(Received for publication, July 31, 1996, and in revised form, October 15, 1996)
From the Howard Hughes Medical Institute, Durham,
North Carolina, the Departments of
Medicine and Biochemistry,
Duke University Medical Center, Durham, North Carolina 27710, and the
¶ Veterans Affairs Medical Center,
Durham, North Carolina 27705
Lysophosphatidic acid (LPA) utilizes a
heterotrimeric guanine nucleotide regulatory (G) protein-coupled
receptor to activate the mitogen-activated protein kinase pathway and
induce mitogenesis in fibroblasts and other cells. A single cell assay
system was used to examine the functional interaction of the LPA
receptor with G proteins in intact mouse fibroblasts, by measuring
LPA-stimulated induction of the immediate-early gene,
c-fos, as read out by a stably expressed
fos-lacZ reporter gene. Pretreatment of these cells with
pertussis toxin at 100 ng/ml almost completely abolished LPA-stimulated
c-fos induction. Western blotting revealed that two
pertussis toxin (PTX)-sensitive G proteins, Gi2 and
G
i3, were present in membranes prepared from these
cells, and Northern blotting confirmed the absence of message for other
PTX-sensitive subunits. Microinjection of an
i1/
i2-specific antibody into living cells
decreased LPA-stimulated induction of c-fos by 60%, whereas introduction of antibodies to either
i3 or
16, a subtype not present in these cells but used as a
control, decreased LPA-stimulated c-fos induction by only
19%. In contrast, the
i1/
i2-specific antibody had no effect on insulin-induced c-fos expression,
which is thought to utilize a G protein-independent mechanism of
signaling. In addition, cellular expression of an epitope-tagged
PTX-resistant mutant of G
i2, but not PTX-resistant
G
i3, restored LPA-stimulated c-fos induction
in cells in which endogenous G protein
subunits were uncoupled from
the receptor by pretreatment with PTX. Together, these results provide
conclusive in vivo evidence that G
i2 is the
PTX-sensitive G protein
subunit which mediates LPA-stimulated c-fos induction and perhaps mitogenesis in these cells.
Lysophosphatidic acid (LPA)1 is a water-soluble phospholipid that is a normal constituent of mammalian serum. It is released from activated platelets (1) and is rapidly generated by growth factor-stimulated fibroblasts (2). LPA has been shown to activate a variety of second messenger pathways, including stimulation of phospholipases C and D and inhibition of adenylyl cyclase (3, 4), and elicits a diverse range of physiological responses including smooth muscle contraction (5), remodeling of the actin cytoskeleton (6), and mitogenesis (3).
LPA is thought to interact with a specific cell surface receptor identified as a 38-40-kDa protein by photoaffinity labeling (7) and, more conclusively, by membrane binding studies (8). Several lines of evidence suggest that this LPA receptor couples to downstream effector molecules through the activation of a guanine nucleotide regulatory (G) protein. For example, pertussis toxin (PTX) abolishes the LPA-induced inhibition of adenylyl cyclase (3) and the ability of guanine nucleotides to regulate LPA binding in rat brain and Swiss 3T3 cell membranes (8). In addition, guanine nucleotides enhance LPA-induced inositol trisphosphate formation in permeabilized cells (9). Finally, LPA has been shown to stimulate high affinity GTPase activity and cholera toxin-catalyzed ADP-ribosylation of Gi in membranes prepared from Rat-1 fibroblasts (10).
LPA stimulates mitogenesis through the activation of p21ras (11, 12) and the subsequent activation of the mitogen-activated protein (MAP) kinase pathway (13). However, in contrast to the activation of p21ras that occurs in response to ligands of receptor tyrosine kinases, such as insulin or epidermal growth factor (reviewed in Ref. 14), the LPA-induced activation of p21ras is abolished by pretreatment with PTX (11, 12). It also appears to be largely independent of the phospholipase C-mediated calcium mobilization induced by LPA, since PTX had no effect on this pathway (3). These findings suggest that a G protein of the Gi family, or Go, regulates the activation of p21ras by LPA. Go is limited in tissue distribution, being found predominately in brain, where it has been shown to be involved in regulating ion channels (15). In contrast, Gi proteins are expressed ubiquitously and are the most abundant G proteins found in many cell types.
The identity of the Gi protein that couples the LPA
receptor to the mitogenic response is unknown. One candidate is
Gi2, since mutationally activated
i2
mimics the Gi-mediated inhibition of cAMP accumulation
caused by LPA (16). In addition, expression of the gip2
oncogene, which encodes a constitutively active form of
G
i2, has been shown to induce neoplastic transformation in Rat-1 cells, but not in NIH 3T3 or Swiss 3T3 cells (17, 18). However, LPA-induced activation of G
i2 has not been
tested directly.
The agonist-stimulated induction of the immediate-early genes, such as
c-fos, precedes the progression of cells through the cell
cycle. Therefore, agonist-stimulated induction of c-fos can be used as a marker for mitogenesis. In this study we used a functional assay to examine receptor-G protein interactions in mouse fibroblasts where the induction of c-fos was read out by a stably
expressed fos-lacZ reporter gene. Inhibitory antibodies
directed against specific G protein subunits were microinjected
into living cells. We assessed the potential inhibitory effect of these
antibodies on the LPA-stimulated induction of c-fos, in
order to investigate which of the Gi subtypes mediates
LPA-stimulated mitogenesis. In addition, using transfection of
PTX-resistant mutants of specific G protein
subunits, we examined
whether cellular expression of a single
subunit was sufficient to
restore LPA-stimulated signaling in cells in which the LPA receptor was
uncoupled from its cognate G proteins by pretreatment with PTX.
