From the Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor St., Glasgow, G4 0NR, Scotland, United Kingdom and § Hanson Institute, Division of Human Immunology, Institute of Medical and Veterinary Science, Frome Rd., Adelaide, SA 5000, Australia
Received for publication, August 21, 2002, and in revised form, December 9, 2002
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ABSTRACT |
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Platelet-derived growth factor (PDGF) and
sphingosine 1-phosphate (S1P) act via PDGF Sphingosine 1-phosphate
(S1P)1 is a bioactive
lysolipid that has been proposed to have both intracellular and
extracellular actions (1). To date, five closely related
G-protein-coupled receptors (GPCR), termed
S1P1-S1P5 (2) (and formerly named EDG1,
EDG5/AGR16/H218, EDG3, EDG6 and EDG8/nrg-1, respectively) have been
identified as high affinity S1P receptors (3-9). Further characterization studies confirmed the S1P1 receptor to be
a GPCR with high affinity for S1P that stimulates p42/p44 MAPK and
inhibits adenylyl cyclase in cells (10-13). The S1P2 and
S1P3 receptors also have high affinity for S1P (14) and are
linked via Gq to phospholipase C and calcium mobilization
and p42/p44 MAPK activation (14, 15) and via G12 and
G13 to Rho-guanine nucleotide factor and Rho
activation. The S1P4 receptor is lymphoid specific and, in
common with the S1P5 receptor, uses Gi/o and
G12 to signal (6, 8).
The S1P1 receptor is implicated in regulating smooth muscle
cell migration, proliferation, and vascular maturation. Insight into
the function of the S1P1 receptor was obtained by studies showing that disruption of the s1p1 gene by
homologous recombination in mice results in extensive intra-embryonic
hemorrhaging and intrauterine (16). This is caused by incomplete
vascular maturation due to the failure of mural cells, vascular smooth
muscle cells, and pericytes to migrate to arteries and capillaries and
to reinforce them properly. Interestingly, the disruption of the
PDGF The question as to which model, sequential versus
integrative signaling, is the major mechanism of action of PDGF and S1P is very important and still remains to be fully investigated. In this
article, we have shown that native functionally active PDGF
receptor-S1P1 receptor complexes are expressed in airway smooth muscle cells. Moreover, PDGF stimulated the phosphorylation of
p42/p44 MAPK, and this phosphorylated p42/p44 MAPK associates with the
PDGF Materials--
All biochemicals including PDGF were from Sigma.
Sphingosine 1-phosphate was from BD Transduction Laboratories
(Oxford, UK). Cell culture supplies and
LipofectAMINETM 2000 were from Invitrogen.
Anti-phospho-p42/p44 MAPK, anti-phospho-PKB, and anti-phospho-p38 MAPK
antibodies were from New England Biolabs (UK). Anti-p42 MAPK, anti-PKB,
anti-horseradish peroxidase-linked anti-phosphotyrosine and anti-p38
MAPK antibodies were from BD Transduction Laboratories.
Anti-PDGF Cell Culture--
The preparation of primary cultures of guinea
pig airway smooth muscle (ASM) cells has been described previously
(23). Their identity was confirmed to be smooth muscle by the presence
of Transfection--
ASM cells were transiently transfected with
antisense/sense S1P1 receptor, sense PDGF Immunoprecipitation--
The medium was removed, and cells were
lysed in ice-cold immunoprecipitation buffer (1 ml) containing 20 mM Tris/HCl, 137 mM NaCl, 2.7 mM
KCl, 1 mM MgCl2, 1 mM
CaCl2, 1% (v/v) Nonidet P-40, 10% (v/v) glycerol, 1 mg/ml
bovine serum albumin, 0.5 mM sodium orthovanadate, 0.2 mM phenylmethylsulfonyl fluoride, leupeptin, anti-pain
pepstatin, and aprotinin (all protease inhibitors were at 10 µg/ml),
pH 8, for 10 min at 4 °C. The material was harvested and centrifuged
at 22,000 × g for 5 min at 4 °C, and 200 µl of cell lysate supernatant (equalized for protein, 0.5-1 mg/ml) was taken
for immunoprecipitation with antibodies (5 µg of
anti-S1P1 receptor antibodies and 40 µl of 1 part
immunoprecipitation buffer and 1 part protein A/G-Sepharose CL4B).
After agitation for 2 h at 4 °C, the immune complex was
collected by centrifugation at 22,000 × g for 15 s at
4 °C. Immunoprecipitates were washed twice with buffer A containing
10 mM Hepes, pH 7, 100 mM NaCl, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 0.5% (v/v) Nonidet P-40) and once in buffer A
without Nonidet P-40. The immunoprecipitates were then combined with
boiling sample buffer containing 62 mM Tris-HCl, pH 6.7, 1.25% (w/v) SDS, 10% (v/v) glycerol, 3.75% (v/v) mercaptoethanol,
and 0.05% (w/v) bromphenol blue. The samples were then subjected to
SDS-PAGE and Western blotting.
p42/p44 MAPK, PKB, and p38 MAPK Assays--
The
phosphorylated forms of p42/p44 MAPK, PKB, and p38 MAPK were detected
by Western blotting cell lysates with the respective anti-phospho-specific antibodies. Anti-p42 MAPK, PKB, and p38 MAPK
antibodies were also used for Western blotting to establish equal
loading of protein in each sample.
Blotting--
Immunoblotting was performed as described by us
previously (19). Immunoreactive proteins were visualized using enhanced chemiluminescence detection.
S1P Measurements--
Cells grown to confluency on multiple
6-well plates (1.2 × 106 cells/well) were
serum-starved overnight and equilibrated in 1 ml of Dulbecco's
modified Eagle's medium/fatty acid-free bovine serum albumin (2 mg/ml)
at 37 °C for at least 1 h before stimulation. [3H]Sphingosine was resuspended with phosphate-buffered
saline containing fatty acid-free bovine serum albumin (2 mg/ml) by
sonication for 10 min. The reactions were started by the addition of
[3H]sphingosine (222,000 dpm/well, final concentration
5~10 nM) and various agonists for the periods of time indicated.
