(Received for publication, September 21, 1995; and in revised form, January 17, 1996)
From the
The edg-1 gene encodes an inducible G protein-coupled
receptor (GPR) homologue that is induced during the in vitro differentiation of human endothelial cells. The aim of this study
was to investigate the G protein-coupling and -signaling properties of
the edg-1 polypeptide. The third cytosolic loop
(i) of edg-1 associates with G
and G
polypeptides in a guanosine
5`-O-(thiotriphosphate)-sensitive manner. Immunoprecipitation
of the edg-1 polypeptide in transfected cells results in the
co-precipitation of G
and G
polypeptides. These data strongly suggest that edg-1 is
capable of coupling to the G
pathway. Overexpression of the edg-1 GPR in human embryonic kidney 293 cells results in the
sustained activation of the MAP kinase activity that is blocked by
pertussis toxin treatment. Moreover, NIH3T3 cells permanently
transfected with edg-1 exhibit enhanced MAP kinase and
phospholipase A
activities. These data suggest that the
G
/mitogen-activated protein kinase pathway is a major
signaling pathway regulated by the orphan receptor edg-1.
Angiogenesis, new blood vessel formation, is a critical
component of many physiological processes such as wound healing and
development. Uncontrolled angiogenesis is associated with numerous
pathological conditions, including diabetic retinopathy, rheumatoid
arthritis, and solid tumor growth(1) . The process of
angiogenesis is initiated by vascular endothelial cells and involves
their orderly migration, proliferation, and differentiation into new
capillary channels. Cultured human umbilical vein endothelial cells
(HUVEC) ()exhibit many of the characteristics of endothelium in vivo and is a widely used in vitro model system to
study the molecular mechanisms of angiogenesis. For example, HUVEC
proliferate in response to angiogenic mitogens, such as fibroblast
growth factor-1 (FGF-1) (2) and vascular endothelial growth
factor(3) , and are growth-arrested by cytokines such as tumor
necrosis factor-
, transforming growth factor-
, as well as
phorbol 12-myristate 13-acetate (PMA)(4, 5) .
Moreover, prolonged treatment of HUVEC with PMA in three-dimensional
collagen or fibrin gels results in the formation of differentiated
capillary-like tubular structures(5, 6, 7) .
To study the early transcriptional events in angiogenesis, we have
cloned several immediate-early genes induced by PMA in
HUVEC(8, 9, 10, 11) . One such gene,
termed endothelial differentiation gene-1 (edg-1), encodes a G
protein-coupled receptor (GPR) homologue(8) . Given that PMA
causes HUVEC to differentiate phenotypically and since immediate-early
genes have been shown to play critical roles in the control of growth
and differentiation, we proposed that edg-1 may be
functionally involved in endothelial cell differentiation(8) .
Recent studies have shown that the edg-1 transcript is also
induced during endochondral ossification which occurs in the developing
skeletal system. ()Mesenchymal cells that are in the process
of differentiating into osteoblasts express high levels of the edg-1 transcript.
The expression patterns and the
promoter structure of the edg-1 gene is similar to the
SPARC/osteonectin gene, which was originally identified from the
activated endothelial cells(13) . These data suggest that the edg-1 GPR is induced during the differentiation of multiple
cell types and suggest that it may regulate signaling events in the
developing cardiovascular system and the skeletal system.
An
important step in the characterization of a GPR is to identify the
associated G protein(s). Such information may help reveal the
downstream signaling pathways. Three approaches have been used to
determine which G protein subtypes can couple to a specific GPR. Kurose et al. (14) reconstituted purified -
and
-adrenergic receptors with recombinant
G
subunits in phospholipid vesicles and measured the
ability of agonists to stimulate GTPase activity. Second, specific
G
subunits have been immunoprecipitated from cellular
extracts and the presence of co-precipitated GPR was measured by ligand
binding assays(15) . Third, co-transfection of receptors and
specific G protein subunit cDNAs into receptor negative cells followed
by the measurement of ligand-activated second messenger pathways have
been used(16) . The principal requirement of each of these
approaches is the knowledge of agonists for the GPRs. However, in the
case of edg-1, the ligands or agonists/antagonists are unknown
at present. Thus, edg-1 is an example of a putative GPR, based
solely on sequence similarity with the known GPR superfamily. We
therefore explored other methodologies for examining the signaling
properties of edg-1.