Fibroblasts derived from fos-lacZ
mice (19) were kindly provided by Dr. Tom Curran, St. Jude Children's
Research Hospital, Memphis, TN. The cells were cultured in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, and antibiotics (100 units/ml penicillin/100
µg/ml streptomycin) and were incubated in a humidified atmosphere of
air with 5% CO2 at 37 °C. Mouse fos-lacZ
fibroblasts stably transfected with the human insulin receptor (see
below) were cultured in the above medium which was also supplemented
with 0.6 mg/ml G418. Mouse fos-lacZ fibroblasts stably
coexpressing the insulin receptor and the PTX-resistant mutant of
Gi3 (see below) were cultured in medium containing all
of the above supplements plus 0.3 mg/ml hygromycin B. All media
reagents except for hygromycin B (Boehringer Mannheim) were obtained
from Life Technologies, Inc.
A stably transfected
derivative of the fos-lacZ cell line was created that
overexpressed the human insulin receptor and resulted in
insulin-stimulated nuclear c-fos expression. The human
insulin receptor cDNA in the SR expression vector (20) was
kindly provided by Dr. Richard Roth, Stanford University, Palo Alto,
CA. fos-lacZ fibroblasts were transfected with a 10:1
mixture of the plasmid containing the human insulin receptor cDNA
(10 µg) and pSV2neo (1 µg; American Type Culture Collection, number
37149, Rockville, MD) by the calcium phosphate method (21). Briefly,
subconfluent cells (50-70% confluence) growing in 60-mm dishes were
incubated for 16 h with the DNA/Ca2+ mixture, washed
twice briefly with Hanks' balanced salt solution, and then incubated
with standard growth medium. Twenty-four h later the cells were split
1:20 and cultured in the presence of 0.6 mg/ml G418. After 7 days,
colonies that appeared to originate from single cells were isolated
from the dishes, expanded, and further subcloned by an additional 1:20
passage. Following expansion of colonies that originated from single
cells, insulin responsive lines were screened by examining
insulin-stimulated
-galactosidase (
-gal) activity in single
cells. Five candidate cell lines were obtained which differed mainly in
the percentage of cell nuclei staining positive for
-gal in response
to 70 nM insulin. Of these, the cell line designated 12B
had the highest percentage of cells staining positive for
-gal. The
microinjection experiments described here were conducted using a single
cell line in which fewer than 1% of the cells demonstrated nuclear
-gal staining in the serum-deprived state, and 75-85% of the cells
exhibited
-gal staining 110 min after stimulation with 70 nM insulin. The
-gal staining was localized to the cell
nucleus, and the pattern of staining was identical to that observed in
immunofluorescence studies utilizing anti-c-fos or
anti-
-gal antibodies (data not shown). Another line derived from
line 12B was generated that stably overexpressed the PTX-resistant mutant of G
i3, termed G
i3C>S. Briefly,
cells were co-transfected with a 10:1 ratio of plasmid containing the
cDNA encoding G
i3C>S in the pRc/CMV expression
vector (22) and a hygromycin plasmid to allow for selection. Cells were
selected for stable transfection with 300 µg/ml hygromycin B by
techniques identical to those described above. Hygromycin-resistant
lines were screened for overexpression of G
i3 by Western
blotting following separation of membrane proteins by high resolution
urea SDS-PAGE (23).
Affinity purified
rabbit polyclonal antibodies to G protein subunits were generously
provided by Dr. Allen Spiegel (National Institutes of Health, Bethesda,
MD); the preparation and specificity of these antibodies has been
described in detail (24, 25). Briefly, antibody AS was raised to a
synthetic decapeptide (KENLKDCGLF) corresponding to the
carboxyl-terminal region of transducin
; its specificity is
i1 =
i2
i3. This
sequence differs by one amino acid from the sequence found in both
G
i1 and G
i2. For G
i1 and
G
i2, no species differences in the relevant decapeptide
have been reported among human, bovine, rat, and mouse (26). Antibody
EC was raised to a synthetic decapeptide (KNNLKECGLY); its specificity
is
i3 >
o
i1 =
i2. Only a single amino acid difference exists between
the human and rat sequences for peptide EC. In the case of antibodies
AS and EC, the peptide antigen was derived from the region of the
protein thought to interact with the receptor (27, 28). Antiserum AR
was raised to a carboxyl-terminal decapeptide of G
16.
This antibody does not cross-react with other known
subunits, and
the
16 species has been found only in hematopoietic cells.2 Buffer exchange and antibody
concentration into 50 mM HEPES-HCl, 40 mM NaCl
(pH 7.3) was accomplished by four rounds of centrifugation in
Centriprep 30 microconcentrators (Amicon, Beverly, MA) according to the
protocol supplied by the manufacturer. Antibody concentration was
determined by measurement of absorbance at 280 nm (29). Monospecific
anti-G
i1 antibody, which was used for Western blotting
only, was raised to a synthetic decapeptide (LDRIAQPNYI) corresponding
to internal sequence 159-168 found in Gi
1 and was
purchased from Calbiochem-Novabiochem Corp. (San Diego, CA). In some
experiments, a G
i1/2 subunit selective rabbit antiserum, number 982, purchased from Calbiochem, was used for immunoblotting. Antibody to the
subunit of Go was purchased from
Upstate Biotechnology Inc. (Lake Placid, NY) and was used for
immunoblotting only.
