(Received for publication, August 22, 1995; and in revised form, November 2, 1995)
From the
Activation of the platelet-derived growth factor (PDGF)
-receptor leads to cell growth and chemotaxis. The PDGF
-receptor also mediates a mitogenic signal, but fails to induce
cell migration in certain cell types. To examine this difference in
signal transduction, a series of point-mutated PDGF
-receptors
were analyzed. Porcine aortic endothelial cells expressing mutant PDGF
-receptors, in which tyrosine residues 768, 993, or 1018 were
changed to phenylalanine residues migrated toward PDGF, whereas
wild-type
-receptors and mutant
-receptors changed at
tyrosine residues 720, 944, or 988 failed to migrate. All mutant
receptors were mitogenically active and their capacity to activate
phosphatidylinositol 3`-kinase and phospholipase C-
was not
different from that of the wild-type receptor. Tyr-768 was found to be
phosphorylated in PDGF-stimulated cells; in the Y768F mutant, there was
a considerable increase in phosphorylation of Ser-767. Tyr-993 was not
phosphorylated, but mutation of this tyrosine residue to a
phenylalanine residue resulted in increased efficiency of
phosphorylation on Tyr-988. Tyr-1018 is known to be an
autophosphorylation site. Phosphorylated Tyr-768 and Tyr-1018 may bind
signal transduction molecules involved in negative modulation of the
chemotactic signaling capacity, whereas phosphorylated Tyr-988 may
mediate increased chemotaxis. Thus our data indicate that the PDGF
-receptor has an intrinsic ability to transduce a chemotactic
signal, and that this signal is counteracted by overriding negative
signals.
Platelet-derived growth factor (PDGF) ()is a potent
mitogen for mesenchymal cells. PDGF plays important roles in the
embryonal development and in wound healing, as well as in the
development of several pathological conditions, such as tumorigenesis
and atherosclerosis (reviewed in (1) and (2) ). PDGF
is a homo- or heterodimeric protein composed of A- and B-polypeptide
chains which elicits its effects by binding to cell surface receptors
designated
- and
-receptors(3, 4, 5) . The PDGF
-receptor is able to bind either A- or B-chains of PDGF, whereas
the
-receptor binds only the B chain.
Binding of PDGF activates
the intrinsic tyrosine kinase activity of the receptors through
receptor dimerization, which leads to receptor autophosphorylation and
to phosphorylation of intracellular substrates (6) . An
important role for receptor autophosphorylation is to present binding
sites for signal transduction molecules containing one or two copies of
so called Src homology 2 (SH2) domains, a conserved non-catalytic
stretch of about 100 amino acids found in signal transduction
molecules, and which mediates the interaction with the
receptor(7) . Nine and three autophosphorylation sites have so
far been identified in the PDGF - and
-receptors,
respectively. Several of these have been shown to interact in a
specific manner with certain SH2 domain-containing proteins (reviewed
in (8) ). For the PDGF
-receptor, two autophosphorylation
sites in the juxtamembrane domain (Tyr-579 and Tyr-581) mediate the
binding of Src family kinases. There are four autophosphorylation sites
in the kinase insert which bind Grb2 (Tyr-716), the regulatory subunit
(p85) of phosphatidylinositol 3`-kinase (PI 3-kinase; Tyr-740 and
Tyr-751), Nck (Tyr-751), and the GTPase activating protein (GAP) of Ras
(Tyr-771). The two autophosphorylation sites in the carboxyl-terminal
tail (Tyr-1009 and Tyr-1021) mediate binding of the SH2
domain-containing phosphatase PTP1D/Syp and phospholipase C-
(PLC-
), respectively. The adapter proteins Shc and Shb seem to
interact with multiple tyrosine residues (9, 37) . In
the case of the PDGF
-receptor, tyrosine residues 754, 988, and
1018 have been identified as autophosphorylation
sites(10, 11) . Among them, Tyr-1018 has been shown to
mediate association of PLC-
(11) . Tyr-731 and Tyr-742 are
important for the binding of PI 3-kinase(12) , and are likely
to be phosphorylation sites, although this has not been directly shown.