Stimulation of cells was stopped by removal of the medium and the
addition of 0.5 ml of ice-cold methanol. Cells were then scraped into
glass vials, and an equal volume of chloroform was added. Samples were
vortexed vigorously and maintained at room temperature for at least 20 min to allow extraction of cellular lipids. The samples were dried
using a SpeedVac centrifuge. Samples were then re-dissolved in 80 µl
of CHCl3:CH3OH (19:1, v/v) and resolved by TLC
using silica gel G60 LK6D TLC plates (Whatman) and solvent system
CHCl3:CH3OH:CH3COOH:H2O
(25:10:1:2, v/v). A [32P]S1P standard was resolved in
parallel. Sample profiles were obtained by excising 1-cm fractions of
the silica and quantification of the radioactivity by liquid
scintillation counting.
[3H]Thymidine
Incorporation--
[3H]Thymidine incorporation was as
described previously by us (24).
Immunofluorecence--
Cells were grown on 12-mm glass
coverslips to 60-90% confluence and transfected as described. After
treatment with S1P and/or PDGF for the times indicated, cells were
fixed in 3.7% formaldehyde in PBS for 10 min then permeabilized in
0.1% Triton X-100 in phosphate-buffered saline for 1 min. Nonspecific
binding was reduced by preincubating cells in blocking solution
containing 5% fetal calf serum, 1% bovine serum albumin in
phosphate-buffered saline for 1 h. Cells were incubated in primary
antibodies (1:100 dilution in blocking solution) for 1-2 h and then
incubated with the appropriate fluorescein isothiocyanate (rabbit)- or
TRITC (mouse)-conjugated secondary antibodies (1:100) for 1 h.
Cells were mounted on glass slides using Vectashield mounting medium
with 4,6-diamidino-2-phenylindole and visualized using a Nikon E600
epifluorescence microscope.
The Existence of Functional PDGF
Fig. 1a shows that the native
PDGF
Several lines of evidence support a model in which association of the
PDGF receptor with the S1P1 receptor enables integrative signaling in response to both PDGF and S1P. First, the treatment of ASM
cells with PDGF-AB stimulated the phosphorylation of p42/p44 MAPK, and
this phosphorylated p42/p44 MAPK associates with the PDGF
In previous studies, we have shown that c-Src is activated by PDGF, and
this can be blocked by pretreating ASM cells with pertussis toxin (25).
Moreover, in the same study we showed that pretreating ASM cells with
PP1, an inhibitor of c-Src, reduced the activation of p42/p44 MAPK in
lysates of ASM cells stimulated with PDGF (25). We now show that the
c-Src inhibitor PP2 ablated the PDGF-dependent
phosphorylation of the p42/p44 MAPK that associates with PDGF
Second, the transfection of a S1P1 receptor antisense
plasmid construct reduced the PDGF-AB- and S1P-dependent
activation of p42/p44 MAPK (% inhibition of PDGF-AB- and
S1P-stimulated p42 MAPK activation (normalized for p42 MAPK levels)
were: PDGF, 48.3 ± 10.6%; S1P, 65 ± 14%,
n = 3, p < 0.05 antisense-treated
agonist response versus agonist response) and had no
significant effect on phorbol ester (PMA)-stimulated p42/p44 MAPK
activation (% inhibition of PMA-stimulated p42 MAPK activation
(normalized for p42 MAPK levels) was 12 ± 20%, n = 3), which does not involve GPCRs (Fig. 2a). The lack of significant
effect of the antisense construct on PMA-stimulated p42/p44 MAPK
activation indicates specificity against S1P1
receptor-mediated responses. The antisense S1P1 receptor plasmid construct reduced S1P1 receptor (molecular mass 45 kDa) expression (Fig. 2b), thereby also validating the
specificity of the anti-S1P1 receptor antibody for
S1P1 receptor. The antisense construct had no effect on
cell viability under the conditions used (data not shown). Finally, S1P
does not cause the release of PDGF, based on previous results obtained
by us, showing that S1P does not stimulate PDGF receptor tyrosine
phosphorylation (24).
Co-internalization of PDGF
We have previously shown that recombinant forms of the PDGF
PDGF also promoted the formation of endocytic vesicles containing
PDGF
A combination of S1P and PDGF produced an increase in the number of
endocytic vesicles containing both recombinant PDGF PDGF-stimulated S1P Formation and p42/p44 MAPK
Activation--
The existence of a PDGF
First, we found that there was a correlation between the ability of
PDGF-BB to stimulate S1P formation and to activate p42/p44 MAPK or to
promote [3H]thymidine incorporation in ASM cells. PDGF-BB
(10 ng/ml) caused a rapid and transient increase in
[3H]S1P production in ASM cells (300% increase in S1P
above basal at peak 3 min of cell stimulation). However, neither
PDGF-AA nor PDGF-AB stimulated S1P formation (Fig.
4a). All of the PDGF subtypes induced the activation of p42/p44 MAPK (fold activations of p42 MAPK
(normalized for p42 MAPK levels) were: control, 1 ± 0.29; PDGF-AA, 5.35 ± 0.42; PDGF-AB, 8.82 ± 1.06; PDGF-BB,
10.52 ± 0.6, n = 3-4, p < 0.05 for PDGF-BB versus PDGF-AB. Therefore, the rank order
activation of p42/p44 MAPK (at 10 ng/ml PDGF) was PDGF-BB > PDGF-AB > PDGF-AA (Fig.