Two confluent
T-175 flasks of mouse fibroblasts were briefly washed twice with
ice-cold phosphate-buffered saline without calcium and magnesium
(PBS, pH 7.2), collected by scraping into ice-cold PBS
, pooled, and
collected by centrifugation at 1,000 × g for 5 min at
4 °C. The cell pellet was resuspended in ice-cold lysis buffer
containing 0.5 mM EGTA, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (Sigma)
and 20 µg/ml leupeptin (Sigma) in 20 mM
Tris-Cl (pH 7.4), homogenized by 20 strokes in a Teflon-glass tissue
grinder (Thomas Scientific, Philadelphia, PA) and centrifuged at
500 × g for 5 min at 4 °C to remove nuclei and
unbroken cells. The supernatant was set aside at 4 °C and the pellet
was subjected to a second round of homogenization and centrifugation.
The combined supernatants were centrifuged at 100,000 × g for 30 min at 4 °C. The resulting pellet was
resuspended in lysis buffer and taken as the membrane fraction. Protein
concentration was measured by a dye binding method (Bio-Rad). Membranes
from bovine brain were kindly provided by W. Koch (Duke University,
Durham, NC). Membrane proteins, or, in some cases, whole cell lysates
were subjected to either electrophoresis on 12% SDS-polyacrylamide
gels according to Laemmli (30), or to high resolution urea SDS-PAGE and
transferred to nitrocellulose (Schleicher & Schuell, Keene, NH) for
immunoblotting according to the procedure in the Bio-Rad Immun-Blot
assay kit. Individual membranes were incubated with anti-Gi
antibodies at the following concentrations: AS, 3.6 µg/ml; EC, 4.3 µg/ml; AR, 2.8 µg/ml; and anti-G
i1 and
-G
o at a 1:1000 dilution. Proteins were visualized using
a horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) at a
1:1500 dilution. Immunoblotting with the EE monoclonal antibody
(Berkely Antibody Co., Berkely, CA), to detect expression of the
transiently transfected G
i2EEC>S construct, which is
the epitope-tagged and PTX-resistant analog of G
i2 (31),
was carried out with a 1:1000 dilution of the primary antibody, and a
1:1000 dilution of the horseradish peroxidase-conjugated goat
anti-mouse IgG (Bio-Rad). Any changes to these protocols are indicated
in the figure legends.
Total RNA was
isolated from mouse fibroblasts or frozen mouse or rat brains by a
modification (32) of the single step acid-guanidinium thiocyanate/phenol/chloroform extraction (33). The RNA was separated on
2.2 M formaldehyde, 1.2% agarose gel, transferred to a
Nytran membrane (Schleicher & Schuell), and used for Northern blot
analysis as described previously (34). The full-length coding rat
cDNAs for Go and G
i1 were excised
from the bacterial expression vector NpT7-5 by complete digestion with
XbaI and partial digestion with NcoI. The 1.13- and 1.29-kb fragments, corresponding to G
i1 and
G
o, respectively, were gel purified and concentrated using the GeneClean kit according to the manufacturers instructions (Bio-101 Inc., La Jolla, CA). The cDNA probes were labeled with [
-32P]dCTP using the Random Primers DNA labeling
system (Life Technologies) and unincorporated nucleotide was removed by
chromatoraphy on a NucTrap column (Stratagene, La Jolla, CA). Filters
were hybridized at 42 °C with denatured probes at 2 × 106 cpm/ml for 45 h, then washed and subjected to
autoradiography at
70 °C. The blots were then stripped in 0.1%
SSPE, 0.1% SDS as described previously (35), and rehybridized for
15 h with a 1.12-kb PstI fragment (2 × 106 cpm/ml) of chicken
-tubulin to ensure equal RNA
loading.