Previous studies have shown that the
third intracellular loop (i) of GPRs are important for G
protein interaction and signal transduction (reviewed in (17) ). Moreover, structural characteristics of G
contact sites on the receptors have been predicted to form
amphipathic
-helices(17) . The i
domain of edg-1 fits this structural model as determined by Helicalwheel
analysis(18) . Therefore, we constructed a fusion protein of
i
and glutathione S-transferase
(GST-i
) and utilized it as an affinity matrix to study G
protein interaction in vitro. In this report, we show that
G
and G
associates with the
GST-i
fusion protein in vitro. Furthermore, we
demonstrate that intact edg-1 binds to G
and
signals via the G
pathway to regulate cellular MAP kinase
activity.
To perform the in vitro binding assay, 750 µg of HUVEC
extracts were incubated with GST fusion protein beads (approximately 10
µg of GST-i fusion protein bound to 165 µl of 75%
glutathione-Sepharose slurry) for 2 h at 4 °C. Beads were then
washed five times with phosphate-buffered saline. Subsequently, bound
G
subunits were detected by
[
P]ADP-ribosylation with pertussis or cholera
toxins. In vitro ADP-ribosylation reactions were carried out
essentially as described(20) . The reactions were terminated by
the addition of 2
SDS sample buffer (4.6% (w/v) SDS, 10% (v/v)
-mercaptoethanol, 20% (w/v) glycerol, 95.2 mM Tris-HCl,
pH 6.8, 0.01% (w/v) bromphenol blue). The
[
P]ADP-labeled proteins were then subjected to
SDS-PAGE on 10% polyacrylamide gels, and autoradiographed.
For
Western analysis, G protein complexes bound to GST-i- or
GST-loaded glutathione-Sepharose 4B beads were separated by SDS-PAGE on
10% polyacrylamide gels. The electrophoretically separated proteins
were then transferred to nitrocellulose paper, blocked with blotto (5%
nonfat dried milk, 50 mM Tris-HCl (pH 8.0), 2 mM CaCl
, 80 mM NaCl, 0.02% NaN
, and
0.2% Nonidet P-40) for 1 h at room temperature with gentle shaking,
followed by incubation for 1.5 h with
anti-G
/G
,
anti-G
/G
(1:500;
Calbiochem-Novabiochem), or G
subtype selective
antibodies (generous gifts of Dr. David Manning, University of
Pennsylvania). After three washes, blots were incubated with
horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin
(1:2000, Cappel, Westchester, PA) and visualized by the
luminescence-based ECL method (Amersham Corp.).
The yeast strain Y190 was simultaneously
transformed with Gal4-i and Act D-G
plasmids using the lithium acetate procedure(22) . After
3 days of growth, cells were lifted with nylon membranes and assayed
for
-galactosidase activity as described elsewhere(23) .
-Galactosidase activity was quantitated using the
luminescence-based assay (Tropix Inc.).
Figure 1:
Pertussis
toxin substrates associate with GST-iin vitro. A, schematic diagram of GST-i
fusion protein and
GST control proteins are shown. B, the GST-i
fusion protein was purified by affinity chromatography on
glutathione-Sepharose. Coomassie Blue staining of purified GST-i
(32 kDa) and GST (28 kDa) is shown. C, HUVEC extracts
were either directly ADP-ribosylated (lanes 1 -3), or
affinity-isolated with GST control protein (lanes 4-6)
or GST-i
(lanes 7-9) followed by
ADP-ribosylation as described under ``Experimental
Procedures.'' Autoradiographs of protein complexes subjected to
[
P]ADP-ribosylation in the absence of toxin (lanes 1, 4, and 7), or presence of
pertussis toxin (PTX) (lanes 2, 5, and 8) or cholera toxin (CTX) (lanes 3, 6, and 9) are shown. These results are representative
of two independent experiments.
Figure 2:
Association of G and
G
with GST-i
in vitro is
GTP
S-sensitive. A, HUVEC extracts were incubated with
glutathione-Sepharose bound GST or GST-i
, followed by
immunoblotting with anti-G
/G
(left panel) or anti-G
/G
(right panel). The position of molecular weight makers
is shown. B, HUVEC extracts were treated with (lanes 2 and 4) or without (lanes 1 and 3) 50
µM GTP
S followed by incubation with
glutathione-Sepharose bound GST-i
. The G
associated with GST-i
was then detected by
immunoblotting with anti-G
/G
(lanes 1 and 2) or
anti-G
/G
(lanes 3 and 4). Data are representative of three independent
experiments.
