(Received for publication, September 8, 1994; and in revised form, January 18, 1995)
From the
Phosphorylated tyrosine residues in receptor tyrosine kinases
serve as binding sites for signal transduction molecules. We have
identified two autophosphorylation sites, Tyr-988 and Tyr-1018, in the
platelet-derived growth factor (PDGF) -receptor carboxyl-terminal
tail, which are involved in binding of phospholipase C-
(PLC-
). The capacities of the Y988F and Y1018F mutant PDGF
-receptors, expressed in porcine aortic endothelial cells, to bind
PLC-
are 60 and 5% of that of the wild-type receptor,
respectively. Phosphorylated but not unphosphorylated peptides
containing Tyr-1018 are able to compete with the intact receptor for
binding to immobilized PLC-
SH2 domains; a phosphorylated Tyr-988
peptide competes 10 times less efficiently. The complex between
PLC-
and the PDGF
-receptor is more stable than that of
PLC-
and the PDGF
-receptor. However, PDGF stimulation
results in a smaller fraction of tyrosine-phosphorylated PLC-
and
a smaller accumulation of inositol trisphosphate in cells expressing
the
-receptor as compared with cells expressing the
-receptor. We conclude that phosphorylated Tyr-988 and Tyr-1018 in
the PDGF
-receptor carboxyl-terminal tail bind PLC-
, but this
association leads to only a relatively low level of tyrosine
phosphorylation and activation of PLC-
.
Platelet-derived growth factor (PDGF) ()is a
polypeptide mitogen that acts on a broad spectrum of cells, including
fibroblasts and glial cells (for reviews, see (1) and (2) ), and that consists of disulfide-linked dimers of A and B
polypeptide chains, forming three possible isoforms, PDGF-AA, -AB, and
-BB. PDGF mediates its biological effects by interacting with two
structurally related receptor types that differ in their interactions
with the PDGF isoforms (for a review, see (3) ). Thus, the
-receptor binds PDGF A- and B-chains, whereas the
-receptor
binds only the B-chain. Binding of ligand induces receptor
dimerization, activation of the receptor tyrosine kinase, and
autophosphorylation on multiple tyrosine residues in the intracellular
region of the receptors (for a review, see (4) ). The
phosphorylated tyrosine residues of the receptor form specific binding
sites for downstream signaling components, containing one or more
100-amino acid residue motifs denoted Src homology 2 (SH2) domains
(reviewed in (5) ). The specificity of the association is
defined both by the amino acid residues surrounding the phosphotyrosine
and the structure of the SH2 domain. The signal transduction molecules
either have endogenous catalytic activities or serve as adaptor
molecules, which lack enzymatic activity but interact with catalytic
components. The SH2 domain-containing proteins that associate with PDGF
-receptors and that have been identified include phospholipase
C-
(PLC-
), the regulatory subunit (p85) of
phosphatidylinositol 3` kinase, members of the Src family of protein
tyrosine kinases, the Ras GTPase activating protein, and most recently
the phosphatase SH-PTP 1D/SH-PTP 2/Syp and the adaptor proteins Grb2,
Nck, Shb, and Shc (reviewed in (3) ). In addition, the two PDGF
receptors interact with other unidentified substrates(6) .
Both - and
-receptors mediate mitogenic signals; however,
signals leading to chemotaxis and membrane edge ruffling are mediated
through the
-receptor, while the
-receptor, in certain cell
types, conveys a negative signal that inhibits chemotaxis. Eight
autophosphorylation sites have been mapped in the PDGF
-receptor:
Tyr-579 and Tyr-581 in the juxtamembrane region(7) , Tyr-740,
Tyr-751, and Tyr-771 in the kinase insert(8, 9) ,
Tyr-857 in the kinase domain (10) , and Tyr-1009 and Tyr-1021
in the carboxyl-terminal
tail(11, 12, 13, 14) . Seven of
these residues are conserved in the PDGF
-receptor at positions
that are homologous with those in the
-receptor. We report the
mapping of two autophosphorylation sites, Tyr-988 and Tyr-1018, in the
carboxyl-terminal tail of the human PDGF
-receptor and show that
these sites are required for the interaction of the
-receptor with
PLC-
. Furthermore, we show that the human PDGF
- and
-receptors differ quantitatively in their abilities to associate
with and phosphorylate PLC-
and to stimulate inositol phosphate
production.