Both PDGF - and
-receptors mediate mitogenic signals upon
ligand stimulation, probably through multiple parallel signal
transduction pathways(13, 14, 15) . On the
other hand, the migratory response of PDGF
-receptor expressing
cells is critically dependent on activation of PI
3-kinase(16, 17) . Although the PDGF
-receptor
also activates PI 3-kinase, this receptor mediates migration in a cell
type-specific manner. Thus, Swiss 3T3 cells and human granulocytes
expressing endogenous PDGF
-receptors (18, 19) and 32D cells expressing the PDGF
-receptors after transfection (12) allows chemotaxis
toward PDGF-AA. In other cell types, such as human foreskin
fibroblasts(20) , human monocytes(19) , rat and baboon
vascular smooth muscle cells(21) , PDGF-AA fails to induce
chemotaxis. In these cell types, which express both PDGF
- and
-receptors, PDGF-AA has also been shown to inhibit chemotaxis
induced by PDGF-AB or -BB(19, 22) . The mechanisms
underlying the cell type-specific effect of the PDGF
-receptor on
chemotaxis is still unknown.
Expression of the PDGF -receptor
after transfection in porcine aortic endothelial (PAE) cells, which
lack endogenous PDGF receptors, allow the cells to migrate toward
PDGF-BB, whereas PAE cells expressing the PDGF
-receptor do not
migrate toward PDGF-AA or -BB(23) , thus offering a suitable
model for analysis of the molecular mechanisms responsible for
-receptor-induced suppression of chemotaxis.
Here, we show that
mutations of three tyrosine residues in the -receptor allows the
transduction of a chemotactic response of PAE cells expressing the
mutant
-receptors. Characterization of the pattern of
autophosphorylation in these mutants implicated the presence of
multiple pathways mediated via the PDGF
-receptor which control
cellular chemotaxis.
Figure 1:
Schematic
illustration of the location of tyrosine residues in the intracellular
domains of the PDGF - and
-receptors and their interacting
SH2 domain-containing proteins. The numbers stand for
positions of the tyrosine residues; numbers in ovals represent uniquely positioned tyrosine residues, whereas numbers in squares indicate tyrosine residues
positioned in regions conserved between the two types of receptors. JM, juxtamembrane domain; TK1, first part of the
tyrosine kinase domain; KI, kinase insert; TK2,
second part of the tyrosine kinase domain; CT, carboxyl-terminal tail.
Figure 2:
Ligand-stimulated kinase activity of the
wild-type , wild-type
, and the tyrosine residue-mutated PDGF
-receptors. PAE cells expressing the wild-type and mutant PDGF
receptors were incubated without(-) or with (+) 100 ng/ml
PDGF-BB for 30 min at 4 °C. The cells were lysed and
immunoprecipitated with anti-receptor antisera. The samples were
subjected to kinase assays as described under ``Experimental
Procedures,'' followed by SDS-PAGE, and
autoradiography.
Figure 3:
Chemotaxis of cells expressing the
wild-type PDGF -receptors, the wild-type, or mutant PDGF
-receptors. Non-transfected PAE cells (
) and PAE cells
expressing the wild-type PDGF
-receptor (
), wild-type
(
), Y720F (
), Y768F (
), Y944F (
), Y988F
(
), Y993F (
), or Y1018F (
) PDGF
-receptors were
analyzed for their migration toward different concentrations of
PDGF-BB. Stimulated random migration (chemokinesis) was estimated as
described under ``Experimental Procedures'' and is referred
to as 100% migration. The data show mean ± S.E. of three
experiments. At least two PAE cell clones for each of the wild-type or
mutant receptors were analyzed and gave similar
results.