5a, left bottom
panel). This was correlated with PDGF
It is important to note from our previous studies that the 50%
inhibitory effect of pertussis toxin on PDGF-stimulated p42/p44 MAPK
activation in ASM cells was achieved with PDGF-AB (25). Therefore,
PDGF-AB in part uses a classical G-protein-coupled receptor signaling
mechanism that does not involve S1P release, since PDGF-AB does not
induce S1P formation. Nevertheless, we formally proceeded with studies
to evaluate the effect of SK inhibitors and overexpression of
recombinant wild type/dominant negative hSK1 on PDGF-BB-stimulated
responses. First, the SK inhibitor tDHS dose dependently inhibited the
PDGF-BB-stimulated [3H]S1P production as well as reducing
basal S1P levels (Fig. 4b). Using 1 µM tDHS,
basal [3H]S1P levels were reduced by ~70%, whereas the
response to PDGF-BB was completely suppressed. tDHS did not affect
[3H]sphingosine uptake into sphingolipids of control or
PDGF-BB-treated cells (Fig. 4c). However, neither tDHS nor
N,N-dimethylsphingosine (DMS, SK inhibitors) (at
10 µM, which ablated S1P formation) had a significant
effect on the PDGF-BB-dependent stimulation of p42/p44 MAPK, p38 MAPK, or PKB (Fig. 5b) (fold activations of p42
MAPK (normalized for p42 MAPK levels) were: control, 0.73 ± 0.56;
PDGF-BB, 9.94 ± 1.33; tDHS, 1.24 ± 0.39; PDGF-BB plus tDHS,
8.95 ± 2.7; DMS, 1.18 ± 0.27; PDGF-BB plus DMS,
9.45 ± 0.02, n = 5 for tDHS and n = 3 for DMS). Higher concentrations of tDHS (30 µM)
consistently inhibited the PDGF-BB-dependent activation of
p42/p44 MAPK. We have previously shown that this is due to its action
on protein kinase C, which is required for PDGF-dependent
stimulation of p42/p44 MAPK in ASM cells (26). Second, neither
FLAG-tagged hSK1WT nor FLAG-tagged dominant negative
hSK1G82D modulated PDGF-BB-stimulated p42/p44 MAPK (fold
activations of p42 MAPK (normalized for p42 MAPK levels) were: control,
1.38 ± 1.4; PDGF-BB, 11.8 ± 2.2; hSK1WT,
1.04 ± 0.26; PDGF-BB plus hSK1WT, 11.8 ± 3.7;
hSK1G82D, 1.12 ± 0.17; PDGF-BB plus
hSK1G82D, 10.9 ± 3.9, n = 9), PKB, or
p38 MAPK activation (Fig. 5c). Overexpression of FLAG-tagged
hSK1WT or FLAG-tagged hSK1G82D in transfected
cells was confirmed by Western blot analysis with anti-FLAG antibodies
(Fig. 5c). Immunofluorescence staining with anti-FLAG
antibodies of FLAG-tagged hSK1WT or FLAG-tagged
hSK1G82D-transfected cells confirmed highly efficient
transfection of ASM cells (Fig. 5d). hSK1WT
activity was increased ~130-fold above basal (Fig. 5e).
Neither form of recombinant kinase affected PDGF-AA-, PDGF-AB- or
PDGF-BB-stimulated [3H]thymidine incorporation (Fig.
5f). Therefore, we conclude that PDGF-BB signaling to
p42/p44 MAPK, PKB, and p38 MAPK does not involve sequential release and
action of S1P in ASM cells.
Sphingosine Kinase Release from Airway Smooth Muscle
Cells--
Despite the lack of evidence to support a model in which
PDGF promotes the release of S1P from ASM cells, hSK1 is exported from
these cells. Indeed, Ancellin et al. (27) also show that SK
is exported from human umbilical vein endothelial cells and HEK 293 cells and may play a role in producing S1P at the cell surface.
Moreover, these authors showed that extracellular-formed S1P induced a
profound angiogenic effect via S1P receptors. We have therefore
evaluated whether FLAG-tagged hSK1WT is exported from ASM
cells. Fig. 6a shows that
FLAG-tagged hSK1WT was exported into the medium. The
transfection of FLAG-tagged hSK1WT into ASM cells did not
compromise cell integrity, as assessed by trypan blue exclusion (data
not shown), and did not cause leakage of other proteins of
similar molecular mass to hSK1, such as p42/p44 MAPK (Fig.
6a).
The exported SK is capable of converting sphingosine to S1P. This was
supported by data showing that a low concentration of exogenous
sphingosine stimulated p42/p44 MAPK activation in ASM cells (Fig.
6b). The activation of p42/p44 MAPK by sphingosine was
significantly increased in ASM cells transfected with S1P1 receptor (data not shown). These findings suggest that export of
endogenous SK may convert sphingosine to S1P, which can subsequently stimulate p42/p44 MAPK. Indeed, the sphingosine-dependent
activation of p42/p44 MAPK was abrogated by overexpression of
FLAG-tagged hSK1G82D (Fig. 6b). However, a role
for exported SK in mediating part of the action of PDGF in ASM cells
was excluded on the basis that PDGF does not promote export of hSK1
(data not shown) and SK inhibitors/hSK1G82D did not
abrogate PDGF receptor signaling (Fig. 5).