In vitro labeling of
wild-type and tyrosine-mutated PDGF -receptors was performed
essentially as follows. PDGF-BB-stimulated and control cells were
solubilized as described above and immunoprecipitated with PDGFR-7. The
protein A-Sepharose beads were washed four times with lysis buffer and
once with 20 mM Tris-HCl, pH 7.5, 150 mM NaCl. In
vitro
P labeling was performed by incubating in a
solution containing 20 mM HEPES, pH 7.5, 10 mM MnCl
, 1 mM dithiothreitol, and 50 µCi
[
-
P]ATP (Amersham; specific activity, 3000
Ci/mmol) for 10 min at 25 °C. The samples were then digested,
immunoprecipitated, and washed as described above, except that the
final wash was with 20 mM Tris-HCl, pH 7.5. The peptide was
eluted from the beads with 8 M urea in 0.1 M Tris-HCl, pH 8.8. Digestion with A. lyticus protease I,
desalting, and Edman degradation was performed as described above.
Immunocomplex kinase assays were
performed as previously described(6) . Essentially, P labeling of the immunoprecipitates, generated as
described above, was performed in a 40-µl reaction volume
containing 20 mM HEPES, pH 7.5, 10 mM MnCl
, 1 mM dithiothreitol, and 5 µCi
[
-
P]ATP (Amersham; specific activity, 3000
Ci/mmol) that was incubated for 10 min at 25 °C. The kinase
reactions were terminated by addition of 40 µl of
twice-concentrated SDS sample buffer and incubated at 95 °C for 4
min. Samples were analyzed by SDS-PAGE followed by cross-linking with
glutaraldehyde and treatment with 1 M KOH at 55 °C for 1 h
to preferentially decrease serine phosphate levels. The gels were then
dried and exposed to Fuji RX film with intensifying screen.
Quantitation of precipitated PDGF
-receptor was performed using a
PhosphorImager (Molecular Dynamics).
Figure 1:
Edman degradation of in vivo labeled carboxyl-terminal PDGF -receptor fragments and
quantification of
P radioactivity in each cycle. A, PAE cells expressing the wild-type
-receptor were
labeled with [
P]orthophosphate, stimulated with
PDGF-BB, lysed, and immunoprecipitated with anti-phosphotyrosine
monoclonal antibodies. The immunoprecipitates were separated by
SDS-PAGE and transferred onto a nitrocellulose filter, and the band
corresponding to the phosphorylated receptor was cut out and treated
with CNBr. The fragments were immunoprecipitated with AbP
1, an
antiserum raised against a synthetic peptide located in the carboxyl
terminus of the
-receptor. B, an aliquot of the purified
CNBr peptide was further degraded with A. lyticus protease I,
which cleaves carboxyl-terminal of lysine residues. The resulting
digest was subjected to Edman degradation followed by measurement of
P radioactivity in each cycle. The amino acid sequences of
the carboxyl-terminal peptides are presented along with the fraction
numbers. Tyr-988 is shown in boldlettertype in A, and Tyr-988, Tyr-1018, Ser-1041, and Thr-1066 are
shown in boldletters in B. Quantification
of the radioactivity in each cycle was performed by use of a
PhosphorImager instrument, as described under ``Materials and
Methods.''
