A novel role for Gqa in a-thrombin-mediated mitogenic

signalling pathways

Alice Gardnera, Polly J. Phillips-Masonb, Daniel M. Rabenc, Joseph J. Baldassarea,*aDepartment of Pharmacological and Physiological Sciences, St. Louis University Medical School,

1402 South Grand Boulevard, St. Louis, MO 63104, USAbDepartment of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106, USA

cDepartment of Physiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

Received 31 August 2001; accepted 13 November 2001

Abstract

a-Thrombin activates several G-proteins including members of the Gq, Gi, and G12/13 families, although the physiological importance of

these proteins is still not completely understood. We specifically investigated the role of Gqa in modulating a-thrombin-induced

mitogenesis. In Gqa1 cells, a stable cell line expressing reduced amounts of Gqa, concentrations of a-thrombin (1 NIH unit/ml), which

induce cell cycle reentry and progression into S phase in wild-type IIC9 cells, do not stimulate phosphatidylinositol (PI) hydrolysis, the rapid

early phase of ERK activity, and transit through G1 into S phase as quantified by cyclin-dependent kinase (CDK)4–cyclin D activity and

[3H]thymidine incorporation. Interestingly, high concentrations of a-thrombin restore these activities and cell cycle progression into S phase.

While, it is well documented that a-thrombin-induced sustained ERK activity mediates important responses for transit through G1 into S

phase, the importance of the rapid, Gq-dependent phase as a prerequisite for a-thrombin-mediated mitogenesis has not been appreciated.

D 2002 Elsevier Science Inc. All rights reserved.

Keywords: PAR, Protease-activated receptor; ERK, Extracellular-regulated kinase; CDK, Cyclin-dependent kinase; GPCRs, G-protein-coupled receptors

1. Introduction

It is well established that a-thrombin is a potent mitogen

in various cell types such as fibroblasts [1], vascular smooth

muscle cells [2], and astrocytes cells [3]. a-Thrombin

induces cell growth in these cells by the activation of

protein-coupled protease-activated receptors (PARs) [4].

Four PARs, PAR1, PAR2, PAR3, and PAR4, have been

cloned and found to be expressed in a variety of tissues [4].

In the case of PAR1, PAR3, and PAR4, these are activated

when a-thrombin clips the amino-terminus domain of the

receptor to unmask a new amino-terminus, which functions

as a tethered ligand. The prototypical thrombin receptor

known as PAR1 has been cloned from human platelets and

hamster fibroblasts [5,6]. Recently, the PAR1 receptor has

been shown to mediate a-thrombin’s ability to induce

mitogenesis [7], as CCL39 fibroblasts from tr � /� knockout

mice, which are missing this receptor, did not reenter the

cell cycle in response to thrombin.

Depending on the cell type, a-thrombin selectively

couples to distinct G-proteins to potentiate growth-factor-

stimulated mitogenesis [8]. For example, in 1321N astrocy-

toma cells, the pertussis toxin-insensitive G12 protein medi-

ates thrombin-induced mitogenesis via a Shc/Ras-dependent

pathway [9]. Conversely, microinjection of Gia2 antibodies

to quiescent Balb3 fibroblasts inhibited thrombin-induced

mitogenesis, indicating that the pertussis toxin-sensitive Gi

family mediated this process in these cells. Furthermore,

dual coupling of the thrombin receptor to pertussis toxin-

insensitive Gq and pertussis toxin-sensitive Gi/o has been

shown to regulate cell growth in the mouse lung fibroblast

CCL39 cell line [10].

a-Thrombin is known to induce the PLC-mediated

hydrolysis of cellular phosphatidylinositol (PI) hydrolysis,

leading to the production of 1,4,5-triphosphate (IP3) and

diacylglycerol (DA) [11,12]. The PAR1 receptor stimulates

PI hydrolysis in a number of cell types [13–15]. For

0898-6568/01/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved.

PII: S0898 -6568 (01 )00279 -0

* Corresponding author. Tel.: +1-324-577-8543; fax: +1-324-577-

8233.

E-mail address: baldasjj@slu.edu (J.J. Baldassare).

www.elsevier.com/locate/cellsig

Cellular Signalling 14 (2002) 499–507

example, in IIC9 cells, a-thrombin-induced PI hydrolysis is

mediated through PAR1 [14]. Evidence indicates that ago-

nist-induced hydrolysis of cellular PI via PI–PLC has an

important role in signal transduction pathways that mediate

mitogenesis [12,16]. In a rat hepatoma cell stably transfected

with antisense PLC constructs, profound effects on cell

growth are observed, namely, a two- to threefold increase

in doubling time and suppressed DNA synthesis [16].