Approximately 48 h prior to the
microinjection experiments, cells were seeded from trypsinized stock
flasks onto single round sterile glass coverslips (Carolina Biological
Supply, Burlington, NC) in 35-mm dishes at a density sufficient to
achieve approximately 50-60% confluence 24 h later. Twenty-four
h after seeding, cells were washed three times with Hanks' balanced
salt solution and incubated for an additional 24 h in Dulbecco's
modified Eagle's medium containing 2 mM glutamine,
antibiotics, and 1% bovine serum albumin (BSA; crystallized and
lyophilized, Sigma) to induce quiescence. Just prior
to microinjection, fresh aliquots of affinity purified G protein subunit antibodies or buffer alone (50 mM HEPES, 40 mM NaCl, pH 7.3) were mixed with a pure rabbit IgG marker
antibody and centrifuged at 100,000 × g for 40 min at
4 °C. The antibodies were present in the injection needle at the
following concentrations: AS, 3.2 mg/ml; EC, 3.9 mg/ml; AR, 2.5 mg/ml;
marker, 1.8 mg/ml. Individual coverslips were removed from the
incubator and injections were made into the cytoplasm of cells over a
period of 30 min, using an Eppendorf microinjection system (Hamburg,
Germany) with a warming stage (37 °C) coupled to a Zeiss Axiophot
microscope (Carl Zeiss Inc., Thornwood, NY). Approximately 100 cells
were injected per coverslip. Cells were immediately stimulated with LPA
or insulin at a final concentration of 2.1 µM or 70 nM, respectively, and returned to the incubator for 110 min. The cells were then washed once with PBS
, fixed for 10 min in
2% paraformaldehyde (Sigma) in a buffer containing
0.1 M PIPES (pH 6.9), 2 mM MgCl2, and 2 mM EGTA, and washed three times with PBS
. For
-gal staining the coverslips were transferred to individual wells of
a 12-well plate, washed once with PBS
, then incubated for 15 h
at 37 °C with 1 mg/ml
5-bromo-4-chloro-3-indoyl-
-D-galactoside (Life
Technologies) in PBS containing 4.6 mM potassium
ferricyanide, 5 mM potassium ferrocyanide, 4.2 mM MgCl2, 0.02% Nonidet P-40 (v/v), and 0.1 mg/ml sodium deoxycholate (Sigma), which yields an
insoluble blue reaction product (36). The cells were washed three times
with PBS
, blocked for 30 min at 25 °C with 5% (v/v) goat serum
(Life Technologies) and 0.5% (w/v) BSA (Fraction
V,Sigma) in PBS
, then incubated with a
fluorescein-conjugated sheep anti-rabbit IgG (Cappel) at 1:500 in
blocking solution for 60 min at 37 °C to identify the injected
cells. The cells were then washed three times with 0.2% (v/v) Tween 20 (Sigma) and 0.5% BSA in PBS
, one time with PBS
,
and the coverslips mounted on glass slides with a 17% (w/v) solution
of Airvol polyvinyl alcohol (Air Products & Chemicals, Inc., Allentown,
PA) and 33% (v/v) glycerol in PBS
. Immunofluorescence microscopy and
light microscopy, using a Zeiss Axiophot microscope, was used to
visualize the injected cells and nuclear
-gal staining,
respectively. Images were recorded using T-Max 400 film (Kodak). These
agonist concentrations and exposure times were found to produce maximal
induction of c-fos in pilot experiments. Percentage of
inhibition was calculated by first determining the number of
fluorescent (injected) cells that stained positive for
-gal divided
by the total number of injected cells. Next, the number of positive
staining buffer-injected cells were counted and divided by the total
number of cells injected. These two ratios were divided, multiplied by
100, and subtracted from 100 to arrive at the percent inhibition. In
pilot experiments, we determined that the number of buffer-injected
cells staining positive for
-gal was similar to the number of
uninjected cells staining positive. Also, the mechanical act of
injection by itself did not induce detectable c-fos
expression.
fos-lacZ
wild-type mouse fibroblasts were seeded at a density of 3 × 104 cells/well into individual wells of 24-well plates that
contained glass coverslips (12 mm diameter), and allowed to attach
overnight. The following day, the cells were transfected with either 1 µg of pSV2CAT or Gi2EEC>S DNA per well by the calcium
phosphate method. 15 h later the coverslips were washed briefly,
first with PBS
and then with PBS
containing 0.5 mM EDTA
and 0.5 mM EGTA, then incubated overnight in Dulbecco's
modified Eagle's medium containing 5% fetal calf serum. The next
morning the media was replaced with Dulbecco's modified Eagle's
medium containing 0.5% BSA and the cells incubated an additional
24-30 h to induce quiescence. The appropriate wells were treated with
200 ng/ml PTX (List Biological, Campbell, CA) for the final 16 h
of the starvation period. The cells were stimulated with 2.1 µM LPA for 110 min, then fixed and stained for
-gal as
described previously. Cells were then washed four times with PBS
,
incubated for 60 min at room temperature in blocking solution
consisting of 0.1% BSA/PBS
, and then incubated for 60 min at
37 °C with a 1:50 dilution of the EE monoclonal antibody or a 1:100
dilution of an anti-CAT Ab (5 Prime
3 Prime, Inc., Boulder, CO).
The coverslips were washed two times in wash buffer (0.2% Triton,
0.1% BSA, PBS
) and then incubated 60 min with a 1:750 dilution of
fluorescein isothiocyanate-conjugated goat anti-mouse IgG in the case
of EE, or a 1:100 dilution of a rhodamine-conjugated goat anti-rabbit
IgG to visualize the CAT protein. The coverslips were washed three
times in wash solution, one time in PBS
, then mounted as described
above. Positive transfectants on the coverslips were identified by
immunofluorescence microscopy, and scored as positive or negative for
-gal staining by light microscopy. The cells used in these
experiments had a higher percentage of cells staining positive for
-gal in the unstimulated state than those used in the microinjection
experiments, possibly because a quality transfection required a
relatively lower initial seeding density. The lower cell density and
the lack of cell-cell contact at the time of starvation may have
increased the time necessary to induce quiescencse. To verify that the
G
i2EEC>S protein expressed in these cells was resistant
to PTX treatment, ribosylated and non-ribosylated
subunits were
separated from each other by high resolution gel electrophoresis.
Briefly, 100-mm dishes of cells that were seeded at a density of
3.5 × 105 cells/dish were transfected with 20 µg of
DNA or treated with the transfection mixture alone, as described
previously. Some dishes were treated with 100 ng/ml PTX for 16-20 h
prior to harvesting the cells for membrane preparation, or,
alternatively, whole cell lysates were prepared by scraping the cells
directly into 2 × sample buffer. Membrane proteins or lysates
were resolved by high resolution urea SDS-PAGE, transferred to
nitrocellulose, and probed with antibodies to the epitope tag and/or
G
i2 as described above.