To
determine the G protein specificity of GST-i, specific
G
subunits were expressed in HEK293 cells by transient
transfection. Cell extracts were prepared and analyzed for their
ability to bind to GST-i
. As shown in Fig. 3,
immunoblot analysis with subunit-specific antisera show that all three
G
subunits as well as the G
subunit
associated with the GST-i
fusion protein but not with the
GST control protein. These data suggest that the i
domain
of edg-1 is capable of binding to the G
family and
G
polypeptides in vitro.
Figure 3:
Interaction of G and
G
with GST-i
in vitro.
G
, G
, G
, and
G
subunits were overexpressed in HEK293 cells by
transient transfection. Cell extracts were affinity-isolated with
glutathione-Sepharose bound GST-i
or GST polypeptides, and
protein complexes were immunoblotted with subtype-specific G
antisera. First panel, Immunoblot of
G
-transfected cells with anti-G
antiseum; second panel, immunoblot of
G
-transfected cells with anti-G
; third panel, immunoblot of G
-transfected
cells with anti-G
; and fourth panel,
immunoblot of G
-transfected cells with
anti-G
. From left, 40 µg of untransfected (lane 1) or G
-transfected (lane 2)
extracts. Lane 3, GST-i
-bound untransfected
extracts; lane 4, GST-bound G
-transfected
extracts; and lane 5, GST-i
-bound
G
-transfected extracts.
Figure 4:
Co-immunoprecipitation of epitope-tagged
full-length edg-1 with G and
G
. HEK293 cells were transiently transfected with the
FLAG epitope-tagged full-length edg-1 and individual
G
cDNAs and cellular extracts were prepared as
described under ``Experimental Procedures.'' The expression
of transfected polypeptides were detected by Western blot analysis with
the M2 antibody (FLAG-edg-1),
G
/G
and
G
/G
antisera. B, cellular
extracts from co-transfected HEK293 cells were immunoprecipitated with
anti-M2 antibody and the G
subunits present in the
immunoprecipitates were detected by Western blot analysis with
G
/G
or
G
/G
antisera in respective lanes.
These results are representative of two independent
experiments.
Cultured HEK293 cells were transiently transfected with edg-1. MAP kinase activity was assayed by an in-gel kinase
assay of extracellular signal regulated kinase-2 (ERK-2)
immunoprecipitates. As shown in Fig. 5A,
immunoprecipitation of HEK293 cells with the ERK-2 antiserum followed
by in-gel kinase assay in MBP-impregnated gels detected a specific band
at 42 kDa. The kinase activity of this band is strongly (approximately
6-fold) induced by treatment with PMA for 15 min (data not shown). This
result indicates that the 42-kDa band is ERK-2. Transient transfection
of the edg-1 expression vector into HEK293 cells, in a
dose-dependent manner, induced the ERK-2 kinase activity (Fig. 5B). The ERK-2 MAP kinase activity was induced at
30-60 h post-transfection and thus appears to be of sustained
kinetics. Pertussis toxin treatment blocked the edg-1-dependent MAP kinase activation. The inhibition of ERK-2
activation in edg-1-transfected cells by pertussis toxin is
not due to the inhibition of transfected gene expression as indicated
in the M2 immunoblots. These data indicate that edg-1 signaling via the G pathway is responsible for
sustained activation of the MAP kinase pathway.
Figure 5: In-gel kinase assay of ERK-2 in edg-1-transfected HEK293 cells. HEK293 cells were transiently transfected with pcDNANeo or pDNFedg-1 (0.2 and 0.8 µg) for 30 and 60 h. At 12 h prior to harvest, cells were incubated with DMEM containing 0.5% FBS in the presence or absence of 100 ng/ml pertussis toxin (PTX). Cell extracts were prepared, immunoprecipitated with anti-ERK-2 antibody and analyzed for MAP kinase activity in MBP-impregnated gels as described under ``Experimental Procedures.'' A, autoradiogram of the MBP-impregnated gel. Cell extracts were also assayed for the expression of the edg-1 polypeptide by immunoblot analysis with the M2 antibody (lower panel). B, the autoradiograms were quantitated by a densitometry. Values shown represent mean ± S.E. from three separate experiments.