We have presented evidence that directly supports a model for
signal integration by PDGF Several lines of evidence were obtained to support the existence of a
functionally active native PDGF Part of the PDGF-stimulated response in ASM cells is insensitive to
pertussis toxin and to S1P1 receptor antisense plasmid construct. Thus, a fraction of the response does not involve
Gi or S1P1 receptors. This is an interesting
finding and is supported by other evidence that suggests that the GPCR
input into signaling by receptor tyrosine kinases is dependent upon
both receptor density and ligand concentration. Thus, high receptor
tyrosine kinase density or ligand concentration has been shown to
surmount the requirement for GPCR input (28, 29). These findings
suggest that receptor tyrosine kinases use at least two distinct
signaling pathways to stimulate p42/p44 MAPK, one of which requires
G-protein, whereas the other is probably initiated by auto-tyrosine
phosphorylation of the growth factor receptor (28, 29). Indeed, our
previous findings showed that the G-protein-dependent
activation of p42/p44 MAPK in response to PDGF occurs via a mechanism
that does not require PDGF-dependent tyrosine
phosphorylation of the PDGF Endocytosis of the PDGF We have also reported that PDGF stimulates a pertussis toxin-sensitive
tyrosine phosphorylation of the Grb-2 associated binding protein, Gab1,
in ASM cells (31). This appears to be dependent upon Gi Others report co-internalization of GPCR-receptor tyrosine kinase
complexes in response to GPCR agonist. For instance, Maudsley et
al. (32) show that isoprenaline induces transactivation of the EGF
receptor, resulting in EGF receptor tyrosine phosphorylation and
complex formation with The formation of a complex between the PDGF Finally, we show that hSK1 is constitutively exported and can support
activation of p42/p44 MAPK by exogenous sphingosine. The export of hSK1
is not regulated by PDGF and, therefore, does not contribute to
signaling by this growth factor. However, it is possible that the
presentation of sphingosine on other cell types and its conversion to
S1P by exported kinase from ASM cells might enable S1P to act with PDGF
on the PDGF There is a possibility that a subfraction of ASM cells undergoes
apoptosis in culture. This raises the intriguing question, Are cell
death mechanisms associated with selective export of SK1 from cells,
thereby reducing its ability to promote cell survival via an
intracellular action? This is possible if there is hindered supply of
sphingosine presented from other cells. However, contact with other
cells that present sphingosine might effectively convert the export of
SK into a cell survival action, since S1P (acting at cell surface S1P
receptors) increases PDGF-stimulated p42/p44 MAPK activation and DNA
synthesis in ASM cells (24).
In summary, our findings provide important information on integration
of G-protein-mediated signals by receptor tyrosine kinases in mammalian
cells by a mechanism that differs from sequential regulation and/or
transactivation of receptor tyrosine kinases by GPCR agonists.
receptor-S1P1 receptor complexes in airway smooth
muscle cells to promote mitogenic signaling. Several lines of evidence
support this conclusion. First, both receptors were
co-immunoprecipitated from cell lysates with specific
anti-S1P1 antibodies, indicating that they form a complex.
Second, treatment of airway smooth muscle cells with PDGF stimulated
the phosphorylation of p42/p44 MAPK, and this phosphorylated p42/p44
MAPK associates with the PDGF
receptor-S1P1 receptor
complex. Third, treatment of cells with antisense S1P1
receptor plasmid construct reduced the PDGF- and
S1P-dependent activation of p42/p44 MAPK. Fourth, S1P
and/or PDGF induced the formation of endocytic vesicles containing both
PDGF
receptors and S1P1 receptors, which was required
for activation of the p42/p44 MAPK pathway. PDGF does not induce the release of S1P, suggesting the absence of a sequential mechanism. However, sphingosine kinase 1 is constitutively exported from cells and
supports activation of p42/p44 MAPK by exogenous sphingosine. Thus, the
presentation of sphingosine from other cell types and its conversion to
S1P by the kinase exported from airway smooth muscle cells might enable
S1P to act with PDGF on the PDGF
receptor-S1P1 receptor
complex to induce biological responses in vivo. These data
provide further evidence for a novel mechanism for G-protein-coupled receptor and receptor tyrosine kinase signal integration that is
distinct from the transactivation of receptor tyrosine kinases by
G-protein-coupled receptor agonists and/or sequential release and
action of S1P in response to PDGF.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor gene in mice results in a similar phenotype (17, 18).
Hobson et al. (16) showed that PDGF-stimulated cell motility
is S1P1 receptor-dependent in human embryonic
kidney 293 cells (HEK 293 cells) and mouse embryonic fibroblasts.
Therefore, a sequential model has been proposed in which PDGF
stimulates S1P synthesis/release, which in turn binds to and activates
the S1P1 receptor to induce activation of Rac, PKB, and
cell motility (16). In contrast, we have reported that the PDGF
receptor forms a complex with the S1P1 receptor in HEK 293 cells transfected with recombinant forms of these receptors (19, 20).
We showed that the association of these recombinant receptor types
enables more efficient tyrosine phosphorylation of Gi
by
the PDGF
receptor kinase, possibly via proximity-induced effects. We
showed that the tyrosine phosphorylation of Gi
was
required to stimulate the p42/p44 MAPK pathway by PDGF or S1P,
consistent with an integrative signal model (19). Freedman et
al. (21) recently confirmed the involvement of Gi in
PDGF
receptor signaling. We also reported that
-arrestin I/GRK2
associate with the PDGF
receptor-S1P1 receptor complex
in HEK 293 cells (19) and that this might represent an important step
regulating p42/p44 MAPK signaling. Freedman et al. (21)
recently confirmed the association of GRK2 with the PDGF
receptor.
receptor-S1P1 receptor complex. These findings show
that the integrative signaling model is a major mechanism of action of
PDGF and S1P in cells. Consistent with an integrative model, we have
also established that these receptors are co-internalized together as a
functional signaling unit to regulate the p42/p44 MAPK pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
receptor antibodies were from Santa Cruz (Santa Cruz,
CA). Reporter horseradish peroxidase-anti-rabbit/mouse IgG antibodies
were from Diagnostics Scotland (Carluke, UK). The PDGF
receptor
plasmid construct was a kind gift from Professor C.-H. Heldin
(Ludwig Institute for Cancer Research, Uppsala, Sweden). Mutagenesis of hSK1WT to produce a catalytically inactive
mutant (hSK1G82D) was reported previously by Pitson
et al. (22).
-actin using smooth muscle-specific mouse anti-
-actin
monoclonal antibodies. Cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% (v/v) fetal calf serum and 10%
(v/v) horse serum. Cells were routinely used at passage 3-4. Cells
were placed in Dulbecco's modified Eagle's medium supplemented with 0.1% (v/v) fetal calf serum and 0.1% (v/v) horse serum for 24 h
before experimentation. HEK 293 cells were maintained in culture as
previously reported by us (19).
receptor,
FLAG-tagged hSK1WT, or FLAG-tagged hSK1G82D
plasmid constructs as required. Cells at 90% confluence were placed in
minimum Eagle's medium containing 2% fetal calf serum and transfected
with 2 µg of plasmid construct after complex formation with
LipofectAMINETM 2000 according to the manufacturer's
instructions. The cDNA-containing media was removed after
incubation for 24 h at 37 °C, and the cells were incubated for
a further 24 h in serum-free medium before agonist additions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Receptor-S1P1
Receptor Complexes in ASM Cells--
We set out to investigate whether
the PDGF
receptor forms a complex with the S1P1 receptor
in cultured ASM cells and whether this represents a functional
integrative signaling unit. ASM cells were chosen for several reasons.