Although PTX has been shown to inhibit
agonist-stimulated activation of Ras and MAP kinase (11, 12), we first
verified that PTX exerted a similar inhibitory effect on LPA-stimulated induction of c-fos. As shown in Fig.
1a, a 24-h period of quiescence was
sufficient to eliminate detectable -gal staining in these fibroblasts; fewer than 1% of the cells showed positive staining at
the end of this time. The addition of 2.1 µM LPA for 110 min resulted in approximately 60-75% of the cells staining positive for
-gal (Fig. 1b). When the cells were pretreated for
16 h with PTX (100 ng/ml), the LPA-induced
-gal staining was
almost completely eliminated (Fig. 1d), although PTX by
itself had no effect on the
-gal staining in quiescent cells (Fig.
1c). These cells also stably expressed the human insulin
receptor and stimulation with 70 nM insulin for 110 min
resulted in positive
-gal staining in 75-85% of the cells (Fig.
1e). The insulin-stimulated induction of c-fos
was completely unaffected by PTX (Fig. 1f), and thus served
as a negative control for the microinjection experiments.
Identification of G
The subunits of the Gi and
Go family contain sites susceptible to ADP-ribosylation
catalyzed by PTX and are therefore candidates for mediating the
inhibitory effect of PTX on LPA-induced c-fos expression. To
determine which
subunits were present in the mouse fibroblasts used
in these studies, we used a panel of
i-specific antibodies to probe cell extracts by Western analysis. Antibody AS has
been shown to have equal specificity for
i1 and
i2. As shown in Fig. 2, this antibody
recognized a single protein in these cells (lane 1). Because
this band could represent
i1 or
i2, or
both, we utilized another antibody which is monospecific for
i1. This antibody failed to cross-react with any protein in equal amounts of fibroblast membranes (lane 4); however,
it did detect a protein in equal amounts of membranes prepared from bovine brain (lane 6). In addition, when as much as 100 µg
of membrane protein was separated using high resolution urea SDS-PAGE, which resolves G
i1 and G
i2, and the
subsequent blot probed with the AS antibody, only a single band was
detected (data not shown). Antibody EC has a high degree of specificity
for
i3, although to some extent it will react with
o. As shown in lane 2, a single band of
immunoreactivity was detected using this antibody. Antibody AR, which
recognizes only G
16, failed to detect any proteins in
mouse fibroblast membranes (lane 3). The
Go-selective antibody used did not recognize any protein in
the mouse fibroblasts (lane 5), while reacting strongly with
an equivalent amount of protein from bovine brain (lane
7).
Although Go and G
i1 were not detected by
Western analysis, in order to ensure that the lack of immunological
detection of these species was not due to its presence at levels less
than the limits of detection, we prepared RNA from these cells to
determine if there was detectable message for G
o and
G
i1. Using full-length coding cDNAs as probes, a
band of approximately 4.5 kb corresponding to G
o was
detected in both rat and mouse brain which we used as a positive
control, but not in the equivalent amount of RNA prepared from mouse
fibroblasts (data not shown). Similarly, the G
i1
cDNA hybridized to a message of approximately 3.5 kb in both rat
and mouse brain, but not mouse fibroblasts (data not shown). The size
of the message detected in both rat and mouse brain using the
G
o and G
i1 probes was consistent with a
previously published report using rat brain RNA (37). Therefore, the
combined evidence obtained by both Northern and Western analysis
suggests that mouse fibroblasts contain little, if any,
G
o and G
i1 mRNA or protein, and
suggest that the predominant
subunits present in the fibroblasts
used in these studies are G
i2 and
G
i3.
The mechanical act of scraping
adherent cells has been proposed to initiate a wounding response, which
includes the induction of c-fos (38, 39). However,
microinjection per se did not induce -gal activity in
this study (data not shown). Injections were made into the cytoplasm of
quiescent fibroblasts with the affinity-purified antibodies AS, EC, and
AR, described above. Each of the antibody solutions contained a pure
rabbit marker IgG as did a solution of injection buffer alone which
served as a standardizing control. As depicted in Fig.
3, panels a, c, e, g, and i show
injected cells that were detected by immunofluorescence staining. The
identical fields of cells are shown following stimulation with LPA or
insulin and subsequent
-gal staining, in panels b, d, f,
h, and j. Injection of antibody AS blocked LPA-induced
-gal activity in each of the three cells shown (panels a
and b); however, the same antibody had no inhibitory effect
on the response to 70 nM insulin (panels g
and h). Each of the representative cells shown that
were injected with antibody EC (panels c and d)
or AR (panels e and f) demonstrate positive
LPA-stimulated
-gal staining. Microinjection of a marker antibody
alone had no measurable effect on the LPA response (panels i
and j).