Figure 2:
Edman degradation of carboxyl-terminal
fragments derived from in vitro labeled wild-type and tyrosine
residue-mutated PDGF -receptors. PAE cells expressing the
wild-type
-receptor, or Y988F or Y1018F mutant receptors, were
stimulated with PDGF-BB, lysed, and immunoprecipitated with PDGFR-7, an
-receptor-specific antiserum. The immunoprecipitates were labeled in vitro using [
-
P]ATP, separated
by SDS-PAGE, and transferred to a nitrocellulose filter; the band
corresponding to the phosphorylated receptor was cut out. The samples
were treated with CNBr, immunoprecipitated with AbP
1 (reactive
with the carboxyl-terminal PDGF
-receptor tail), and further
digested with A. lyticus protease I. The digests were
subjected to Edman degradation followed by measurement of
P radioactivity in each cycle. The amino acid sequences of
the carboxyl-terminal peptides are presented along with the fraction
numbers; Tyr-988 and Tyr-1018 are shown in boldletters.
To confirm that Tyr-988 and Tyr-1018
are autophosphorylation sites, we used site-directed mutagenesis to
change the residues individually to phenylalanine residues. The mutated
receptors were stably expressed in PAE cells (see below), which were
stimulated with PDGF-BB, immunoprecipitated with an
-receptor-specific antiserum (PDGFR-7), and phosphorylated in
vitro. The receptors were purified and fragmented with CNBr,
followed by isolation of the carboxyl-terminal peptide, which was
further digested with A. lyticus protease I, as described
above. Edman degradation was performed on the peptides followed by
analysis of
P radioactivity in the different fractions. As
shown in Fig. 2, radioactivity from the wild-type receptor was
detected at cycles 8 and 17. In the Y988F mutant receptor,
radioactivity was found at cycle 17 but not at cycle 8, whereas in the
Y1018F mutant receptor, radioactivity was detected at cycle 8 but not
at cycle 17, as expected. The results demonstrate that Tyr-988 and
Tyr-1018 are autophosphorylation sites both in vivo and in
vitro.
The ability of the wild-type and mutant PDGF
-receptor to transduce mitogenic signals was investigated by a
[
H]thymidine incorporation assay. We have
previously shown that the wild-type
-receptor responds
mitogenically to PDGF-AA and PDGF-BB stimulation (6) . As seen
in Fig. 3, both wild-type and mutant receptor-expressing cell
lines were found to respond, in a similar dose-dependent manner, to
PDGF-BB stimulation by increased incorporation of
[
H]thymidine. Non-transfected PAE cells were, as
expected, negative. The slight deviation by the mutant receptor cell
lines, in response to lower concentrations of PDGF, compared with that
of wild-type receptorexpressing cells, was not due to a lower affinity
of PDGF (data not shown); it is possible that the differences are
related to the slightly lower receptor number on the mutant
receptorexpressing cells. Thus, the tyrosine-mutated receptors are able
to induce DNA synthesis.
Figure 3:
Stimulation of
[H]thymidine incorporation by PDGF-BB in cells
expressing wild-type and tyrosine residue-mutated PDGF
-receptors.
Serum-starved PAE cells expressing the wild-type
-receptor (closedcircles), Y988F (opensquares), Y1018F (closedsquares)
mutated receptors, and non-transfected control cells (opencircles) were incubated with
[
H]thymidine at the indicated concentrations of
PDGF-BB for 24 h. Trichloroacetic acid-precipitable radioactivity was
determined as described under ``Materials and Methods'' and
expressed as percent of the incorporation in control
cultures.
Figure 4:
Tyrosine phosphorylation and association
of PLC- with ligand-activated wild-type and tyrosine
residue-mutated PDGF
-receptors. PAE cells expressing the
wild-type
-receptor or Y988F or Y1018F mutated receptors were
incubated with or without 100 ng/ml PDGF-AA or PDGF-BB for 1 h at 4
°C. The cells were solubilized, and the lysates were subjected to
immunoprecipitation using the receptor antiserum PDGFR-7 (A)
or the PLC-
antiserum (B). The immunoprecipitates were
separated by SDS-PAGE and transferred to nitrocellulose membranes. The
blots were probed with the phosphotyrosine monoclonal antibody PY20,
and the reactions were visualized using the ECL Western blotting
detection system. The filter shown in panelB was
stripped and reprobed with PLC-
antiserum (C) and, after
a second stripping, reprobed with PDGF
-receptor antiserum (D). Maybe due to the low sensitivity of the PDGF receptor
antiserum in immunoblotting, several components appeared to be
immunoreactive in the Y1018F sample (rightside of panelD) in a ligand-independent manner. None of
these components had a migration rate corresponding to the
-receptor and could be regarded as unspecific. The migration
positions of the PDGF
-receptor and PLC-
are indicated, as
well as the migration positions of molecular weight standards (myosin,
200,000; phosphorylase b, 97,000), run in parallel. ip, immunoprecipitation.