To further confirm
the in-gel kinase assay, the edg-1 expression vector and the
hemagglutinin epitope (HA)-tagged ERK-2 cDNA were co-transfected into
HEK293 cells, and MAP kinase activity of the HA immunoprecipitates was
assayed. As shown in Fig. 6, edg-1 transfection
activated the MAP kinase activity. In addition, the edg-1-induced activation of the HA-ERK-2 was suppressed by
pertussis toxin. These data indicate that edg-1 signals via
the G pathway to induce the ERK-2/MAP kinase activity.
Figure 6: Immune complex kinase assay of ERK-2 in edg-1-transfected HEK293 cells. HEK293 cells were transiently transfected with pcDNANeo or pDNFedg-1 (0.2 and 0.8 µg) along with HA-ERK2 cDNA (0.2 µg) for 30 and 60 h, respectively. At 12 h prior to harvest, cultures were incubated with DMEM containing 0.5% FBS in the presence or absence of 100 ng/ml pertussis toxin (PTX). Cell extracts were prepared, immunoprecipitated with HA antibody, followed by immune complex kinase assay. The autoradiogram of MBP phosphorylated by anti-HA immunoprecipitates is shown in A. The migration positions of molecular weight markers as well as MBP are indicated. The autoradiogram is representative of three independent experiments with similar results. B, the phosphorylation of MBP was quantitated by densitometry. Values shown represent mean ± S.E. from three separate experiments.
Figure 7:
Enhanced MAP kinase and phospholipase
A activities in NIH3T3 cells stably transfected with edg-1. A, NIH3T3 cell clones transfected with human edg-1 cDNA or vector alone were isolated and the expression of
the transfected cDNA was assessed by Northern blot analysis. E, edg-1 clone; M, pMexNeo vector clone. B, activation of MAP kinase in stably transfected NIH3T3
cells. Extracts from serum-starved cells were prepared,
immunoprecipitated with anti-ERK-2 antibody, and the immune complex
kinase assay using MBP as substrate was conducted. Phosphorylated MBP
was qunatitated by densitometry. Values shown represent the mean
± S.E. from two separate experiments. C, activation of
phospholipase A
activity in stably transfected NIH3T3
cells. Assay of phospholipase A
activity was carried out by
measuring arachidonic acid release from prelabeled cells as described.
Data represent mean ± S.E. of triplicate values from a
representative experiment that was repeated three
times.
The process of angiogenesis is initiated by vascular endothelial cells and involves their orderly migration, proliferation and differentiation into new capillary channels(1) . While considerable efforts have been focused on the study of proliferative events, little is known about the nonproliferative aspects of angiogenesis. Since (i) HUVEC exhibit many of the characteristics of endothelium in vivo and (ii) prolonged treatment of HUVEC grown on collagen or fibrin gels with PMA results in growth arrest and the formation of differentiated capillary-like tubular structures(5, 6) , PMA-induced early response genes may play critical roles in the nonproliferative aspects of angiogenesis. The edg-1 transcript was isolated as a PMA-inducible early response gene from HUVEC(8) . The cDNA encodes a polypeptide with 382 amino acids that possess many structural features of a GPR(8) . Sequence alignment of edg-1 with other GPRs indicate that it belongs to the GPR family group I(38) . It is most closely related to other orphan receptors AGR-16 (39) and rat orphan GPR H218(40) , which were isolated by homology cloning of receptors expressed in the cardiovascular system and the brain, respectively. This subfamily of orphan receptors are closely related in structure to the cannabinoid and melanocortin receptor subfamilies(41, 42) . However, the ligands that are capable of interacting with the edg-1 GPR are unknown at present. The cloning of edg-1, however, raises several questions, such as (i) what is the ligand for edg-1, (ii) what signaling pathways are regulated by edg-1, and (iii) how the induction of edg-1 expression relates to angiogenesis.
Determination of the function of orphan receptors has been onerous due to the intrinsic difficulty in the identification of ligands. For example, the mas oncogene, which was isolated due to its transforming capacity, is still uncharacterized with respect to ligands and signaling properties(43) . Due to the advent of homology cloning by PCR, many orphan receptor sequences have become available. However, ligands for only a few of these orphan receptors have been found to date, for example, the cannabinoid and orphanin receptors(42, 44) . In this report, we present a novel approach to define the signaling properties of the orphan receptor edg-1.