The overall inhibitory effect of each of the antibodies on LPA-induced
-gal activity was quantitated and is summarized in Table
I. Antibody AS decreased the number of positive
responses by an average of 57.5 ± 0.5% (±S.E.,
n = 3). Small but detectable decreases of about 19.1 ± 9.8% and 19.1 ± 9.9%, respectively, were observed for both
antibodies EC and AR. As can be seen in Figs. 1 and 3, the intensity of
-gal staining was somewhat variable among individual cells. Our
system of scoring positive cells, i.e. detection of any blue
staining in the nucleus is considered positive, may underestimate the
overall inhibitory capability of each of the antibodies, since no
possibility is allowed for partial inhibition of the response. These
data suggest that antibody AS, which recognizes the G
i2
protein present in these fibroblasts, is capable of functionally
inhibiting the LPA-stimulated activation of the G protein, and that
G
i2 is the predominant G protein species that mediates
LPA-stimulated c-fos induction in these cells.
|
Although the G protein
subunit antibodies used for microinjection showed reasonably good
specificity in recognizing their corresponding protein in
immunoblotting, we considered that there could be significant
nonspecific inhibitory effects of these antibodies when used in the
in vivo studies described here. In light of this possibility, we utilized an approach that has previously been used
successfully to examine PTX-sensitive signaling pathways in
vivo (22, 40, 41), and introduced PTX-resistant
subunits into
these cells by transfection, where the contribution of endogenous G
protein
subunits could be eliminated by treatment of the cells with
PTX. First, we attempted to create cell lines that stably overexpressed
the PTX-resistant mutants of G
i2 or G
i3, as well as their wild-type counterparts, with the reasoning that signaling in cells expressing these mutants could be systematically restored and assayed following treatment of the cells with PTX. Transient transfection of these constructs would yield only a small
population of cells that expressed the PTX-resistant protein, and would
not be amenable to the single cell assay system described here, because
the specific cells that expressed the protein could not be identified.
One hundred clones resistant to hygromycin were expanded and assayed
for overexpression by Western blot analysis following separation of
membrane proteins using high resolution urea SDS-PAGE. A single
PTX-resistant G
i3 clone (designated 12B/48) demonstrated
elevated levels of immunoreactivity to the G
i3 antibody,
EC, compared to the wild-type cells (Fig. 4A, upper band in each lane). The high resolution gel system
separated the 41-kDa G
i3 and the 40-kDa
G
i2 subunits; and as shown in Fig. 4A,
antibody EC shows considerable cross-reactivity with the
i2 species. This cross-reactivity, although stronger
than expected, nonetheless provided a convenient internal control
demonstrating that an equivalent amount of membrane protein was loaded
in each lane, based on similar levels of
i2
immunoreactivity. Additional blots with this clone using the
G
i1/2-specific AS antibody following separation on high
resolution gels, confirmed that the levels of G
i2 in
this clone were similar to those in control cells (data not shown).
Experiments using this stable PTX-resistant cell line were then carried
out to determine if the G
i3 species of
subunit could
restore LPA-induction of c-fos in cells that were pretreated
overnight with PTX; however, LPA did not induce c-fos in
these cells (data not shown). We were unsuccessful in isolating a clone
that overexpressed either the wild-type or the PTX-resistant mutant of
the G
i2 protein. This suggests that the levels of
expression of this protein may be very highly regulated in the cells
used in this study.
Based on the above finding, that the PTX-resistant form of
Gi3 did not restore the LPA induction of
c-fos, and given the inability to test the PTX-resistant
form of G
i2 using stable expression, we devised a
transient transfection protocol that took advantage of earlier work
(31) whereby a monoclonal antibody epitope was introduced into an
internal site of the PTX-resistant form of G
i2. This
approach allowed us to transfect and analyze a population of cells in
which transfected G
i2 could be identified against a
cellular background of endogenous G proteins. Epitope-tagged G
i2C>S was readily detected in lysates prepared from
transfected cells by immunoblotting using the EE antibody (Fig.
4B, lane 1), and migrated at the same position as a protein
detected by blotting with the AS antibody, which reacts with
G
i2 in these cells (Fig. 4B, lane 3). The EE
antibody was highly specific for the EE epitope; the antibody did not
react with any protein in an equivalent amount of lysate from wild-type
cells (Fig. 4B, lane 2). To confirm that the transfected
construct resulted in the expression of a protein that was resistant to
PTX in these cells, we utilized high resolution urea SDS-PAGE to
separate ribosylated G
i2 from unmodified
G
i2. PTX-catalyzed ADP-ribosylation results in a reduced
migration rate of G
i2 through this gel system, as is
demonstrated in Fig. 4C. In membranes prepared from untransfected PTX-treated cells, a single band of slower migrating immunoreactivity is detected (lane 2) compared to
untransfected cells that were not exposed to PTX (lane 1).
In contrast, in an equal amount of membrane protein prepared from
transfected cells, a single band appears in the lane prepared from
non-PTX-treated cells (lane 3), while PTX treatment results
in two bands of immunoreactivity (lane 4); the upper band
corresponds to endogenous ribosylated G
i2, and the
lower, faster migrating band corresponds to the PTX-resistant form of
G
i2. These results also demonstrate that introduction of
the EE epitope into the G
i2 cDNA did not interfere
with membrane localization of the expressed protein.