Figure 4:
A,
thin-layer chromatography of PI 3-kinase reaction products from
unstimulated or ligand stimulated cells. Cells expressing the wild-type
PDGF -receptors, wild-type PDGF
-receptors, Y768F, Y993F, or
Y1018F mutant PDGF
-receptors were incubated without(-) or
with (+) 50 ng/ml PDGF-BB for 5 min at 37 °C. After
incubation, the cells were lysed and immunoprecipitated with
anti-receptor antiserum. The immunoprecipitates were subjected to PI
3-kinase assays and the PI 3-kinase reaction products were analyzed by
thin-layer chromatography followed by autoradiography. The positions of
phosphatidylinositol phosphate and the origin (ORI) are
indicated. B, tyrosine phosphorylation of PLC-
after
PDGF-BB stimulation of PAE cells expressing wild-type or tyrosine
residue-mutated PDGF receptors. Cells expressing the wild-type PDGF
-receptors, wild-type PDGF
-receptors, Y768F, Y993F, or
Y1018F mutant PDGF
-receptors were incubated without(-) or
with (+) 100 ng/ml PDGF-BB for 30 min at 4 °C. After
incubation, the cells were lysed and immunoprecipitated with
anti-PLC-
monoclonal antibody (left panels) or with
anti-receptor antiserum (right panel). The immunoprecipitates
were separated by SDS-PAGE and transferred onto nitrocellulose
membranes. The blots were probed with either anti-phosphotyrosine
monoclonal antibody PY20 or with anti-PLC-
antibody and then
visualized using ECL Western blotting detection system (Amersham).
Migration positions of the PDGF
-receptors (
R), PDGF
-receptors (
R), and PLC-
are indicated. Density
of the detected bands were measured by densitometric scanning. The
relative densities of the tyrosine phosphorylated PLC-
/tyrosine
phosphorylated PDGF receptor for the wild-type
-, wild-type
-, Y768F
-, Y993F
-, and Y1018F
-receptors were
1.1, 0.074, 0.095, 0.098, and 0.000, respectively. C,
formation of inositol phosphates in PAE cells expressing wild-type or
tyrosine-residue mutated PDGF receptors. Cells labeled with myo-[
H]inositol (1.5 µCi/ml) for 48
h were incubated in presence of vehicle (open bars), 50 ng/ml
PDGF-BB (solid bar), or 5% FCS (shaded bars) for 15
min at 37 °C. The samples were quenched by addition of 30%
trichloroacetic acid. The total inositol phosphates were separated by
anion exchange chromatography and the amount measured by scintillation
counting. The values represent means ± S.D. of triplicate
determinations.
It has been suggested that the SH2 domain-containing proteins
PLC- and RasGAP are involved in modulation of the chemotactic
response(17, 31) ; PLC-
in a positive and RasGAP
in a negative manner. We examined complex formation and tyrosine
phosphorylation of these signaling proteins upon receptor activation.
The different cell types were stimulated with PDGF-BB for 30 min on ice
and then lysed. The lysates were immunoprecipitated either with
anti-PLC-
antiserum or with anti-receptor antiserum, separated by
SDS-PAGE, transferred onto nitrocellulose membranes; the membranes were
then immunoblotted with the anti-phosphotyrosine antibody PY20. Upon
ligand stimulation of wild-type PDGF
-receptor, wild-type
-receptor, and the mutant Y768F and Y993F
-receptors,
PLC-
became tyrosine phosphorylated and was found in complex with
the autophosphorylated receptors. Phosphorylation of PLC-
was not
detected upon activation of mutant Y1018F PDGF
-receptors, in
agreement with the recent characterization of Tyr-1018 as the major
PLC-
binding site in the PDGF
-receptor(11) .