Stimulation with PDGF-AA and PDGF-BB also induced a low level of
tyrosine phosphorylation of PLC- in the wild-type receptor cells
and in the Y988F mutant receptor-expressing cells (Fig. 4B). In addition, a 170-kDa component most likely
corresponding to the autophosphorylated
-receptor was
co-immunoprecipitated with PLC-
in the wild-type and Y988F mutant
receptor-expressing cells. In contrast, the Y1018F mutant receptor
failed to associate with or phosphorylate PLC-
. Reprobing the
filter in Fig. 4B with PLC-
antiserum showed
similar levels of PLC-
in all lanes (Fig. 4C),
demonstrating that the difference in tyrosine phosphorylation of
PLC-
was not due to differences in the amount of PLC-
immunoprecipitated from the various cell lines. To show that the
component coprecipitating with PLC-
indeed was the
-receptor,
the filter used in Fig. 4, B and C, was
stripped again and reprobed with PDGF
-receptor antiserum (Fig. 4D). These results indicate that in
vivo, the Y988F mutant had reduced capacity to associate with
PLC-
, whereas the Y1018F mutant had almost completely lost its
capacity to associate with PLC-
, irrespective of whether PDGF-AA
or PDGF-BB were used for stimulation.
To quantify the fraction of
phosphorylated wild-type, Y988F, and Y1018F receptors that associate
with PLC-, in vitro kinase assays were performed. The
different cell lines were stimulated with PDGF-BB, lysed, and
immunoprecipitated with PY20 or PLC-
antiserum. The immune
complexes were then incubated with [
-
P]ATP
and analyzed by SDS-PAGE, followed by autoradiography. As seen in Fig. 5, stimulation with PDGF-BB increased autophosphorylation
of the wild-type receptor, as well as receptor-mediated phosphorylation
of PLC-
. PDGF-BB stimulation of the Y988F and Y1018F mutant
receptor-expressing cells induced the kinase activity of the mutant
receptors, but the extent of complex formation with PLC-
was
changed. The total pool of phosphorylated receptors, as well as the
pool of phosphorylated receptors co-immunoprecipitated with PLC-
,
was quantified for each cell line using a PhosphorImager instrument.
About 55% of the phosphorylated wild-type receptors were
co-immunoprecipitated with PLC-
(compare lanes2 and 4). Changing Tyr-988 to Phe reduced the pool of
phosphorylated receptors associated with PLC-
to 35% of the total
phosphorylated receptor pool (compare lanes6 and 8). The association with PLC-
to the Y1018F receptor was
drastically reduced, since only 3% of these phosphorylated receptors
were coprecipitated with PLC-
(compare lanes10 and 12). Repeated experiments gave similar figures for
the reduction in complex formation between PLC-
and the mutant
receptors. These data indicate that Tyr-1018 is the major binding site
for PLC-
and that Tyr-988 could serve as a minor binding site.
Figure 5:
Quantification of the association of
PLC- to ligand-activated wild-type and tyrosine residue-mutated
PDGF
-receptors. PAE cells expressing the wild-type
-receptor
or Y988F or Y1018F mutant receptors were incubated with or without 100
ng/ml PDGF-BB for 1 h at 4 °C. The cells were solubilized, and the
lysates were subjected to immunoprecipitation using the phosphotyrosine
monoclonal antibody PY20 or the PLC-
antiserum. Kinase assays were
performed on the immune complexes, and the samples were analyzed by
SDS-PAGE. The gel was treated with 1 M KOH at 55 °C for 1
h prior to autoradiography. The 170-kDa
-receptor band was
quantitated using a PhosphorImager instrument. ippt,
immunoprecipitation.