To investigate whether G protein heterotrimers containing only
Gi2 are capable of coupling the LPA receptor to the
induction of c-fos, we conducted experiments on
coverslip-plated cells that were transiently transfected with either
the epitope-tagged and PTX-resistant G
i2 construct or
with a CAT plasmid. Following transfection, the cells were starved in
the absence or presence of PTX and then stimulated with LPA. At each
condition tested, the coverslips were examined by immunofluorescence
microscopy following staining with an antibody to the EE epitope or to
CAT (Fig. 5A), to visualize positive
transfected cells. Each transfected cell was also examined by light
microscopy to determine whether c-fos had been induced. We
used the expressed CAT protein as a marker that enabled us, in parallel
experiments, to quantitate the extent of PTX-mediated inhibition of
LPA-stimulated c-fos induction that would be expected in the
absence of the putative rescue DNA. In Fig. 5A, panels o and
p demonstrate the ability of the PTX-resistant mutant of
Gi2 to restore the LPA-induction of c-fos in
cells pretreated with PTX. In contrast, panels g and h demonstrate that LPA-stimulated c-fos induction
remained inhibited in CAT expressing cells that were pretreated with
PTX. Representative fields of cells at each condition tested are shown
in Fig. 5A. As summarized in Fig. 5B, LPA
increased the percentage of cells staining positive for
-gal
2.6-fold (from 18 to 47%) in CAT transfected cells, and as expected,
the stimulation due to LPA was significantly attenuated in cells that
were pretreated with PTX (16% positive in the absence of LPA
versus 20.4% positive after treatment with LPA). In
contrast, in cells expressing the PTX-resistant mutant of
G
i2, PTX pretreatment had relatively little effect on the percentage of cells staining positive for
-gal when stimulated with LPA (2.1-fold increase in the absence of PTX versus
1.9-fold in the presence of PTX).
In this study we investigated the functional interaction of the
LPA receptor with G proteins in intact mouse fibroblasts. Because
LPA-induced mitogenesis in mouse fibroblasts can be blocked by PTX, we
undertook this investigation to determine which of the PTX-sensitive G
protein subtypes was involved in this response. Western blotting
revealed that two PTX-sensitive G protein subunits, G
i2 and G
i3, were present in membranes
prepared from the mouse fibroblasts used in this study. These data are
consistent with those reported for Balb3T3 mouse fibroblasts (26). The absence of message for other PTX-sensitive G proteins, namely G
i1 and G
o, was confirmed by Northern
analysis.
Microinjection of specific G protein subunit antibodies suggested
that the LPA receptor selectively activated G
i2, based
on the approximate 60% inhibition of LPA-stimulated c-fos induction seen following microinjection of a G
i2
specific antibody. In light of the ability of PTX to almost completely abolish LPA-stimulated c-fos induction, the reason for the
incomplete ablation of c-fos induction in all of the cells
injected with the antibody is not clear. It is conceivable that
variable amounts of antibody were delivered to individual cells, and
that a small number of unbound G
i2 molecules would be
capable of eliciting a detectable response. Another possible
explanation is related to the experimental protocol used in this study.
The time lag between the injection of antibody into the cells and the
subsequent addition of agonist was somewhat variable, i.e.
cells injected first were exposed to antibody for up to 40 min while
those injected last were exposed for 10 min. The injected antibody may
require longer than 10 min to bind the G protein with full saturation. Levels of inhibition greater than that seen with the G
i2 antibody are possible in these cells; for example, injection of an
inhibitory Ras antibody blocked both LPA- and insulin-stimulated c-fos expression by approximately
80%.3 The inability of the
G
i2 antibody to block insulin-induced c-fos
expression serves as a negative control, since PTX is generally ineffective at blocking insulin-stimulated c-fos induction
and/or mitogenesis (present data and Refs. 42 and 43). We were
unsuccessful in our attempt to restore the LPA response by
microinjecting purified GDP-bound G
i2 or
G
i3 protein into PTX-treated wild-type cells. This
approach may have been unsuccessful because the subunits could have
been modified by PTX immediately upon delivery to the cell, or possibly
because the protein was not localized to the proper site within the
cell or was not present in sufficient amounts.
We cannot rule out the possibility that there could be appreciable
nonspecific effects of the antibodies when used in the microinjection
experiments. Indeed, our results using high resolution gel
electrophoresis to separate Gi2 and G
i3
indicate that antibody EC detects not only G
i3, but also
G
i2, by immunoblotting, as has been described (25).
Therefore, it is possible that part or all of the 19% inhibition of
LPA-stimulated c-fos we observed with antibody EC may
reflect an effect on receptor-G
i2 coupling, rather than
a general nonspecific effect on the signaling pathway. Results from
other studies suggest that antibody EC is capable of inhibiting
G
i3-mediated signaling. For example, antibody EC
inhibited 5-hydroxytryptamine1A-stimulated
phospholipase C activity and attenuated receptor-mediated inhibition of
adenylyl cyclase to a much greater extent than did the AS antibody in
HeLa cells, which contain at least 10 times more G
i3
protein than G
i2 (44, 45). In Chinese hamster ovary
cells, antibody AS and antibody EC partially inhibited, to a similar
extent,
2-adrenergic-mediated inhibition of adenylyl
cyclase, and in combination completely reversed the inhibition (46).
Despite these observations, it may remain difficult to unequivocally
assess the relative roles of the Gi proteins in mediating
specific signaling pathways by the use of antibodies, especially in
cells that contain more than one Gi species.