Structure-function analysis of GPRs has defined
several features of the GPRs that are essential for specificity in
signaling(17) . While the structure of the i domain
plays a major role in determining the specificity of G protein
coupling, other intracellular loops (e.g. the C-terminal
portion of intercellular loop 2 and the C-terminal tail) also
contribute to the specificity(30, 31) . For example,
the intercellular loop 2, i
, and C-terminal domains of
rhodopsin are able to interact with transducin independently (32) . The i
domain of edg-1 is only 34
residues in length and contains the G
-activator motif
(BBXXF) as well as other potential regulatory
sites(45) . We expressed the i
domain of edg-1 as a C-terminal GST fusion protein and used it as an affinity
matrix to characterize G proteins that interact with it. Toxin labeling
studies clearly indicate that the pertussis toxin substrates
(G
and G
) but not the cholera toxin
substrates (G
) are capable of associating with the edg-1-i
. While we have not ruled out association
of other G proteins (e.g. G
, G
,
G
, and G
) with i
, our data
clearly show that the G
and G
family
of proteins bind to edg-1-i
. The interaction
between G
and edg-1-i
in this in vitro binding assay is highly specific, since (i) the
G
subunits are unable to interact with GST protein and
(ii) the interaction was suppressed by GTP
S. In addition, these
data also suggested that the i
domain alone in the
C-terminal context of the soluble GST fusion polypeptide is of
sufficient affinity and specificity to physically associate with the G
protein
subunits.
While the in vitro association
experiments proved that the GST-i and the G
associates specifically, they do not, however, demonstrate direct
interaction. Thus, the yeast two-hybrid system was used to demonstrate
direct physical interaction between the edg-1-i
domain and the G
polypeptide. The i
domain was expressed as a C-terminal fusion protein with the DNA
binding domain of the transcription factor GAL4 (GAL4-i
)
and the N-terminal deleted form of the rat G
polypeptide was expressed as a C-terminal fusion with the
transactivation domain of the GAL4 protein (Act D-G
).
Interaction of two proteins results in the transactivation of the
GAL4-LacZ reporter gene in the host Y190 cells (21) . While the
GAL4-i
alone and Act D-G
alone did not
transactivate the LacZ reporter gene, co-expression of both plasmids
strongly transactivated the LacZ expression, suggesting that the edg-1-i
and the G
polypeptides
interact directly. The two-hybrid system has been used previously to
demonstrate direct interaction between the G protein subunits and the
downstream kinases in the yeast mating pathway(46) . To our
knowledge, this is the first demonstration of interaction of a
signaling domain of the GPR with an
subunit of a G protein. Due
to the relative ease of the readout, this system holds promise as a
genetic method to delineate sequences involved in the receptor/G
protein interaction.
Chimeric receptor studies have indicates that
multiple cytosolic domains of GPRs cooperate to determine specificity
in G protein coupling. For example, when the i domain of
the G
-coupled
-adrenergic receptor was
inserted into the corresponding region of the G
-coupled M1
muscarinic receptor, the resulting chimeric receptor stimulated both
G
and G
pathways(47) . Thus, the
i
domain alone is not sufficient to switch the G
protein-coupling characteristics. Further chimeric studies have shown
that replacement of all the cytosolic loops is sufficient to convert
the G protein-coupling
specificity(30, 31, 32) . While the i
domain of edg-1 binds to G
and
G
, it is important to establish which G proteins are
capable of coupling to intact edg-1 GPR. Thus, the
epitope-tagged full-length edg-1 polypeptide was co-expressed
with the individual G
and G
polypeptides in
HEK293 cells. In this system, the edg-1 polypeptide associated
with G
and G
. The association of
G
and G
with full-length edg-1 was not observed in transiently transfected HEK293 cells. Thus,
multiple domains in the cytosolic loops of edg-1 are likely to
be involved in determining the specificity in coupling to G
and G
polypeptides. These data demonstrate that edg-1 is a G
-linked receptor.
Recent data have
supported the two-state model of receptor function; in this model
receptors are in equilibrium between active (R*) and inactive (R)
conformations(33) . Thus, overexpression of GPRs should
increase the concentration of the R* state and thereby lead to
constitutive signaling(34, 35) . Recent studies from in vitro transient transfection studies with orphan receptors (48) and in vivo transgenic studies with the
adrenergic receptors (34, 35) support this model.