The signalling properties of the thrombin receptor(s) are

mediated in part by coupling to a variety of G-proteins. The

pertussis toxin-insensitive G12 and G13 interact with the

activated thrombin receptor in astrocytoma cells [17]. In

contrast, the thrombin receptor couples to the Gq/11 and the

Gi/o family of G-proteins in neuroblastoma SH-EP cells and

fibroblasts [8,18]. Studies of prototypical G-proteins dem-

onstrate that activation of the thrombin-activated PAR1

receptor and subsequent PI hydrolysis is via coupling to a

specific G-protein, namely Gq [19]. In COS-7 cells, a

chimeric receptor carrying the thrombin receptor’s second

cytoplasmic loop-activated PI hydrolysis in response to a

nonthrombin agonist by coupling directly to Gq [19].

The mitogen-activated protein kinases (MAPKs) are

activated by a variety of growth factors in the process of

mitogenesis [20–22]. MAPKs are activated by sequential

phosphorylation of a three-kinase module. This cascade

comprises a MAPK (e.g. ERK1/2), a MAPK kinase (e.g.

MEK1), and a MAPK kinase kinase, such as Raf-1 [23].

During mitogenesis, the extracellular-regulated kinase

(ERK) pathway is activated by receptor tyrosine kinases

and G-protein-coupled receptors (GPCRs) [24]. a-Throm-

bin-induced activation of ERK is known to be essential for

its mitogenic action [25]. However, the specific role of the

G-proteins coupling a-thrombin to the ERK cascade have

presently not been delineated.

Balancing the growth-stimulatory and -inhibitory signals

that a cell receives regulates the transition between prolifera-

tion and quiescence. Cell cycle G1 progression and entry into

S phase are controlled by a series of sequential regulatory

events mediated by the cyclin/cyclin D-dependent kinase

complexes [26–29]. The cyclin-dependent kinases (CDKs)

CDK4 and CDK6 are activated by the D-type cyclins [26,30].

Early events in the cell cycle show that expression levels of

D-type cyclins are stimulated by growth factors and their

signal transduction cascades [30,31]. Specifically, the ERK

pathway has been shown to regulate expression of cyclin D

[32]. In addition to cyclin binding, CDK activity is regulated

by the binding of CDK inhibitors and posttranslational

modifications [33,34]. Cyclin D–CDK4 plays a crucial role

in reversing the inhibitory action of the retinoblastoma (Rb)

family of proteins by phosphorylation [35,36]. During early

to mid-G1 phase, the phosphorylation of Rb by CDK4–

cyclin D releases histone deacetylation inhibition [37,38] and

induces cyclin E expression [60,61].

In this paper, we show that reduction of the amounts of

Gqa in IIC9 cells stably transfected with antisense cDNA

for Gqa results in the inability of a-thrombin to stimulate

cell growth. Furthermore, the Gqa mutant (Gqa1) exhibits

altered inositol phosphate levels, CDK activity, and altered

activation of the rapid phase of ERK. The data in this work

is the first to show that the a-thrombin-stimulated acute

phase of ERK activation, mediated by Gqa, appears to be

important for mitogenesis.

2. Materials and methods

2.1. Cells and cell culture

IIC9 cells, a subculture of Chinese hamster embryo

fibroblasts, were grown and maintained in Dulbeco’s modi-

fied Eagle’s medium (DMEM) (Life Technologies, Grand

Island, NY) containing 10% (v/v) foetal calf serum and

2 mM L-glutamine (Sigma, St. Louis, MO) and 100 U

penicillin/ml 100 mg streptomycin/ml. All chemicals were

purchased from Sigma, unless specified otherwise. Subcon-

fluent cultures were growth-arrested by washing twice with

serum-free DMEM (Life Technologies) and incubated for

two days in serum free DMEM containing 1 mg/ml bovine

serum albumin plus 20 mM HEPES, pH 7.4 (basal media).

Growth-arrested cells were washed twice and equilibrated in

basal media for 30 min prior to addition of agonist.

a-Thrombin was added to growth-arrested cells at 1 U/ml

in all experiments.

2.2. Generation of Gqa-deficient cells

Gqa antisense transfectants were generated and main-

tained as described previously [39]. Briefly, 5 mg of

pcDNA3 containing Gqa cDNA (a generous gift from Dr.

R. Reed) in an antisense orientation to the cytomegalovirus,

as shown in Fig. 4, was transfected into subconfluent IIC9

cells using Lipofectamine (Life Technologies) according to

the manufacturer’s protocol. As a control, pcDNA3 without

an insert was used. The transfected cells were incubated for

16–18 h in Opti-MEM (Life Technologies) mixture at 37 �C.The media was replaced with DMEM plus 10% serum,

and cells were grown for 48 h to allow expression of the

neomycin-resistant gene. The transfected cells were subcul-

tured into 100-mm culture dishes and were grown for

several weeks in selection media containing 500 mg/ml

G418 (Boehringer Mannheim, Indianapolis, IN). G418-

resistant clones were isolated with cloning rings, and the

transfected clones were maintained in DMEM containing

10% serum plus 250 mg/ml G418.