Eriksson et al.(11) also showed that a higher extent
of complex formation between the receptor and PLC-
is observed
upon activation of the PDGF
-receptor compared to the
-receptor; however, tyrosine phosphorylation and consequently
activation of the catalytic activity of PLC-
, is considerably more
efficient in activated PDGF
-receptor cells than in PDGF
-receptor cells. There was no difference in the efficiency of
tyrosine phosphorylation of PLC-
between the wild-type
-receptor and the Y768F and Y993F mutant
-receptors as
assessed by densitometric scanning of the immunoblots in Fig. 4B. In order to examine whether the efficiency of
induction of the enzymatic activity of PLC-
differed between the
wild-type and the chemotactic mutant
-receptors, PDGF-induced
formation of inositol phosphates was measured (Fig. 4C). Cells were labeled with myo-[
H]inositol for 48 h, incubated with
or without 50 ng/ml PDGF-BB for 15 min at 37 °C, and the samples
were extracted by addition of 30% trichloroacetic acid to the cell
monolayer. Total inositol phosphates were collected using AG 1-X8
formate resin. Upon PDGF-BB stimulation, the total amount of
radioactively labeled inositol phosphates increased to 160% of the
control value in the wild-type
-receptor expressing cells, while
the wild-type
-receptor expressing cells showed only a slight
increase. The difference in the level of increase in total inositol
phosphates between the PDGF
- and
-receptors is consistent
with results previously described for primary human fibroblasts (11) and rat vascular smooth muscle cells(32) . Like in
the wild-type
-receptor, the extent of accumulation of total
inositol phosphates were negligible in the Y768F, Y993F, or Y1018F
chemotactic mutant
-receptors. Thus, it is not likely that
PLC-
catalytic activity is involved in mediation of chemotaxis of
the mutant
-receptor cells.
Upon ligand stimulation of the
wild-type PDGF -receptor expressing cells, RasGAP was tyrosine
phosphorylated and in complex with the receptor, as judged from
coimmunoprecipitation of the receptor when a RasGAP antiserum was used.
However, neither activation of the wild-type nor the chemotactic mutant
PDGF
-receptors induced phosphorylation or association of RasGAP
with the receptors (data not shown). This is in agreement with previous
reports by Heideran et al.(33) and Bazenet et
al.(34) , in which RasGAP is described to be a substrate
for the PDGF
-receptor but not for the
-receptor.
In
conclusion, these observations argue against modulation of PDGF
-receptor-mediated tyrosine phosphorylation or activation of PI
3-kinase, RasGAP, or PLC-
, as a reason for the gain of chemotactic
ability of the Y768F, Y993F, and Y1018F mutant
-receptors.
Figure 5:
A, two-dimensional tryptic phosphopeptide
maps of the wild-type and Y768F PDGF -receptors. Cells expressing
the wild-type or Y768F PDGF
-receptors were labeled in vivo with [
P]orthophosphate, stimulated with
PDGF-BB, and immunoprecipitated with anti-receptor antiserum. The
immunoprecipitates were separated by SDS-PAGE and transferred onto
nitrocellulose membranes. The bands corresponding to the PDGF
-receptors were cut out, digested with trypsin in situ,
oxidized, and again extensively digested with trypsin. The resulting
digests were separated electrophoretically at pH 1.9, followed by
ascending chromatography. Radioactive phosphopeptides were visualized
by autoradiography. The position of a phosphopeptide spot present in
the wild-type
-receptor map but not in the Y768F
-receptor
map is indicated by an arrowhead. In each case, the origin is
marked with an open triangle. B, Edman degradation
elution profile and phosphoamino acid analysis (inset) of the
phosphopeptide indicated by an arrowhead in the wild-type PDGF
-receptor map in A. The phosphopeptide was extracted from
TLC plate, subjected to Edman degradation, and the
P
radioactivity in the fractions generated in each cycle was measured.
The amino acid sequence of the tryptic peptide containing Tyr-768 is
presented along with the fraction numbers. Migration positions of
serine and tyrosine in the phosphoamino acid analysis are indicated as S and Y, respectively. C, elution profile by
Edman degradation and phosphoamino acid analysis of the tryptic
peptides from the wild-type (upper panel) and Y768F (lower
panel) PDGF
-receptors immunoprecipitated with YSD antiserum.