Since edg-1 is an inducible receptor, regulation of signaling
pathways by modulation of receptor numbers may be of physiological
significance. Our data suggest that the fraction of the overexpressed edg-1 receptors that are in the activated (R*) state are
capable of coupling to the G and G
polypeptides for productive signaling. The G
family
of polypeptides are known to activate a number of cellular signal
transduction pathways such as inhibition of the adenylate cyclase,
activation of phospholipase A
(37, 49) ,
induction of MAP kinase activity (36) and the activation of ion
channels(50) . We thus examined the activity of basal and
forskolin-induced cAMP levels and the basal release of
[
H]arachidonic acid release (phospholipase
A
activity) from HEK293 cells transfected with edg-1. Both the cAMP and phospholipase activities were not
altered by overexpression of edg-1 with and without the
G
and G
polypeptides (data not shown). In
contrast, cellular MAP kinase activity was strongly induced by edg-1 transfection and is attenuated by pertussis toxin. It is
unclear why the cellular cAMP levels and phospholipase activities are
unaltered by these treatments in HEK293 cells. It is possible that edg-1 regulates the MAP kinase activity in a constitutive
manner and that regulation of other pathways require the presence of
the ligand(s). Alternatively, appropriate effector isoenzymes that
respond to the edg-1 signal may not be present in HEK293
cells. Nevertheless, these data do confirm that edg-1-dependent MAP kinase activation occurs via the
G
pathway in HEK293 cells. Interestingly, the MAP
kinase activity is induced in a sustained manner by edg-1 transfection. In PC-12 phreochromocytoma cells, sustained MAP
kinase activation is essential for neurite extension and
differentiation(51, 52) . Because edg-1 expression is associated with differentiation of endothelial cells in vitro(8) , these data are consistent with a
functional role for edg-1 in cellular differentiation.
In
stably transfected NIH3T3 cells, edg-1 overexpression is
associated with MAP kinase activation. In contrast to HEK293 cells, the edg-1 expressing NIH3T3 cells also exhibited enhanced
phospholipase A activity. Because the cellular
phospholipase A
enzyme is phosphorylated and activated by
MAP kinase(37) , these data suggest that edg-1 signaling via the MAP kinase is involved in the induction of
phospholipase A
activity in NIH3T3 cells.
The MAP kinase
pathway is a widely used signaling system that regulates cell growth,
differentiation and
apoptosis(51, 52, 53, 54, 55, 56, 57) .
Both GPRs as well as tyrosine kinase receptors induce the MAP kinase
activity in a number of cell
types(36, 49, 53, 54, 55, 56, 57, 58, 59) .
In fibroblasts and neuronal cells, MAP kinase activation is known to
induce cell cycle traverse and differentiation,
respectively(51, 52, 53, 54, 55, 56, 57, 59) .
For example, lysophosphatidic acid and thrombin are known to induce
fibroblast proliferation via G-coupled
receptors(36, 60, 61) . In PC-12 cells, the
activation of the MAP kinase pathway regulates differentiation by
inducing neurite outgrowth(51, 52) . Recent studies in
early Xenopus development have indicated that MAP kinase
activation by the FGF receptor can account completely for the
mesoderm-inducing capacity of the FGF polypeptides(12) . These
studies highlight the importance of the MAP kinase pathway in the
control of cell growth and differentiation. The edg-1 polypeptide was originally isolated because it was induced during
the in vitro differentiation and growth arrest of endothelial
cells(8) . While the physiological function of edg-1 is unknown at present, data presented in this report indicate that
overexpression of edg-1 results in constitutive activation of
the MAP kinase activity by the G
pathway. Whether such a
mechanism plays a functional role in endothelial cell differentiation
awaits further experimentation.
In conclusion, our data indicate
that (i) the edg-1-i domain associates with
G
and G
polypeptides, (ii) the edg-1 receptor associates with G
and
G
, and (iii) overexpression of edg-1 induces
MAP kinase activity in HEK293 and NIH3T3 cells and (iv) edg-1 overexpression is associated with enhanced phospholipase A
activity in NIH3T3 cells. These data provide a basis for further
understanding of the function of the inducible orphan receptor edg-1.