2.3. Western blot analysis

Individual colonies from the stable transfections were

screened for the expression of appropriate proteins by

immunoblotting with Gqa polyclonal antibody (Calbio-

chem, La Jolla, CA) and tested against the parental cell line

IIC9 without insert (positive control). Expression of Gi2a,

A. Gardner et al. / Cellular Signalling 14 (2002) 499–507500

Gi3a, and Gqa antisense cDNAs were immunoblotted with

HA-12CA5 monoclonal antibody (Boehringer Mannheim)

or Ga polyclonal antibodies (Calbiochem), respectively.

Serum-deprived cell cultures were stimulated in the absence

or presence of a-thrombin or EGF, washed in cold 1�PBS, lysed, on ice, in 30 ml of solubilization buffer (25 mM

HEPES, 300 mM NaCl, 2 mM ethylenediaminetetraacetic

acid (EDTA), 1.5 mM MgCl2, 1% Triton X-100, 0.5 mM

phenylmethylsulfonylfluoride, 0.1 mM Na3VO4, 10 mg/ml

leupeptin, 10 mg/ml aprotinin, and 10 mg/ml pepstatin), and

then subjected to brief sonication. Following centrifugation

(Eppendorf 5415C, 14,000 rpm, 4 �C), an equal volume of

Laemmli loading buffer was added. Protein concentrations

of lysates were determined by Bio-Rad Protein Assay (Bio-

Rad), as recommended by the manufacturer. Protein lysates

(10–15 mg) were resolved by 10% SDS–polyacrylamide

gel electrophoresis, and separated proteins were transferred

to polyvinylidine (PVDF) membranes (Immobilon, Bed-

ford, MA). The blot was incubated overnight, at 4 �C, inwash buffer (20 mM Tris, pH 8.0, 150 mM Na Cl, 0.01%

Tween 20) containing 10% dry milk (w/v) and was then

washed. The membranes were probed with the appropriate

antibody, and after washing, they incubated with a second-

ary antibody (HRP-conjugated goat antimouse IgG or anti-

rabbit, Bio-Rad). Immunoreactive bands were visualized by

enhanced chemiluminescence (ECL) Western blotting sys-

tem (Amersham), as recommended by the manufacturer.

2.4. Phosphoinositide hydrolysis assay

Phosphoinositide hydrolysis was determined essentially

as described by Hung et al. [40]. IIC9 cells were seeded into

six-well plates (Corning) at a density of approximately 105

cells/well and were grown to subconfluence (approximately

50%). Cells were washed twice with basal media and were

then incubated for 24–48 h with basal media containing no

myoinositol plus 1–2 mCi/ml [3H]Myoinositol (DuPont/

NEN, Boston, MA, USA) for 24 h at 37 �C. LiCl (20

mM) was added to the cells 1 min prior to addition of a-thrombin, and cells were incubated at 37 �C. After 15 min,

the cells were rinsed once in cold PBS, and total inositol

phosphates were extracted in 600 ml of cold 4% HClO4 (v/v)

for 30 min at 4 �C. To each supernatant, 50 ml of phenol red/60 mM HEPES was added, neutralized with NH4OH, and

then centrifuged. The supernatants were applied to a 0.5-ml

column of Dowex AGIX8 (200–400 mesh size), formate

form (Bio-Rad). Columns were washed with 3 ml water

(� 3), followed by 3 ml 0.05 M ammonium formate/0.005

M borax (� 3). [3H]Inositol phosphates were eluted with

3 ml of 0.1 M formic acid/1.8 M ammonium formate, and

1 ml quantified by scintillation counting.

2.5. Immune-complex kinase ERK assay

Subconfluent (80%), growth-arrested IIC9 cells were

stimulated with a-thrombin (1 U/ml) for 5 min at 37 �C.