The amino acid sequence of the YSD-immunoprecipitated peptide is
presented along with the fraction numbers. Results of the phosphoamino
acid analysis on the peptides are shown on the right-hand side of each panel.
If both Tyr-762 and Tyr-768 are autophosphorylation sites, one would expect a peptide containing phosphorylated Tyr-762 to be recognized in the Y768F receptor map. This peptide would be expected to become more positively charged and hydrophobic compared to the corresponding wild-type peptide, due to replacement of the phosphorylated tyrosine residue with a phenylalanine residue. Thus, the peptide is expected to migrate more toward the right (toward the anode) upon electrophoresis, and upwards upon chromatography, compared to the wild-type peptide. In the particular experiment shown in Fig. 5A, ascending chromatography was run extensively to obtain high resolution of the Tyr-768-containing spot in the wild-type receptor map, which resulted in loss of some phosphopeptides from the TLC plate. When the peptides were run for a shorter time in the ascending chromatography, at least two phosphopeptide spots were found in the upper right corner of the Y768F receptor map but not in the wild-type receptor map (data not shown). One of the two peptides was phosphorylated on serine and radioactivity appeared at cycle 8, the other showed tyrosine and serine phosphorylation and radioactivity appeared at cycles 3 and 8, as judged from phosphoamino acid analysis and Edman degradation (data not shown). These phosphopeptides most likely represented the peptides containing Tyr-762 and Phe-768.
In order to confirm phosphorylation on tyrosine
residues 762 and 768, and to further examine serine phosphorylation in
the peptide, tryptic digests from the in vivoP-labeled wild-type and Y768F PDGF
-receptors
were immunoprecipitated with an antiserum which specifically recognizes
Tyr-768 and the surrounding amino acid residues (YSD antiserum). The
immunoprecipitates were eluted and subjected to Edman degradation. As
shown in Fig. 5C, upper panel, radioactive
peaks were detected at cycles 3, 8, and 9 in the wild-type receptor
fragment, corresponding to Tyr-762, Ser-767, and Tyr-768. Phosphoamino
acid analysis of the immunoprecipitated peptide revealed
phosphorylation strongly on tyrosine and weakly on serine. In the
corresponding analysis of the Y768F PDGF
-receptor (Fig. 5C, lower panel), peaks of phosphorylation were
seen at cycles 3 and 8, which corresponded to Tyr-762 and Ser-767.
Radioactivity in cycle 9 most likely represents trailing from cycle 8.
The result of phosphoamino acid analysis of the immunoprecipitated
Y768F peptide revealed a dramatic increase in the ratio of serine
phosphorylation to tyrosine phosphorylation compared to that of the
corresponding peptide from the wild-type receptor. The ratios of the
radioactive content of cycle 8 to cycle 3 were 0.26 for the wild-type
peptide and 1.57 for the Y768F peptide, which suggested that
substitution of Tyr-768 to a phenylalanine residue resulted in
considerable enhancement of phosphorylation on Ser-767.
Figure 6:
A, two-dimensional tryptic phosphopeptide
maps of the wild-type, Y993F, and Y1018F PDGF -receptors. Cells
expressing the wild-type, Y993F, or Y1018F PDGF
-receptors were
labeled in vivo with [
P]orthophosphate,
stimulated with PDGF-BB, and immunoprecipitated with anti-receptor
antiserum. The immunoprecipitates were separated by SDS-PAGE and
transferred onto nitrocellulose membranes. The bands corresponding to
the PDGF
-receptors were cut out, digested with trypsin in
situ, oxidized, and again extensively digested by trypsin. The
resulting digests were separated electrophoretically at pH 1.9,
followed by ascending chromatography. Radioactive phosphopeptides were
visualized by autoradiography. An intense spot evident in the Y993F map
is indicated by an arrow. Arrowheads indicate the
positions of the phosphopeptides containing Tyr-1018. In each case, the
origin is marked with an open triangle. B, Edman
degradation elution profile and phosphoamino acid analysis (inset) of the phosphopeptide indicated by the arrow in the Y993F map in A. The phosphopeptide was extracted
from a TLC plate, subjected to Edman degradation, and the
P radioactivity in the fractions generated in each cycle
was measured. The amino acid sequences of all the possible PDGF
-receptor-derived tryptic peptides, containing a tyrosine residue
in the seventh position, are presented along with the fraction numbers. Asterisk (*) indicates the Phe-993
residue.