To confirm the involvement of Tyr-988 and Tyr-1018 in PLC-
binding, competition experiments were performed. The ability of
unphosphorylated and phosphorylated synthetic peptides to compete with
receptor binding to the PLC-
SH2 domains was tested as follows.
PDGF
-receptors were labeled with
P through
stimulation of the PDGF
-receptor-expressing cells with PDGF-BB,
followed by lysis and immunoprecipitation using PY20. In vitro kinase assay was performed on the immobilized immunoprecipitate in
the presence of [
-
P]ATP. Phosphorylated
receptors were eluted with phenylphosphate, and, after desalting,
aliquots were incubated with Sepharose-coupled fusion protein composed
of glutathione S-transferase linked to a stretch covering the
two PLC-
SH2 domains. Incubations were performed in the presence
and absence of different concentrations of unphosphorylated and
phosphorylated synthetic peptides corresponding to residues
980-996 and 1007-1026. After washing the beads, the extent
of association between the receptor and the PLC-
SH2 fusion
protein was estimated by SDS-gel electrophoresis, autoradiography, and
quantification using a PhosphorImager (Fig. 6). Fig. 6shows that both types of phosphorylated peptides were able
to compete with the intact
-receptor for binding to the PLC-
SH2 domain fusion protein. The 1018(P) peptide displayed about 10 times
higher affinity for the fusion protein compared with the 988(P)
peptide. The unphosphorylated peptides were not able to compete to any
significant extent.
Figure 6:
Competition between synthetic peptides and P-labeled
-receptor for binding to PLC-
SH2
domains. PAE cells expressing the PDGF
-receptor were stimulated
with PDGF-BB and lysed, and receptors were immunoprecipitated with
phosphotyrosine antibodies, followed by in vitro kinase assay.
P-Labeled receptors were eluted with phenylphosphate, and
the abilities of the receptors to interact with a bacterial fusion
protein containing both PLC-
SH2 domains were analyzed in the
presence and absence of synthetic unphosphorylated and phosphorylated
peptides at the indicated concentrations. The extent of binding of
P-labeled
-receptor was analyzed by SDS gel
electrophoresis and quantitated using a
PhosphorImager.
Figure 7:
Abilities of the wild-type PDGF - and
-receptors interact with and phosphorylate PLC-
. Cultures of
PAE cells expressing either
- or
-receptors were incubated
with or without 100 ng/ml PDGF-BB for 1 h at 4 °C. The cells were
solubilized, and a glycoprotein fraction was collected using wheat germ
agglutinin Sepharose 6B (A) or the lysates were
immunoprecipitated by the anti-PLC-
antiserum (B). The
samples were separated by SDS-PAGE and transferred to nitrocellulose
membranes. The blots were probed with the phosphotyrosine monoclonal
antibody PY20, and the reactions were visualized using the ECL Western
blotting detection system. The filter shown in panelB was stripped and reprobed with PLC-
antiserum (C) as
a control for equal loading. The migration positions of the PDGF
receptors and PLC-
are indicated, as well as the migration
positions of molecular weight standards (myosin, 200,000; phosphorylase b, 97,000) run in parallel. The molecular nature of the band
around 130 kDa, seen in panelA for samples derived
from the PDGF
-receptor cells, is
unknown.