The medium was removed, and cells were washed twice in

cold PBS and scraped into 150 ml of ice-cold ERK lysis

buffer (20 mM Tris–HCl, pH8, 10% glycerol, 1% Triton

X-100, 1 mM NaF, 1 mM sodium vanadate, 50 mM

b-glycerolphosphate, 10 mg/ml aprotinin, 10 mg/ml pepstatin,

and 10 mg/ml leupeptin). The lysates were briefly sonicated,

and insoluble material was pelleted by centrifugation at

14,000� g at 4 �C for 5 min. Protein concentrations were

determined using the Bio-Rad Protein Assay, according to

the manufacturer’s specifications. For the immunoprecipita-

tions, 2 mg of either ERK1 polyclonal antibody (Santa Cruz

Biotechnology, Santa Cruz, CA) or monoclonal antibody

12CA5, raised to a peptide from influenza HA1 protein

(Boehringer Mannheim), was added to the supernatants and

incubated with gentle rocking at 4 �C for 4 h. The complexes

were then incubated for 2–3 h with either protein A-Sepha-

rose for polyclonal antibodies, or protein G-Sepharose for

monoclonal antibodies. ERK1 immune complexes were

washed twice with 1 ml of cold lysis buffer, twice with

1 ml of cold LiCl buffer (0.5 M LiCl, 100 mM Tris–HCl,

pH 7.6), and once with 1 ml of cold assay buffer (20 mM

Tris–HCl, pH 7.5, 50 mM b-glycerolphosphate, 1.5 mM

ethyleneglycol-bis{b-aminoethylether}N,N,N00,N00-tetra-

acetic acid (EGTA), 1 mM dithiothreitol). ERK1 immune

complexes were pelleted for 2 min at 14,000� g and

resuspended in 20 ml of reaction buffer per sample (20 mM

Tris–HCl, pH 7.3, 10 mM MgCl2, 50 mM ATP), 10 mgmyelin basic protein (MBP) per sample, and 2.5–5.0 mCi of[g32P]ATP. The samples were incubated for 45 min at 30 �C,and the reaction terminated by the addition of 5 ml of

4�Laemmli sample buffer. They were then boiled for

5 min and subjected to SDS–PAGE. The gels were dried,

and ERK activity was quantified by PhosphoImager analysis

(Molecular Dynamics).

2.6. [3H]Thymidine incorporation

Wild-type IIC9 or Gqa cells were plated in 12-well plates

at 2.5� 104 cells/well in DMEM (Life Technologies) with

10% foetal calf serum (Sigma). After 24 h, cells were

washed twice in DMEM and incubated for 48 h in DMEM

containing 1 mg/ml bovine serum albumin (Sigma, radio-

immunoassay grade), 2 mM L-glutamine (Sigma), 100 U

penicillin, 100 mg streptomycin/ml, and 20 mM Na HEPES,

pH 7.4 (Washington University Tissue Culture Facility,

MO). Following 20 h of incubation with either serum

(10%, v/v) or a-thrombin (1 U/ml), or EGF (100 ng/ml),

1 mCi/ml [3H]thymidine (DuPont/NEN) was added, and the

incubation was continued for an additional 3 h. Cells were

washed twice with cold PBS, and [3H]thymidine incorpora-

tion was determined as trichloroacetic acid (TCA)-insoluble

radioactivity by liquid scintillation counting as previously

described [32]. Briefly, DNAwas precipitated by incubating

the cells for 30 min with 1 ml of cold 5% (v/v) TCA. The

TCA-precipitated DNA was washed twice with cold 5%

TCA and solubilized with 500 ml of 2% (w/v) sodium

A. Gardner et al. / Cellular Signalling 14 (2002) 499–507 501

bicarbonate/0.1 M NaOH. Cells were scraped, and the lysate

was neutralized with 100 ml of 5% TCA. The TCA-pre-

cipitated [3H]DNA was quantified by scintillation counting.

3. Results

3.1. Reduced amounts of Gqa result in apparent growth

A number of studies implicate a critical role that Gqaplays in the regulation of cell growth [41–43]. To invest-

igate the importance of Gqa in cell growth, we isolated a

clonal cell line in which a full-length Gqa cDNA construct

in the antisense orientation is expressed (Fig. 1A). Western

blot analysis with antibodies directed against Gqa show a

60% reduction in the amounts of Gqa in the Gqa-reducedclones, Gqa1 (Fig 1B) and Gqa2, as well as four independ-

ently isolated Gqa-reduced clones (data not shown). In

contrast to Gqa, no change in the amounts of the other

Ga subunits, expressed in IIC9 and Gqa1 cells, is detected

(Fig. 1C). In the presence of 10% serum IIC9, cells double

within 24 h. Interestingly, the Gqa1 cells show a marked

increase in the time to transit the cell cycle with a doubling

time of approximately 36–40 h (Fig. 2). To investigate

the ability of the Gqa1 cells to enter into the cell cycle

and progress into S phase, we determined the effect of

a-thrombin on [3H]thymidine incorporation in Gqa1 cells

20 h after a-thrombin addition. In IIC9 cells, addition of low

mitogenic concentrations of a-thrombin (1 NIH unit/ml)

stimulates a four- to sixfold increase in [3H]thymidine

incorporation as compared with unstimulated IIC9 cells

(Fig. 3). Interestingly, a-thrombin fails to stimulate a

significant increase in [3H]thymidine incorporation in

Gqa1 cells (Fig. 3). The Gqa1 cells, however, have not lost

the ability to reenter the cell cycle and transit through G1, as

serum (10%) induces a similar increase in [3H]thymidine

incorporation in the Gqa1 cells as compared to IIC9 cells.