We argued that Tyr-988
could be phosphorylated to an increased extent in the Y993F PDGF
-receptor, because of the closeness of these residues. To examine
this possibility, the following experiment was performed. The
wild-type, Y988F, Y993F, and Y1018F PDGF
-receptors were labeled in vivo, autophosphorylated, and immunoprecipitated according
to the procedure described above. The receptor proteins were then
chemically cleaved with cyanogen bromide resulting in peptide fragments
of relatively large sizes. A group of fragments from the
carboxyl-terminal tail of the receptors (amino acid residues 981 to
1072) were collected by immunoprecipitation using a specific antiserum
raised against amino acid residues 1042 to 1061 of the
-receptor
(SQT antiserum). The immunoprecipitated fragments were further digested
by incubation with trypsin and then analyzed by two-dimensional
phosphopeptide mapping. The tryptic map derived from the
immunoprecipitated cyanogen bromide fragment showed five radioactive
phosphopeptide spots (Fig. 7A), designated spots 1 to 5 (Fig. 7B). In the map from Y1018F
-receptors,
spots 1 and 2 were missing as compared to the wild-type
-receptor
map. Edman degradation of peptides 1 and 2 in the wild-type receptor
map showed tyrosine phophorylation at the seventh amino acid from the
amino terminus (data not shown), in agreement with the notion that the
peptides contained phosphorylated Tyr-1018. The spots 3, 4, and 5 were
lacking in the Y988F
-receptor map, suggesting that these spots in
the wild-type
-receptor map represented peptides containing
Tyr-988. Interestingly, the radioactive content of spots 4 and 5 from
the Y993F
-receptor was 50% higher as compared with the
corresponding spots derived from the wild-type
-receptor. The
radioactive contents of spots 1 and 2 were the same in the two cases.
These results indicate that Tyr-988 is phosphorylated with increased
stoichiometry following mutation of Tyr-993 to a phenylalanine residue,
without affecting the phosphorylation efficiency on Tyr-1018.
Figure 7:
A,
two-dimensional tryptic phosphopeptide maps of cyanogen bromide-cleaved
fragments derived from the carboxyl-terminal tail of wild-type, Y988F,
Y993F, and Y1018F PDGF -receptors. Cells expressing the wild-type,
Y988F, Y993F, or Y1018F PDGF
-receptors were labeled in vivo with [
P]orthophosphate, stimulated with
PDGF-BB, and immunoprecipitated with anti-receptor antiserum. The
immunoprecipitates were separated by SDS-PAGE and transferred onto
nitrocellulose membranes. The band corresponding to the PDGF
-receptor was cut out, cleaved with cyanogen bromide, and
immunoprecipitated with SQT antiserum. The immunoprecipitated fragment
was oxidized and digested with trypsin. The resulting digests were
separated electrophoretically at pH 1.9, followed by ascending
chromatography. Radioactive phosphopeptides were visualized by
Bio-imaging analyzer BAS 2000 (Fuji photo film). The origin of each
sample has been trimmed away in the figure, but is located outside the
lower-left corner of each panel. B, quantification of
radioactivity in the phosphopeptide spots in the tryptic maps in A. The intensities of the phosphopeptide spots (designated
spots 1 to 5) in A were measured using BAS
2000. In order to compare the intensity values of the spots between the
different receptor maps, the value for spot number 1 was fixed to 100
in each map. Intensities of other spots were calculated relative to the
values for spot number 1. Arrows indicate the recorded major
changes in phosphorylation.