First, these cells express high levels of S1P1 receptor
(24). Second, pretreatment of cells with pertussis toxin reduced
PDGF-AB- and S1P-stimulated activation of p42/p44 MAPK by 50 and
100%, respectively (24, 25). Under the conditions used in these
studies, we showed that pertussis toxin completely inactivates
Gi
. Therefore incomplete blockade of PDGF receptor
signaling suggests that the receptor uses both Gi-dependent and -independent pathways to
regulate p42/p44 MAPK. Thus, a substantial fraction of the PDGF
receptors might be associated with a GPCR in these cells, since
pertussis toxin acts by uncoupling GPCRs from their respective
G-protein to prevent signaling. Third, S1P and PDGF-AB use a common
signaling pathway involving Gi-regulated Grb-2-associated
phosphoinositide 3-kinase, which lies up-stream of p42/p44 MAPK
activation (24, 25). Fourth, a combination of submaximal S1P and
PDGF-AB induced a synergistic activation of p42/p44 MAPK (24). Fifth,
S1P is co-mitogenic with PDGF-AB (24). These data are consistent with
the possibility that the S1P1 receptor might be associated
with and exert cross-talk regulation with the PDGF receptor.
receptor is indeed associated with native S1P1
receptors in ASM cells. Thus, the PDGF
receptor (molecular
mass 180 kDa) and S1P1 receptor (molecular mass 45 kDa)
were co-immunoprecipitated from cell lysates with anti-S1P1
receptor antibodies. Neither receptor was immunoprecipitated when the
antibodies were omitted from the procedure. Stimulation of ASM cells
with PDGF-AB did not promote further formation of the PDGF
receptor-SIP1 receptor complex, suggesting that the complex
is preformed in these cells. Transfection of ASM cells with recombinant
PDGF
receptor did not increase the amount of PDGF
receptor
present in the anti-S1P1 receptor immunoprecipitates (Fig.
1a), suggesting that the endogenous S1P1
receptor is limiting for complex formation with the PDGF
receptor
(Fig. 1a). S1P1 receptor was also
co-immunoprecipitated with PDGF
receptor using anti-PDGF
receptor
antibodies (data not shown). The interaction of the S1P1
receptor with the PDGF receptor exhibits specificity, since
S1P1 receptors do not associate with the EGF receptor
tyrosine kinase and there is no cross-talk regulation between EGF and
S1P in terms of activating p42/p44 MAPK (Ref. 19 and data not shown). EGF signaling also differs from that of PDGF in that EGF-stimulated p42/p44 MAPK activation is insensitive to blockade by pertussis toxin
and, therefore, appears to be independent of GiPCRs (19, 26).
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Fig. 1.
The association of S1P1 receptors
with PDGF receptors and phosphorylated p42/p44
MAPK. ASM cells were pretreated with the c-Src inhibitor PP2 (10 µM, 10 min) before stimulation with or without PDGF-AB
(10 ng/ml, 10 min) as indicated. In certain cases, cells were
transfected with PDGF
receptor plasmid construct. a,
Western blots (WB, showing the co-immunoprecipitation of
PDGF
receptor (molecular mass 180 kDa) and S1P1 receptor
(molecular mass 45 kDa) from ASM cell lysates using
anti-S1P1 receptor antibodies (Ab).
Anti-S1P1 receptor immunoprecipitates were resolved on
SDS-PAGE and Western-blotted with anti-PDGF
receptor and
anti-S1P1 receptor antibodies. ASM cells in which
recombinant PDGF
receptor (PDGF
R) were
transfected were also used to show that endogenous S1P1
receptors are limiting for the binding of PDGF
receptor.
b, Western blots showing the PDGF-dependent
phosphorylation of p42/p44 MAPK and association with the PDGF
receptor-S1P1 receptor complex. Also shown is the effect of
pretreating cells with the c-Src inhibitor, PP2, on receptor-associated
phosphorylated p42/p44 MAPK and PDGF
receptor-S1P1
receptor complexes. Anti-S1P1 receptor immunoprecipitates
were resolved on SDS-PAGE and Western-blotted with anti-PDGF
receptor and anti-phosphorylated p42/p44 MAPK antibodies. This figure
also shows a Western blot of the corresponding effects of PP2 on the
PDGF-dependent phosphorylation of p42/p44 MAPK and PDGF
receptor levels in total cell lysates. These are representative results
of an experiment performed three times. IP,
immunoprecipitation; T/F, transfection.
receptor-S1P1 receptor complex. Thus, substantially more phosphorylated p42/p44 MAPK was co-immunoprecipitated with the PDGF
receptor-S1P1 receptor complex using anti-S1P1
receptor antibodies from cells treated with PDGF compared with controls (Fig. 1b). p42 MAPK was also detected in
anti-S1P1 receptor immunoprecipitates from lysates of
PDGF-treated cells using anti-p42 MAPK antibodies to probe the Western
blots (data not shown). Similar results were obtained when S1P was used
to stimulate cells (data not shown).
receptor-S1P1 receptor complex (Fig. 1b).
Similar results were observed in cell lysates (Fig. 1b). PP2
did not reduce the amount of the PDGF
receptor associated with the
S1P1 receptor in anti-S1P1 receptor
immunoprecipitates nor did it affect PDGF
receptor levels in cell
lysates (Fig. 1b).
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Fig. 2.