3.2. IP levels in Gqa1 cells are abolished using mitogenic

concentrations of a-thrombin

Previous studies in IIC9 cells demonstrate that a-throm-

bin induces rapid activation of phospholipase C (PI–PLC)-

mediated phosphoinositide hydrolysis, presumably via Gqa.We reasoned that if PI–PLC activity is dependent on

activation of Gqa, then a-thrombin-induced PI hydrolysis

should be markedly reduced in Gqa1 cells (Fig. 4). In wild-

type IIC9 cells, mitogenic concentrations of a-thrombin (1

NIH unit/ml) induce a fourfold increase in phosphoinositide

amounts above basal levels (Fig. 4). However, this level of

a-thrombin is ineffective in the Gqa1 cells (Fig. 4). We next

examined whether increased concentrations of a-thrombin

can induce PI hydrolysis. Addition of 10 NIH units/ml

results in increases in the Gqa1 cells that are similar to

those seen in IIC9 cells with only 1 NIH unit/ml a-thrombin

(Fig. 4). A dose–response analysis clearly demonstrates that

the reduction of Gqa results in a marked decrease in the

potency without any decrease in efficacy (Fig. 4), indicating

that the increasing amounts of a-thrombin can compensate

for the loss of Gqa.Our data clearly demonstrate that a reduction in amounts

of Gqa results in the failure of low doses of a-thrombin to

Fig. 1. Western blot analysis of Gqa antisense ablation. (A) Schematic

representation of the Gqa antisense construct. EcoRI fragment of Gqa from

pGEM-2/Gqawas subcloned into a vector plasmid, pcDNA3, in an antisense

orientation. The 30 end of the Gqa cDNA was immediately adjacent to the

cytomegalovirus promoter under which control the antisense was tran-

scribed. (B and C) Membrane proteins were separated on SDS–PAGE gel

(12.5%), and membranes were probed with an antibody against Gqa, Gi2a,Gi3a Gsa Goa, and G13a at 1:1000 dilution, for 1 h, as described in the

Materials and Methods. G-protein bands were detected by ECL.

Fig. 2. Gqa1 cells display a marked increase in cell cycle transit time. IIC9

cells were plated at approximately 20,000 cells/well in six-well dishes in

DMEM media containing 10% FCS. On the following day, cells were

washed with calcium- and magnesium-free PBS and fed with DMEM plus

a-thrombin (1 U/ml). Media were changed daily. To evaluate growth, cells

were trypsinized, resuspended, and counted with a Coulter counter. All

growth curves are representative of at least two separate experiments.

A. Gardner et al. / Cellular Signalling 14 (2002) 499–507502

induce cell cycle reentry and progression through G1.

Because high doses of a-thrombin (10 NIH units/ml) restore

PI hydrolysis, we reasoned that high doses would rescue

a-thrombin’s ability to stimulate growth. In support of this

idea, addition of 10 NIH units/ml stimulates cell cycle entry

and progression through G1 as determined by [3H]thymi-

dine incorporation (Fig. 5). Interestingly, high concentra-

tions of a-thrombin (10 NIH units/ml) induce amounts of

[3H]thymidine incorporation comparable to that seen in IIC9

cells when activated by low doses (1 NIH unit/ml) of

a-thrombin (Fig. 5). In agreement with the previous data,

the dose–response analysis for a-thrombin-induced thymi-

dine incorporation clearly demonstrates that the reduction of

Gqa results in a marked decrease in the potency without any

decrease in efficacy, indicating that increasing amounts of

a-thrombin can compensate for the loss of Gqa.

3.3. Gqa1 cells exhibit altered activation of the rapid phase

of ERK activity

To understand the mechanism for the growth-deficient

phenotype of the Gqa1 cells, we examined whether these

cells have altered signalling pathways known to be import-

ant for growth. Previous studies in a number of fibroblast

Fig. 3. Gqa1 cells display aberrant DNA sythesis. Quiescent Gqal cells were stimulated with or without a-thrombin (2 U/ml, 20 h) or serum (20 h). Following

removal of the mitogen, [3H]thymidine (1 mCi/ml) added for 3 h. The effects of Gqa ablation on thymidine incorporation were analyzed, as described in the

Materials and Methods. Means ± S.D. of three independent experiments performed in triplicate are presented.

Fig. 4. Inositol phosphate levels are aberrant in antisene-ablated Gqa1

cells. Wild-type IIC9 and Gqa1 cells were labeled with myo-[3H] inositol

(1 mCi/ml) for 24 h and then incubated in serum-free media supplemented

with 20 mM LiCl in the absence or presence of 0.01, 0.1, 0.5, 1.0, 2.0, 5.0,

or 10.0 U/ml thrombin for 15 min at 37 �C. IP3 levels were quantified as

described in the Materials and Methods. Triplicate samples were

performed on each set of experiments, which yielded the means ± S.D.

of three independent experiments.