It is well established that PDGF -receptor expressing
cells migrate efficiently toward PDGF-BB. Data based on
-receptors
lacking the PI 3-kinase binding site(16, 17) , as well
as treatment with the PI 3-kinase inhibitor wortmannin and expression
of a mutated PI 3-kinase p85 subunit, (
)indicate that PI
3-kinase activity is crucial for
-receptor-mediated chemotaxis.
The ligand-stimulated PDGF
-receptor also activates PI 3-kinase,
but fails to induce chemotaxis in certain cell types. We show that
point-mutated
-receptors can acquire the capacity to mediate
chemotaxis of PAE cells, in which the wild-type
-receptor is
chemotactically inactive. Our data imply that the lack of wild-type
-receptor-mediated chemotaxis in the PAE cells and other cell
types is not due to a lack of ability to activate the signal
transduction pathways leading to directed migration. Rather, one or
more negative signals are also activated by the
-receptor which
suppress the migratory response. Since PI 3-kinase was active in the
ligand-stimulated chemotactic mutants, the negative signals are likely
to act downstream of PI 3-kinase. PLC-
, which has been described
to be involved in positive regulation of chemotaxis in certain cell
types (16, 17) is most likely not responsible for
modulation of
-receptor-mediated chemotaxis, since the Y1018F
mutant, which is unable to bind and activate PLC-
, was able to
mediate chemotaxis. RasGAP has been inferred to negatively modulate
PDGF
-receptor mediated chemotaxis(17) . Neither the
wild-type, nor the chemotactically active mutant
-receptors,
associated with or phosphorylated RasGAP, arguing against a role for
RasGAP in
-receptor signaling.
The PDGF -receptor can thus
emit both positive and negative signals influencing the migratory
response. The capacity to emit these signals appear to depend on the
cellular environment. Interestingly, the negative signals can affect
migration initiated by other ligand-receptor complexes. We have
previously shown that activation of the PDGF
-receptor leads to
inhibition of PDGF
-receptor-mediated chemotaxis, in a
dose-dependent manner(19) . Koyama et al.(21, 22) described that activation of the PDGF
-receptor in vascular smooth muscle cells led to inhibition of
chemotaxis induced by the PDGF
-receptor, or by receptors for
fibronectin and smooth muscle cell-derived growth factor. It is likely
that the negative signals acting on the
-receptor-induced
chemotaxis and on chemotaxis induced by other types of receptors are
the same.
Koyama et al.(21, 22) showed
that PDGF -receptor-induced inhibition of fibronectin-mediated
chemotaxis was attenuated by treatment of the vascular smooth muscle
cells with the protein kinase C (PKC) inhibitor staurosporine
suggesting that PKC is a mediator of PDGF
-receptor negative
signaling. However, another PKC inhibitor H-7, and the PKC activators
12-O-tetradecanoylphorbol-13-acetate and SC-9, had no effect
in their study, which together with the rather low specificity of
staurosporine for PKC(35) , may suggest involvement of a
different serine/threonine kinase in the negative signaling.
Diliberto et al.(36) showed that PDGF-BB
(activating both - and
-receptors) was more potent than
PDGF-AA (activating only
-receptors) in stimulating Ca
fluxes in Balb/c3T3 fibroblasts. In addition, preincubation of
the cells with PDGF-AA lead to inhibition of the PDGF-BB-induced
increase in the intracellular Ca
levels, in part
through PKC-dependent mechanisms. It is an interesting possibility,
which remains to be tested, that differences in the modulation of
intracellular Ca
levels account for the differences
in chemotactic signaling between the wild-type
-receptor and
-receptor mutants.