PDGF and S1P stimulation of p42/p44 MAPK in
ASM cells. ASM cells were pretreated with antisense
S1P1 plasmid construct (24 h) before stimulation with
PDGF-AB (10 ng/ml, 10 min) or S1P (5 µM, 10 min) or
phorbol ester (PMA, 1 µM, 10 min).
a, Western blots showing the effect of an antisense
S1P1 plasmid construct on PDGF or S1P- or
PMA-dependent activation of p42/p44 MAPK. b,
Western blots showing the effect of antisense S1P1 plasmid
construct on S1P1 receptor expression. Blots were stripped
and reprobed with antibodies that react with p42 MAPK to ensure equal
protein loading. These are representative of an experiment performed
3-4 times. C, control.
Receptor-S1P1 Receptor
Complexes in Endocytic Vesicles--
One prediction of the proposed
PDGF
receptor-S1P1 receptor complex association model is
that stimulation of cells with PDGF is expected to cause the
co-internalization of both PDGF
and S1P1 receptors into
the same endocytic vesicles. ASM cells were transfected with Myc-tagged
S1P1 receptor to capture PDGF
receptor and to allow
detection of S1P1 receptors with anti-Myc tag antibodies. Under these conditions, stimulation of cells with PDGF promoted the
formation of endocytic vesicles containing both PDGF
receptor and
Myc-tagged S1P1 receptor (yellow vesicles,
Fig.
3A, panels 6a and 6b). Inhibitors of clathrin-mediated endocytosis
(e.g. concanavalin A and monodansylcadervine) blocked the
internalization of the PDGF
receptor-S1P1 receptor
complex in response to PDGF (data not shown). We excluded the
possibility that PDGF elicits the release of S1P (see later), which
might have acted on S1P1 receptors, causing their
recruitment to the same endocytic vesicles as those containing PDGF
receptors. The treatment of cells with S1P also induced
co-internalization of S1P1 and PDGF
receptors, although
the sensitivity of detection was considerably reduced (data not shown).
This is explained by the fact that overexpression of recombinant
S1P1 receptors probably results in the expression of a
significant amount of non-associated S1P1 receptor, which might effectively buffer the PDGF
receptor-S1P1 receptor
complex pool from S1P.
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Fig. 3.
Co-internalization of PDGF
receptors and S1P1 receptors in endocytic vesicles in
response to S1P and/or PDGF. A, photomicrographs
showing immunofluorescent staining of ASM cells (transfected with
Myc-tagged S1P1 receptor) with anti-Myc tag and
anti-PDGF
receptor antibodies. Stimulation of ASM cells with PDGF
(10 ng/ml, 5 min) is shown. Panels 1-3 are control cells;
panels 4-6 are PDGF-stimulated cells. Panels 1 and 4 are immunostained with PDGF
receptor antibodies
(green) to detect endogenous receptor; panels 2 and 5 are immunostained with anti-Myc tag antibodies
(red), and panels 3a and 6a are merged
panels showing localization of both receptors (yellow).
Panel 3b is a magnified area showing control cells, whereas
panel 6b is a magnified area showing discrete endocytic
vesicles containing both receptors formed in response to PDGF.
B, photomicrographs showing immunofluorescent staining of
HEK 293 cells (transfected with Myc-tagged S1P1 receptor
and PDGF
receptor) with anti-Myc tag and anti-PDGF
receptor
antibodies. Stimulation of HEK 293 cells with PDGF (10 ng/ml, 5 min) or
S1P (5 µM, 5 min) or both. Panels a-d are
control cells; panels e-h are S1P-stimulated cells;
panels i-l are PDGF-stimulated cells; panels m-p
are PDGF plus S1P-stimulated cells. Panels a, e,
i, and m are immunostained with anti-PDGF
receptor antibodies (green); panels b,
f, j, and n are immunostained with
anti-Myc tag antibodies (red); panels c,
g, k, and o are merged panels showing
co-localization of both receptors (yellow); panel
d is a magnified area showing co-localization of both receptors to
the plasma membrane in control cells. Panels h,
l, and p are magnified areas of respective cells
showing discrete endocytic vesicles containing both receptors formed in
response to PDGF and/or S1P.
receptor
and S1P1 receptor form functional associated signaling complexes in HEK 293 cells (19). Because the levels of the PDGF
receptor-S1P1 receptor complex are very high in these cells
due to overexpression of the two proteins, we predicted that this system might show increased sensitivity in terms of co-internalization of both receptors in response to S1P. Thus, overexpression of both
receptors to increase their association might surmount the buffering
effect of non-associated S1P1 receptors on S1P observed in
ASM cells. Consistent with this, we found that the stimulation of
transfected HEK 293 cells with S1P induced an increase in the number of
endocytic vesicles containing both recombinant PDGF
receptors and
Myc-tagged S1P1 receptors. PDGF also induced
co-internalization of both receptors to the same endocytic vesicles
(Fig. 3B, yellow vesicles, panels h
and l).
receptors that were not associated with Myc-tagged S1P1 receptors (green vesicles, Fig.
3B, panel l). These endocytic vesicles might
contain recombinant PDGF
receptor that is either free or associated
with endogenous S1P1 receptors, for which transcript is
expressed in HEK 293 cells (data not shown).
receptors and
Myc-tagged S1P1 receptors compared with that induced with each agent alone (Fig. 3B, yellow vesicles,
panel p). This is entirely consistent with a model in which
combined stimulation of cells with PDGF and S1P will increase the
number of PDGF
receptor-S1P1 receptor complexes occupied
with either or both ligands, leading to an increased number of
ligand-bound PDGF
receptor-S1P1 receptor complexes being
internalized within common endocytic vesicles. These findings represent
an entirely novel mechanism for co-internalization of receptor tyrosine
kinase/GPCRs.
receptor-S1P1
receptor complex in ASM cells and the functional evidence provided here
support a model in which S1P and PDGF use an integrative signaling
mechanism in these cells. Nevertheless, it was necessary to formally
evaluate whether part of the response to PDGF is mediated via the
release of S1P, which might act on S1P1 receptors in a
sequential manner. We therefore compared the effect of (i) different
PDGF isoforms and (ii) sphingosine kinase inhibitors and recombinant
wild type and a dominant negative mutant hSK1 on PDGF-stimulated S1P
formation, p42/p44 MAPK, PKB, and p38 MAPK activation.