Fig. 5. DNA synthesis in Gqa1 cells is rescued by high mitogenic

concentrations of thrombin. Quiescent Gqa1 cells were stimulated with or

without a-thrombin (0.01, 0.1, 0.5, 1.0, 2.0, 5.0, or 10.0 U/ml) for 20 h.

Following removal of the mitogen, [3H]thymidine (1 mCi/ml) was added for

3 h. The effects of Gqa ablation on thymidine incorporation were analyzed,

as described in the Materials and Methods. Triplicates were performed on

each set of experiments, which yielded the means ± S.D. of three

independent experiments.

A. Gardner et al. / Cellular Signalling 14 (2002) 499–507 503

cell lines [22,44,45] found that ERK activation is critical for

cell entry and progression through G1. Furthermore, studies

from our laboratory demonstrate that inhibition of a-throm-

bin-stimulated ERK activation blocks G1 progression in

IIC9 cells (unpublished observations). To ascertain whether

the reduced amounts of Gqa expressed in Gqa1 cells results

in inhibition of ERK activity, we examined the effect of low

mitogenic concentrations of a-thrombin on endogenous

ERK activity, as evidenced by the degree of phosphoryla-

tion of MBP (Fig. 6). As shown in Fig. 6, addition of 1 NIH

unit/ml of a-thrombin to IIC9 cells induces a rapid fivefold

increase in ERK activity, as indicated by the robust degree

of phosphorylation of MBP, followed by a lower, sustained

increase. We and others have shown, in a variety of cells,

that a-thrombin-stimulated ERK activation is biphasic,

with a rapid, initial phase within the first few minutes

of activation, followed by a late sustained phase of

ERK activity lasting for at least 4 h [8,39,44,63]. In agree-

ment with previous data, the rapid peak of ERK activity

in IIC9 wild-type cells is observed within 5–10 min after

a-thrombin addition (Fig. 6). However, in the Gqa1 cells, 1

NIH unit/ml a-thrombin stimulates only a two- to threefold

increase above basal levels, approximately 50% of that seenin IIC9 cells (Fig. 6). Interestingly, sustained ERK activity

is similar to the sustained activity reported in IIC9 cells

(Fig. 6). These data suggest that the rapid increase in ERK

activity is dependent in part on Gqa. Because high concen-

trations of a-thrombin restore PI hydrolysis, we reasoned

that high concentrations of a-thrombin could also restore

the rapid phase of ERK activity in the Gqa1 cells to the level

found in IIC9 cells (Fig. 7). Consistent with this reasoning,

high doses of a-thrombin stimulate a four- to fivefold

increase in ERK activity measured 5 min after a-thrombin

addition (Fig. 7). These results clearly suggest that the rapid

increase in a-thrombin-induced ERK activation is depend-

ent on Gqa, while sustained activation is unaffected.

3.4. Gqa1 cells exhibit altered CDK activity

Previous results show that a-thrombin, and PDGF,

induce cyclin D1 protein expression and CDK4–cyclin

D1 activity in IIC9 cells [44,46]. Furthermore, our labor-

atory, and others, also found that inhibition of mitogen-

induced ERK activation prevents the expression of cyclin

D1, CDK4–cyclin D1 activity, and G1 progression [44,46].

To identify a potential downstream cell cycle target in

the Gqa1 cells, we quantified the levels of cyclin D1.

As determined by Western blot analysis, low doses of

a-hrombin stimulate similar amounts of cyclin D1 protein

expression in IIC9 and Gqa1 cells (Fig. 8A). Our observa-

tions indicate that reduction of the amounts of Gqa does not

affect cyclin D1 expression. We next examined whether the

CDK4–cyclin D1 activity is altered in these cells. While

addition of low amounts of a-thrombin increases CDK4–

cyclin D1 levels within 4 h in IIC9 cells, only after 8 h is a

comparable increase seen in the Gqa1 cells (Fig. 8B).

Fig. 6. ERK-1 activation is aberrant in Gqa-antisene-deficient Gqa1 cells.

Quiescent IIC9 and Gqa1 cells, serum deprived for 48 h, were stimulated

with thrombin (1 U/ml, 5 min for the initial peak or 4 h for the sustained

peak) and ERK activity determined as described in the Materials and

Methods. ERK1 complexes were immunoprecipitated from lysates

containing equal protein, and kinase activity was tested by its ability to

phosphorylate MBP in vitro as described in the Materials and Methods.

ERK1 activity was quantitated using a Molecular Dynamics Phosphor-

Imager. Data are represented as fold increase relative to basal (normalized

to one). Duplicates were performed on each set of experiments, which

yielded the means ± S.D. for three independent experiments.