Figure 8:
Formation of inositol phosphates (InsP) in PAE cells transfected with both
- and
-receptors (P
:1) or human fibroblasts (AG
1523). Cultures pre-labeled with 1 µCi/ml myo-[
H]inositol for 48 h under
serum-free conditions were incubated in the presence of vehicle (openbars), 50 ng/ml PDGF-AA (stripedbars), 50 ng/ml PDGF-BB (solidbars),
or 10% FCS (shadedbars) for 30 min at 37 °C. The
samples were quenched by addition of acidified methanol, and the
water-soluble and lipid fractions separated. The inositol phosphates
were separated by anion-exchange chromatography, and the total amount
of cpm eluted as inositol phosphate (InsP), inositol
1,4-bisphosphate, and inositol 1,4,5-trisphosphate were related to the
amount of radioactivity in the corresponding lipid fraction in 10
cells. -Fold stimulation under the different conditions are
shown, with the basal level in unstimulated cells for each cell line
set to 1. The values represent means ± S.E. from two experiments
with triplicate samples.
To investigate whether the -receptor
requires the presence of the
-receptor to mediate a release of
inositol trisphosphates, we used PAE cells co-expressing both receptor
types (P
:1, (18) ), as well as human fibroblasts, AG
1523, in which both receptor types are expressed. In both cell types, a
consistent response was seen, with a limited accumulation of inositol
phosphate after stimulation with PDGF-AA, which activates the
-receptor, as compared with the levels seen after stimulation with
PDGF-BB, which activates both the
- and the
-receptor (Fig. 8). The magnitude of the response seen after stimulation
with PDGF-BB was lower in P
:1 cells than in AG 1523, probably
due to the fact that the P
:1 cells express about 10,000
-receptors per cell, whereas AG 1523 express about 100,000
-receptors per cell.
We have identified two autophosphorylation sites, Tyr-988 and
Tyr-1018, near the carboxyl terminus of the human PDGF -receptor,
which are phosphorylated both in vivo and in vitro. These residues serve as binding sites for PLC-
, as shown by
the reduced levels (Y988F mutant receptor), or close to complete loss
(Y1018F mutant receptor) of complex formation and tyrosine
phosphorylation of PLC-
in the PDGF-stimulated mutant receptor
cell lines in vivo. The mutant receptors are functional
kinases in vivo (see Fig. 4A) and are as
efficient as the wild-type
-receptor in their interactions with
other SH2 domain-containing proteins. (
)Synthetic peptides
encompassing Tyr-988(P) or Tyr-1018(P) were able to compete with the
intact receptor for binding to the PLC-
SH2 domains; the
Tyr-988(P) peptide competed with considerably lower efficiency.
Crystallographic analysis of the carboxyl-terminal SH2 domain of
PLC-
in complex with a peptide from the PLC-
binding site in
the PDGF
-receptor (containing Tyr-1021), showed that the
phosphotyrosine and the following six residues make specific contact
with the SH2 domain(37) . The amino acid sequences following
Tyr-1018 in the PDGF
-receptor and Tyr-1021 in the
-receptor
are identical (Table 1). PLC-
binding sites in other
receptor tyrosine kinases, such as the FGF
receptor-1(38, 39) , the hepatocyte growth factor
receptor(40) , the nerve growth factor receptor(41) ,
and the low affinity binding sites in the PDGF
- and
-receptors (Tyr-988 and Tyr-1009, respectively; see Table 1), are also related and consist mostly of hydrophobic
residues, and proline residues are often found in position +5 or
+6. The EGF receptor carboxyl-terminal tail has been shown to
contain both high affinity binding (Tyr-992) and low affinity binding
(Tyr-1068) binding sites(33) . The association of
PLC-
with the EGF receptor, however, is complex as
mutagenesis of individual autophosphorylation sites suggests that other
autophosphorylation sites may compensate for the loss of primary sites (42) . Interestingly, the low affinity binding sites in the
PDGF receptors appear to be structurally related to the binding sites
in the hepatocyte growth factor and nerve growth factor receptors,
since all have hydrophobic residues (T, I, V, or L) in position +1
and V in position +3 carboxyl-terminal of the phosphorylated
tyrosine. Since the contribution of the
-receptor Tyr-988
autophosphorylation site in PLC-
binding is relatively small, the
role of this site in PLC-
activation is unclear. It is possible
that also other SH2 domain-containing proteins bind to this site.