Given the potential limitation of the antibody approach, we conducted
experiments in cells that either stably or transiently expressed
PTX-resistant Gi3 or G
i2 proteins, and
examined whether expression of either of these mutant proteins restored LPA signaling following pretreatment of the cells with PTX, which eliminated the contribution of endogenous G protein
subunits. The
PTX-resistant proteins lack the critical carboxyl-terminal cysteine
required for modification catalyzed by PTX, and have been demonstrated
to remain functional in cells treated with PTX (22, 40, 41). Our
studies revealed that G protein heterotrimers containing only mutant
G
i2 protein were sufficient to restore the activation of
c-fos by LPA, and that expression of mutant G
i3 did not. The PTX-resistant G
i3
cDNA that was overexpressed in these cells encodes a functional
protein, because in separate experiments, transient transfection of
this construct restored 5-hydroxytryptamine1A-stimulated
Na/H exchange in fibroblasts pretreated with
PTX.4
LaMorte et al. (26) have demonstrated that pretreatment of
Balb3T3 cells with PTX decreased serum-stimulated DNA synthesis by
approximately 30%. They proposed that Gi2 mediates
serum-stimulated mitogenesis, since an antibody specific for
G
i2, when microinjected into these cells, decreased
serum-stimulated DNA synthesis by 37%. Total inhibition of
serum-stimulated DNA synthesis would be somewhat surprising, since
other ligands in serum, such as platelet-derived growth factor, can
activate the MAP kinase pathway without the mediation of G proteins
(47). However, the inhibitory effects seen by LaMorte et al.
(26) using either PTX or G
i2 antibody may also reflect
inhibition of the LPA effect, since LPA is a component of serum.
Previous data have suggested that LPA-stimulated mitogenesis is
independent of the PTX-insensitive Gq-mediated calcium
mobilization (3). The simplest explanation, based on the results of
this study, is that Gi2 is the only G protein that is
necessary for restoration of c-fos inducibility, although
our studies were not designed to test the contribution of
non-PTX-sensitive G proteins. These results may be in contrast to
thrombin-stimulated DNA synthesis in fibroblasts, which requires both
G
i2 and G
q (48).
The functional strategy used here to examine receptor-G protein
coupling relied on the expression of c-fos, a nuclear
protein. Previous reports have proposed a role for Gi or
Gi-like proteins in post-receptor events, such as mitosis,
based on the ability of insulin or epidermal growth factor to induce a
redistribution of G subunits from the plasma membrane to perinuclear
sites (49), or based on the observation that G protein
subunits
have been detected in the nuclei of certain cells (50). We believe that the mechanism studied here, in which G protein antibodies inhibited signaling, was due to an inability of the receptor to activate its
cognate G protein, because the antibodies used were raised against
COOH-terminal peptides located in a region of the G protein thought to
interact with receptors (27, 28). Likewise, expression of defined
PTX-resistant
subunits restored signaling, presumably because the
subunit recombines with free
subunits in the membrane, and
interacts with the receptor as the holoprotein. It was on the basis of
this proposed mechanism that we conducted our immunoblotting studies
with membrane protein, and disregarded cytoplasmic and nuclear
proteins. However, epitope-tagged G proteins like the one used here may
be important reagents for examining possible alternative or additional
mechanisms of action for G proteins in mitogenesis.
The principal finding of this study, that LPA interacts selectively
with Gi2 to induce c-fos, is supported by the
following additional evidence: 1) mutationally activated
i2 mimics LPA-mediated inhibition of forskolin- or
prostaglandin E1-stimulated adenylate cyclase (16), and 2)
expression of GTPase-inhibited
i2 alters normal growth
control in some cell types but not in others (17, 18).
G
i2 also couples several other well known receptors to
downstream effector molecules. For example, Gi2 has been
reported to mediate
2-adrenergic inhibition of adenylyl
cyclase in platelet membranes (25) and Rat-1 fibroblasts (51). In
contrast, not all receptors sensitive to PTX proceed through
G
i2. For example, G
i3 can couple
angiotensin II receptors to inhibition of adenylyl cyclase in rat
hepatocytes (52), and also regulates multiple effector enzymes in
Chinese hamster ovary cells stimulated by m2-muscarinic agonists (22).
Recent experiments have revealed that the principal stimulatory agent
of MAP kinase by G protein-coupled receptors is the heterodimer
(53, 54, 55). To determine whether the
complex was involved in the
pathway studied here, we microinjected a
-sequestering peptide
into cells and assessed the effect on LPA-stimulated induction of
c-fos. We were unable to detect any inhibitory effect of the
binding peptide on LPA-stimulated c-fos expression.
Possible explanations for this result are that we were not able to
completely titrate the activating molecules with the protein, or that
LPA induction of c-fos is not mediated by the
subunits, but perhaps by G
i or some other unknown mechanism. In support of this possibility are results from experiments in Rat-1 cells in which the activated
i2 subunit itself
has been shown to induce neoplastic transformation (17) and
constitutively activate the MAP kinase cascade (56). These findings may
reveal differing regulatory mechanisms utilized by different cells used to control proliferation.
In summary, we have demonstrated here by two complementary approaches
that of the PTX-sensitive G protein subunits, heterotrimers containing only the
i2 subunit are sufficient for the
activation of an LPA-stimulated signaling pathway which results in the
transcription of the c-fos gene. Although several reports
which are based on the results of experiments using broken cell
preparations have raised questions about the assumption that different
subunits mediate specific signaling processes (57, 58, 59), the results from this study support the proposal that specific G protein subtypes mediate specific signaling events.
We thank all members of the Blackshear
laboratory for helpful discussions, and Drs. A. Spiegel for providing G
protein antibodies, T. Curran for fos-lacZ mouse
fibroblasts, R. Roth for the human insulin receptor cDNA, H. Bourne
and P. Wilson for the Gi2EEC>S cDNA, E. Peralta and
T. Hunt for G
i2C>S and G
i3C>S cDNA, S. Graber for purified G
i2 and G
i3, W. Koch for providing the
binding domain peptide of the
-adrenergic receptor kinase and for bovine brain membranes, and P. Casey for the G
o and G
i1 plasmids and for
helpful discussions.