Fig. 7. ERK-1 activation in Gqa1 cells is rescued by high mitogenic

concentrations of thrombin. Quiescent IIC9 and Gqa1 cells, serum deprived

for 48 h, were stimulated (0.01, 0.1, 0.5, 1.0, 2.0, 5.0, or 10.0 U/ml) for 5

min, and ERK activity was determined as described in the Materials and

Methods. ERK1 was immunoprecipitated, and kinase activity was tested by

its ability to phosphorylate MBP. Data are represented as fold increase

relative to basal (normalized to one). Duplicates were performed on each

set of experiments, which yielded the means ± S.D. for three independ-

ent experiments.

A. Gardner et al. / Cellular Signalling 14 (2002) 499–507504

Interestingly, high concentrations of a-thrombin result in

an increase in CDK4–cyclin D1 activity within 4 h, similar

to IIC9 cells (Fig. 8B). These data show that the rapid

increase in ERK activation is important for CDK4–cyclin

D1 expression.

4. Discussion

a-Thrombin is a potent inducer of a variety of cellular

responses including phosphoinositide hydrolysis [14], ERK

activation [8], and DNA synthesis [47,48] in a variety of cell

types. Studies from several laboratories [40,49,50] dem-

onstrate that a-thrombin activates several G-proteins includ-

ing members of the Gq, Gi, and G12/13, although the

physiological importance of these proteins is still not

completely understood. We specifically investigated the role

of Gqa in modulating a-thrombin-induced mitogenesis. We

show in Gqa1 cells, a stable cell line expressing reduced

amounts of Gqa, that concentrations of a-thrombin (1 NIH

unit/ml), which induce cell cycle reentry and progression

into S phase in wild-type IIC9 cells, do not stimulate PI

hydrolysis, the rapid early phase of ERK activity, and transit

through G1 into S phase as quantified by CDK4–cyclin D

activity and [3H]thymidine incorporation. Interestingly, high

concentrations of a-thrombin (10 NIH units/ml) restore

these activities and cell cycle progression into S phase.

While it is well documented that a-thrombin-induced sus-

tained ERK activity mediates important responses important

for transit through G1 into S phase, the importance of the

rapid, Gq-dependent phase has not been appreciated.

A question naturally arises as to whether the same PAR-

type receptor mediates a-thrombin signalling events in IIC9

cells, or whether these cells utilize two distinct thrombin

receptors. PAR1, PAR3, and PAR4 thrombin receptors are

all activated by a-thrombin [4]. Non-PAR1-blocking anti-

bodies block platelet activation by low, but not high,

concentrations of a-thrombin [51]. Similar results are found

in transgenic mice in which PAR1 is knocked out [7]. These

data suggest the presence of an additional thrombin receptor

distinct from the PAR1 type. Indeed, further work indicated

that a low-affinity PAR4 receptor was present in mouse

platelets that mediated responses at higher concentrations of

a-thrombin [7]. However, a-thrombin’s effects in IIC9 cells

appear to be mediated by PAR1. Enterokinase treatment of

IIC9 cells expressing a mutant PAR1 receptor, which con-

tains an enterokinase site in place of the a-thrombin site,

stimulates PI hydrolysis [52], ERK activation, and G1

transit (Baldassare JJ, Chen J, Raben DM, unpublished

observation), suggesting that a-thrombin mediates mito-

genesis through activation of PAR1. Furthermore, Northern

blot analysis of IIC9 cells, using low-stringency hybridiza-

tion conditions, indicated that only one species of thrombin

receptor is expressed [52]. Consistent with these data that

PAR1 is the only a-thrombin-responsive species in fibro-

blasts is the observation that the fibroblasts from PAR1-

deficient mice do not respond to a-thrombin, whereas

platelets from these mice do respond [7].

While a-thrombin induces a marked increase of phos-

phoinositide hydrolysis in wild-type mice, this activity is

completely lost in transgenic mice in which PAR1 is

knocked out [7]. Studies from a number of laboratories

indicate that a-thrombin-induced phosphoinositide release

is mediated specifically by Gqa [19]. We report that

reduction of Gqa results in a marked reduction of the

potency of a-thrombin but no loss in efficacy. Additionally,

loss of phosphoinositide hydrolysis correlates with partial

loss rapid ERK activation and deficient growth in these

cells. There is a precedence that agonists that regulate

cell growth also activate phosphoinositide metabolism

[12,16,43]. Evidence indicates that a-thrombin-stimulated

phosphoinositide release correlates with the induction of

immediate early genes and proliferation of pulmonary artery

smooth muscle cells [12]. Furthermore, work by Nebigil

demonstrated that suppression of PI-specific PLCb, by

expression of PLCb mRNA antisense, inhibited protein

kinase C (PKC), Ras, and ERK activity, as well as growth

in FTO-2B cells [16]. Additionally, in clones with sup-

pressed PLC activity, cellular levels of PIP2 were elevated

and correlated with growth inhibition in these cells [16].