However, the tyrosine phosphatase SH-PTP ID/SH-PTP 2/Syp, which binds
to Tyr-1009 in the PDGF
-receptor(14) , does not appear to
bind with high efficiency to Tyr-988 in the
-receptor (not shown). (
)
PLC- catalyzes the hydrolysis of
phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol
1,4,5-trisphosphate, two second messengers involved in the activation
of protein kinase C and in the release of Ca
from
internal cellular stores, respectively (reviewed in (43) ).
PLC-
was recently implicated in mitogenic signal transduction,
since restoration of its binding site in a PDGF
-receptor mutated
at multiple tyrosine residues was accompanied by a restoration of the
mitogenic signaling capacity of the mutant receptor(44) .
However, we show that mutant PDGF
-receptors, in which either
Tyr-988 or Tyr-1018 were replaced with phenylalanine residues, mediated
an increased DNA synthesis in response to PDGF-AA, comparable with that
for the wild-type receptor. This is in agreement with a report by Hill et al.(45) who observed increased DNA synthesis in
the absence of PLC-
activation after treatment of PDGF-stimulated
murine fibroblasts with genistein. Intact mitogenic signaling was also
reported for mutants of PDGF
-receptors (13) and FGF
receptors(38, 39) , which were unable to mediate
PLC-
binding and phosphorylation. The recently characterized
signal transduction pathway involving the SH2 domain-containing protein
Grb2, which couples receptor tyrosine kinases to Ras, is believed to
represent a major mitogenic pathway(46, 47) . It is
possible that multiple parallel mitogenic pathways exist in the cell
and that PLC-
is part of one such pathway.
PLC- becomes
phosphorylated on tyrosine residues in response to PDGF or EGF
stimulation(48, 49) . Tyrosine phosphorylation has
been shown to increase the enzymatic activity of PLC-
(36) and to reduce its binding to the activated epidermal
growth factor receptor(50) . Our results indicate that the
interaction of PLC-
with the PDGF
- and
-receptors are
qualitatively different. A stable complex between PLC-
and the
autophosphorylated
-receptor could readily be detected, but the
degree of PLC-
phosphorylation was low. In contrast to this, and
in agreement with results from previous studies (11, 13) , only a small fraction of the activated
-receptor pool occurred in complex with PLC-
at each single
moment. However, efficient phosphorylation of PLC-
on tyrosine
residues was produced by the
-receptor. Most likely, the
phosphorylated and activated PLC-
was then released from the
receptor complex. As a consequence, there was a higher production of
inositol phosphates in the
-receptor-expressing cells than in the
-receptor-expressing cells. Higher production of inositol
phosphates in response to PDGF-BB (which binds to both
- and
-receptors) than to PDGF-AA (which binds only to
-receptors)
was also recorded for PAE cells co-expressing both receptor types after
transfection and for human foreskin fibroblasts, AG 1523, which
endogenously express the two PDGF receptor types. When 32D
hematopoietic cells expressing
- or
-receptors were compared,
the receptors induced similar extents of phosphorylation of
PLC-
(51) , and the stimulation of inositol phosphate
production was equally efficient for the two receptors(52) .
Swiss 3T3 cells endogenously expressing both PDGF receptor types also
respond equally well to PDGF-AA and -BB in the formation of inositol
phosphates(53) . On the other hand, vascular smooth muscle
cells, which express PDGF
- and
-receptors, accumulate
inositol phosphate in response to PDGF-BB but not PDGF-AA(54) .
In agreement, different populations of vascular smooth muscle cells
examined for PDGF-induced Ca
fluxes were either
unresponsive or only partially responsive to treatment with PDGF-AA, as
compared with PDGF-BB(55) . In this work, we provide an
explanation for the molecular mechanisms underlying these observations,
since the efficiency with which PDGF
-receptor mediates tyrosine
phosphorylation of PLC-
, and consequently, inositol phosphate
production, is considerably lower than that of the PDGF
-receptor.