/
receptor
auto-tyrosine phosphorylation (Fig. 5a, left
mid-panel) and PDGF-stimulated [3H]thymidine
incorporation (Fig. 5a, right panel). Fig.
4a shows the dose response of S1P production (peak at
3 min of cell stimulation) induced by PDGF-BB and the lack of effect of
PDGF-AA and PDGF-AB. Half-maximal and maximal stimulation of
[3H]S1P production was observed at 1.5 ± 0.6 and 6 ng/ml PDGF-BB, respectively (Fig. 4a). We therefore further
investigated whether the increased efficacy of PDGF-BB on p42/p44 MAPK
activation could be accounted for by potential release of S1P by
PDGF-BB.
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Fig. 4.
The PDGF stimulation of S1P production in ASM
cells. a, dose response of PDGF-stimulated S1P
production in ASM cells. Cells were incubated with PDGF-AA
(triangles) or PDGF-AB (circles) or PDGF-BB
(squares) or EGF (diamonds) and
[3H]sphingosine for 3 min. Results are expressed as
percentage of [3H]S1P formed above control cells and are
the means ± S.D. for n = 3 experiments. Basal
[3H]S1P formed was 2000-6000 dpm/1.2 × 106 cells. b, the effect of tDHS on basal
(triangles) and PDGF-BB-stimulated (squares) S1P
production after 3 min. c, the effect of tDHS on
[3H]sphingosine uptake in control (triangles)
and PDGF-BB-stimulated cells (squares). In b and
c, results are expressed as dpm of [3H]S1P or
dpm of [3H]sphingolipids formed per 1.2 × 106 cells, respectively, and are means ± S.D.
(n = 3).
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Fig. 5.
PDGF stimulation of p42/p44 MAPK, p38 MAPK,
PKB, PDGF receptor tyrosine phosphorylation, and
[3H]thymidine incorporation in ASM cells. ASM cells
were treated with or without PDGF-BB or PDGF-AA or PDGF-AB (all at 10 ng/ml) for 3 min. a, Western blots showing the effect of
PDGF-BB, PDGF-AB, and PDGF-AA on p42/p44 MAPK activation or PDGF
receptor tyrosine phosphorylation (left panel). The
right panel shows the effect of the different PDGF sub types
on [3H]thymidine incorporation (PDGF-AA
(triangles), PDGF-AB (circles), and PDGF-BB
(squares)). b, Western blots showing the effect
of tDHS (10 µM, 10 min) or
N,N-dimethylsphingosine (DMS, 10 µM, 10 min) on PDGF-BB-dependent activation
of p42/p44 MAPK, PKB, or p38 MAPK activation. c, Western
blots showing the effect of FLAG-tagged hSK1WT or
hSK1G82D on PDGF-BB-dependent activation of
p42/p44 MAPK, PKB, or p38 MAPK activation. The analysis with anti-FLAG
antibodies proved that recombinant hSK1WT and
hSK1G82D (molecular mass 45 kDa) are overexpressed in
transfected cells. d, photomicrograph showing
immunofluorescent staining with anti-FLAG antibodies of ASM cells
transfected with FLAG-tagged hSK1WT and
hSK1G82D to demonstrate highly efficient transfection of
the cell population. i and ii,
vector-transfected; iii and iv, FLAG-tagged
hSK1WT-transfected; v and vi,
FLAG-tagged hSK1G82D-transfected; e, histogram
showing the increase in hSK1WT activity in transfected ASM
cells; f, histogram showing the effect of vector (open
bars), hSK1WT (filled bars), and
hSK1G82D (hatched bars) on PDGF-AA- PDGF-AB-,
and PDGF-BB-stimulated [3H]thymidine incorporation. Blots
were stripped and reprobed with antibodies that react with p42 MAPK
(a-c) or PDGF /
receptors (a) to ensure
equal protein loading. These are representative results of an
experiment performed 3-9 times.
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Fig. 6.
Export of hSK1WT from ASM
cells. ASM cells were transfected with FLAG-tagged
hSK1WT and hSK1G82D before stimulation with
sphingosine (1 µM, 10 min). a, Western blot
showing export of FLAG-tagged hSK1WT (molecular mass 45 kDa) from ASM cells. No leakage of p42 MAPK was detected. b,
Western blot showing the effect of sphingosine on p42/p44 MAPK in
vector- versus FLAG-tagged hSK1G82D-transfected
cells. Blots were stripped and reprobed with antibodies that react with
p42 MAPK to ensure equal protein loading. These are representative
results of an experiment performed three times.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor-S1P1 receptor
complexes in ASM cells. We have also demonstrated that PDGF does not
promote the release of S1P to stimulate p42/p44 MAPK, PKB, or p38 MAPK in a sequential manner in these cells. The sequential model may be
operative in other cell types where PKB is very sensitive to low levels
of S1P release. hSK1WT is constitutively exported from ASM
cells and is not regulated by PDGF. Interestingly, the enzyme converts
exogenous sphingosine to S1P, which in turn stimulates the p42/p44 MAPK pathway.