Our data indicate that several mechanisms
are operating in negative modulation of PDGF -receptor negative
signaling. Phosphorylation of Tyr-768, which is surrounded by the motif
PASY768KKK, appears to allow negative modulation of chemotaxis, maybe
by binding a specific signal transduction molecule. This signal
transduction molecule would be activated by the
-receptor but not
by the
-receptor, since there is no tyrosine residue with a
similar environment in the
-receptor. It is, however, also
possible that the gain of chemotactic ability of the Y768F
-receptor mutant was due to the increased phosphorylation of
Ser-767 in the mutant receptor expressing cells, which could lead to
positive modulation of chemotaxis. Analysis of a double mutant,
simultaneously changed at Ser-767 and Tyr-768, will allow us to
differentiate between these two alternatives. Tyr-762 was also shown to
be an autophosphorylation site in our study. Although this residue is a
positional homologue of Tyr-771 in the
-receptor, which is the
binding site for RasGAP, the
-receptor does not bind RasGAP.
Distinct surrounding motifs for the two tyrosine residues, RSLY762DRP
in the
-receptor and SSNY771MAP in the
-receptor, probably
account for the difference in binding of RasGAP. The
-receptor
Tyr-993 is not phosphorylated, but we could show that there was
increased phosphorylation of Tyr-988 in the Y993F mutant receptor
expressing cells. Tyr-988 is surrounded by the motif DNAY988IGV which
is not found in the
-receptor. It is possible that the increased
phosphorylation of Tyr-988 in the Y993F mutant receptor cells allows
positive modulation of chemotaxis, by a more efficient recruitment of a
specific signal transduction molecule.
The mechanism by which the
Y1018F -receptor mutant allowed chemotaxis is not clear. It is not
likely that the loss of PLC-
binding resulted in the chemotactic
phenotype, since PLC-
has been shown to act positively (17) or to be without effect (16) on
-receptor-mediated chemotaxis. Moreover, activation of the
catalytic activity of PLC-
is not prominent in ligand-stimulated
wild-type or mutant
-receptor expressing PAE cells (Fig. 5)
or in other cell types expressing endogenous receptors, such as
vascular smooth muscle cells (32, 35) or human
foreskin fibroblasts(11) . It is possible that a signal
transduction molecule distinct from PLC-
can also bind to
phosphorylated Tyr-1018 and mediate the negative signal for chemotaxis.
The different potential pathways for modulation of PDGF
-receptor-induced chemotaxis are summarized in Fig. 8. We
suggest that autophosphorylated tyrosine residues 768 and 1018 serve to
mediate negative regulation of chemotaxis, downstream of PI 3-kinase.
In contrast, it is possible that Tyr-988 mediates a signal for positive
regulation. The negative signaling for modulation of chemotaxis appears
to act on the level of directed migration only, since both membrane
edge ruffling, which can be regarded as an integral part of the
cellular motility response(16) , as well as random migration,
were induced to the same level in ligand-stimulated wild-type and
mutant
-receptor PAE cells, as in the wild-type PDGF
-receptor expressing cells. (
)This is in agreement with
the observation by Koyama et al.(21) that PDGF-AA
inhibited PDGF-BB-induced chemotactic activity but not random migration
of baboon smooth muscle cells. Identification of the signal
transduction molecules that interact with the phosphorylation sites
determined in this work, Tyr-768 and Tyr 988, and elucidation whether
molecules other than PLC-
interact with phosphorylated Tyr-1018,
may reveal the basis for the cell-type specific pattern of
-receptor negative signaling. Such efforts should also broaden our
understanding of how directed movement of cells is controlled.
Figure 8:
Schematic illustration of possible modes
of modulation of chemotactic signaling by the PDGF -receptor. Numbers indicate the positions of tyrosine residues. (P) indicates that the tyrosines are phosphorylatable. +,
positive modulation; -, negative modulation; PI3-K,
phosphatidylinositol 3`-kinase. Mutation of the phosphorylatable
tyrosine residues 768 or 1018 to phenylalanine residues relieves a
putative negative regulatory mechanisms, whereas mutation of Tyr-993
causes an increased autophosphorylation of Tyr-988 possibly resulting
in an enhanced chemotactic signaling.