significantly blocked rPLD1 activation by Galpha 13 (Fig. 2). Dominant negative N19RhoA and N17Rac-1 alone partially but significantly blocked rPLD1 activation and, in combination, completely blocked the effect (Fig. 3). These data support a requirement for guanine nucleotide exchange factor-dependent regulation of Rho family G proteins such as Rho and Rac in this response (34). Cytochalasin D treatment of the cells did not block the response (not shown), suggesting that the inhibitory effects of C3 toxin, N19RhoA, and N17Rac are not simply due to a general disruption of signaling responses as a consequence of altered cytoskeletal organization or function induced by these treatments. All together, these data are consistent with the involvement of Rho family G proteins in the signaling pathway between Galpha 13 and rPLD1.

We showed that rPLD1 was also activated by mutationally active Galpha q, but not by Galpha i2 or Galpha s. This observation is consistent with reports of PLD activation by Gq-coupled receptors and PKC-activating phorbol esters (1). rPLD1 is stimulated by the alpha - and beta -isozymes of PKC in vitro2 and by phorbol ester when expressed in COS-7 cells (8). Our results indicate that rPLD1 is selectively activated by alpha -subunits of the G12 and Gq families, although beta gamma -subunit mediated regulation of rPLD1 or activation of other PLD isoforms by heterotrimeric G proteins other than Gq and G12 family members remain a possibility.

Galpha 13Q226L expression triggers actin stress fiber formation and focal adhesion assembly (26), JNK activation (27-29), sodium-proton exchange (23, 24), serum response element-dependent gene transcription (13), and apoptosis (30) by pathways also inhibited by dominant negative Rho family G proteins. For several reasons, rPLD1 activation is likely to occur either upstream of these effects or by an entirely separate pathway. First, Rho family G proteins are direct regulators of rPLD13 (5), while the Galpha 13-mediated effects listed above are likely to be downstream of a cascade of Rho-initiated signals. Second, cytochalasin D disrupts cytoskeletal responses, but did not inhibit rPLD1 activation (not shown). Apoptotic responses and gene induction typically require hours to develop, while rPLD1 is activated within minutes by fluoride-stimulated Galpha 13 (Fig. 1). Finally, activation of the JNK cascade by Galpha 13 in COS-7 cells is blocked by N17Ras, but not N19RhoA (27-29), while rPLD1 activation is blocked by N19RhoA, but not N17Ras (Fig. 3).

Phospholipases are key components of transmembrane signal transduction pathways. PLD-catalyzed production of phosphatidylcholine-derived messengers could mediate critical downstream responses in G13 signaling. Further study is needed to more precisely define the role of PLD in G13-mediated responses. We suggest that G13 may play a role in the regulation of Rho-responsive PLD activity by G protein-coupled receptors.

    FOOTNOTES

* This work was supported by Grants DK 47448 and DK 07563 from the National Institutes of Health.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 Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed. Tel.: 615-322-6494; Fax: 615-322-4381; E-mail: john.exton{at}mcmail.vanderbilt.edu.

1 The abbreviations used are: PC, phosphatidylcholine; PLD, phospholipase D; PA, phosphatidic acid; PKC, protein kinase C; PIP2, phosphatidylinositol 4,5-bisphosphate; PBS, phosphate-buffered saline; JNK, c-Jun N-terminal kinase.

2 S. K. Park, D. S. Min, and J. H. Exton, unpublished observations.

3 The related Galpha 12Q229L was also found to activate rPLD1 (S. G. Plonk and J. H. Exton, unpublished observations).

4 Acute activation of rPLD1 was also observed in thromboxane A2-treated COS cells expressing thromboxane A2 receptors (S. G. Plonk and J. H. Exton, unpublished observations).

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Abstract
Introduction
Procedures
Results
Discussion
References

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