Fig. 8. CDK4–cyclin D activity is rescued by high mitogenic concen-

trations of thrombin. (A) IIC9 cells or Gqa1 cells were stimulated with 1 U/

ml of thrombin for the times indicated. Membrane proteins from either IIC9

cells or Gqa1 cells were separated on SDS–PAGE gel (10.0%), and

membranes were probed with an antibody against cyclin D1 at 1:1000

dilution, for 1 h, as described in the Materials and Methods. Lanes 1, 3, and

5 are wild-type IIC9 cells, and lanes 2, 4, and 6 are Gqa1 cells data. G-

protein bands were detected by ECL. (B) Quiescent IIC9 and Gqa1 cells,

serum deprived for 48 h, were stimulated with thrombin (1 or 10 U/ml) for

5 min, and ERK activity was determined as described in the Materials and

Methods. CDK4–cyclin D activity was immunoprecipitated, and kinase

activity was tested. Results are represented as fold increase relative to basal

(normalized to one). Data are representative of at least three independ-

ent experiments.

A. Gardner et al. / Cellular Signalling 14 (2002) 499–507 505

These data further support our observations that Gq-coupled

phosphoinositide hydrolysis is a prerequisite for a-throm-

bin-stimulated mitogenesis.

ERK regulation by GPCRs is a common phenomenon

and is necessary and adequate for the regulation of prolif-

eration in cell systems [53]. ERK activation by receptors

linked to pertussis toxin-sensitive Gai proteins is mediated

by bg subunits [54] and preferentially coupled to activation

of Ras and PI3 kinase [55]. In the majority of cases, the

activation of ERK by pertussis toxin-sensitive Gia proteins

is PKC independent, which is indicative of a lack of

receptor-mediated PI hydrolysis [56,57]. Conversely, per-

tussis toxin-insensitive Gqa proteins are generally associ-

ated with the activation of ERK by a Raf–PKC-dependent

route. In mouse lung fibroblasts, the mechanism by which

a-thrombin activates the rapid phase of ERK has been

shown to be dependent on two pathways [8]. In one

pathway, the PAR1 thrombin receptor utilizes PKC and is

c-Raf dependent, and the second is Gia dependent and

c-Raf independent [8]. Our observations in IIC9 wild-type

cells indicate that the rapid phase of ERK activation by

a-thrombin is modulated by two pathways: one is Gqadependent (Baldassare JJ, Gardner A, unpublished observa-

tions), and the other is pertussis toxin sensitive and Ras

dependent and is mediated by bg subunits (Baldassare JJ,

Gardner A, unpublished observations). Importantly, our data

suggests that cooperative signalling of Gqa- and Gia-mediated pathways for the proficient activation of the

ERK cascade is a prerequisite for the efficient commence-

ment of the a-thrombin mitogenic cascade, as Gqa1 cells

were not fully proficient at mediating the activation of the

rapid phase of ERK and subsequent thymidine incorpora-

tion, even though the sustained phase of ERK was normal in

these cells. The notion that dually coupled GPCRs require

cooperation of pertussis toxin-sensitive and -insensitive

pathways in the efficient activation of mitogenic cascades

has recently been addressed [53]. Blaukat et al. [53]

demonstrated that the bradykinin B2 receptor required the

cooperative signalling of Gqa- and Gia-mediated pathways

for the proficient activation of the ERK cascade, indicating

that this concept is a potential mechanism by which GPCRs

regulate various cellular responses.

The indication that reduced levels of Gqa result in a

marked decrease of the a-thrombin mitogenic signalling

pathways suggests that the mitogenic potency of the path-

way is diminished. In support of this suggestion, high doses

of a-thrombin restored PI hydrolysis, as well as the activ-

ities of ERK and CDK4–cyclin D, and cell cycle progres-

sion into S phase. However, the mechanism resulting in this

phenomenon remains to be determined. An answer may lie

in the recent paper by DeFea et al. [62], who demonstrated

that agonist activation of the Gq-coupled PAR2 receptor

induces the formation of a multiprotein signalling complex

that is required for the appropriate intracellular targeting of

activated ERK and, therefore, its mitogenic potential. They

showed that b-arrestin-dependent endocytosis of the signal-

ling complex, containing internalised PAR2 receptor, raf-1,

and ERK, was required for ERK activation. Other GPCRs

have been found to have a similar mechanism for the

activation of ERK [58,59]. Therefore, in the case of Gqa1

cells, it is conceivable that a decreased quotient of internal-

ised receptor exists in the multiprotein signalling complex,

which leads to an overall decreased level of ERK activity

and growth potential. In contrast, when high levels of a-thrombin are utilized, a greater number of PAR1 receptors

are recruited to the multiprotein signalling complex, thus

normalizing the signalling mechanism, namely, the required

level of ERK activity and the consequent rescue of a-thrombin’s mitogenic potential.

Therefore, our observations suggest that decreased Gqalevels could result in decreased coupling efficiency to a

signal transduction pathway that functions as an early,

essential signal linked to the induction of cell proliferation.

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