receptor-S1P1 receptor complex that can support integrative signaling by S1P/PDGF in ASM
cells. First, the PDGF
receptor and S1P1 were
co-immunoprecipitated from cell lysates with specific
anti-S1P1/PDGF
receptor antibodies. Second, the
treatment of ASM cells with PDGF stimulated the phosphorylation of
p42/p44 MAPK, and this phosphorylated p42/p44 MAPK associates with the
PDGF
receptor-S1P1 receptor complex. To our knowledge, this is the first report showing that phosphorylated p42/p44 MAPK is
present in a complex with receptor in response to growth factor. Current models have suggested that after ligand binding and the initiation of signaling the receptor is subsequently disconnected from
events such as association of activated Raf-MEK (Raf-MAPK kinase 1)
with p42/p44 MAPK. The close association of phosphorylated p42/p44 MAPK
with the PDGF
receptor-S1P1 receptor complex and attendant protein components is entirely compatible with an integrative signaling response to PDGF and S1P. Third, the treatment of cells with
antisense S1P1 receptor plasmid construct (which lowered S1P1 receptor expression) reduced the PDGF- and
S1P-dependent activation of p42/p44 MAPK. These findings
support the requirement for cross-talk regulation between both
receptors. They also suggest that the S1P1 receptor might
be partially constitutively active and capable of releasing active
G-protein subunits for tyrosine phosphorylation by the PDGF
receptor
kinase, as observed in HEK 293 cells (19). Fourth, S1P and PDGF
promoted the co-internalization of PDGF
receptor and
S1P1 receptor in the same endocytic vesicles. The ability
of S1P to induce internalization of the PDGF
receptor is important
as it establishes further evidence for close association between the
S1P1 receptor and the PDGF
receptor. This model is, therefore, distinct from the sequential model, in which PDGF causes the
release of S1P that subsequently acts at S1P1 receptors in a manner that is completely independent of the PDGF receptor. Indeed,
the results presented here clearly show an interaction between S1P and
PDGF receptors that is independent of PDGF-stimulated S1P release from cells.
receptor (19). Moreover, we showed that
overexpression of Gi suppresses PDGF
receptor tyrosine
phosphorylation while increasing PDGF-stimulated activation of p42/p44
MAPK (19). This is supported by studies from Freedman et al.
(21), who have recently shown that GRK2-catalyzed phosphorylation of
the PDGF
receptor suppresses PDGF-stimulated auto-tyrosine
phosphorylation of the PDGF
receptor. Thus, G-protein input may
switch growth factor receptor signaling such that stimulation of
p42/p44 MAPK is less dependent upon recruitment of proteins to tyrosine
phosphates on the growth factor receptor and is more dependent upon a
Gi
/GRK2/
-arrestin I-mediated pathway. Taken together
with the findings presented in Alderton et al. (19) and in
this current paper we propose that PDGF
receptor endocytic signaling
mediated by G-protein might be initiated by GRK2 and
-arrestin I
that are recruited indirectly to the PDGF
receptor via its
association with the S1P1 receptor. In this regard,
Watterson et al. (30) have recently shown constitutive
association of GRK2 with the S1P1 receptors.
receptor signal complexes is required for
activation of p42/p44 MAPK (31), and our results suggest that this is
driven by a GiPCR-dependent mechanism. Thus,
increased internalization of PDGF
receptor-S1P1 receptor
complexes in response to combined stimulation with S1P and PDGF will
improve the efficiency with which PDGF (and vice versa, S1P) stimulates
the p42/p44 MAPK pathway.
and c-Src (25, 31). In previous studies, we have shown that c-Src is
activated by PDGF, and this can be blocked by pretreating ASM cells
with pertussis toxin (25). Furthermore, there is an indication that
GRK2 is regulated by c-Src (21), thereby providing a functional link
between the PDGF
receptor and GRK2. The tyrosine phosphorylation of
Gab1 promotes the binding of phosphoinositide 3-kinase 1a (PI3K1a),
which in turn induces association of dynamin II to the
PI3K1a·Gab1·Grb-2 complex. This process appears to be
essential for clathrin-mediated endocytosis of PDGF receptor signal
complexes that include Raf-MAPK kinase 1 for re-localization with and
activation of cytoplasmic p42/p44 MAPK (31). In this regard, dynamin II
might function to promote "pinching off" of the endocytic
vesicles containing active PDGF
receptor-S1P1 receptor
signal complexes for subsequent activation of p42/p44 MAPK in the cytoplasm.
-adrenergic receptors. Under these conditions, isoprenaline induces the co-internalization of
-adrenergic receptors and EGF receptors to the same endocytic
vesicles. However, Maudsley et al. (32) report that EGF
alone does not induce internalization of the
-adrenergic receptor.
This contrasts with our findings, where we show that PDGF induces
co-internalization of both PDGF
receptor and the GPCR,
S1P1. In addition, the internalization of the PDGF
receptor in response to S1P is significant because this does not
involve PDGF release. We conclude that integrative signaling and
co-internalization of both PDGF
receptor and S1P1 receptor in response to PDGF and/or S1P is a novel important mechanism. It is clearly a different mechanism compared with receptor tyrosine kinase transactivation by GPCR agonists.
receptor and
S1P1 receptor may be a prototypical example for other
growth factor receptors. This is supported by several studies showing
that a number of growth factors use classic GPCR-mediated signaling
pathways to stimulate p42/p44 MAPK in mammalian cells (33, 34).
receptor-S1P1 receptor complex to induce
biological responses in vivo. This would also represent a
novel form of intercellular communication.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Bioscience and Biotechnology Research Council and The Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
¶ To whom correspondence should be addressed. Tel.: 44-141-5482659; Fax: 44-141-5522562; E-mail: n.j.pyne@ strath.ac.uk.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M208560200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
S1P, sphingosine
1-phosphate;
S1P1, S1P receptor;
ASM cells, airway smooth
muscle cells;
DMS, N,N- dimethylsphingosine;
EDG, endothelial differentiation gene;
Gi, inhibitory
G-protein;
Gi, alpha sub-unit of Gi;
Gs, stimulatory G-protein;
GPCR, G-protein coupled
receptor;
GRK, GPCR kinase;
HEK cells, human embryonic kidney cells;
MAPK, mitogen-activated protein kinase;
PDGF, platelet-derived growth
factor;
PKB, protein kinase B;
SK, sphingosine kinase;
tDHS, DL-threo-dihydrosphingosine;
TRITC, tetramethylrhodamine isothiocyanate;
EGF, epidermal growth factor;
PMA, phorbol 12-myristate 13-acetate.
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