(Received for publication, January 26, 1995)
From the
To determine the molecular basis for the transforming function
of platelet-derived growth factor (PDGF)-A in NIH/3T3 cells, we have
constructed chimerae consisting of the extracellular domain of the
human CSF-1R (fms) linked to the cytoplasmic domain of the
PDGF receptor (
R) containing a series of deletion or point
mutations. The ability of fms/
R chimerae to mediate
CSF-1-dependent anchorage-independent growth, focus formation, and
chemotaxis of NIH/3T3 cells was then examined. Our results provide
evidence that a domain encompassing amino acid residues 977-1024 of the
PDGFR is required for ligand-dependent focus formation, but not
chemotaxis or anchorage-independent growth, and that tyrosine residues
within this domain constitute the major binding site for phospholipase
C
. Therefore, our findings suggest that: (i) the focus forming
function of
PDGFR correlates well with the ability of the receptor
to bind phospholipase C
, and (ii) the mechanism of focus formation
mediated by
PDGFR may be distinguished from that required for
chemotaxis or anchorage-independent growth.
Platelet-derived growth factor (PDGF) ()is a
multifunctional molecule that does not only regulate DNA synthesis and
cell division but also induces a variety of other biological effects
that are implicated in neoplastic diseases, atherosclerosis, tissue
repair, and inflammatory responses(1, 2) . PDGF
comprises dimers of PDGF-A and PDGF-B chains encoded by distinct
genes(3, 4) . PDGF dimeric isoforms have been shown to
bind with different affinities to two different but related receptor
molecules, designated
PDGFR and
PDGFR, which are encoded by
distinct genes(5, 6) . The PDGF-A homodimer mediates
biological functions that include chemotaxis, anchorage-independent
growth, and focus formation by activating the tyrosine kinase activity
of
PDGFR in NIH/3T3
fibroblasts(7, 8, 9, 10) .
Accumulating evidence indicates that many of the effector molecules
involved in the PDGF-mediated signaling pathway interact with the
kinase insert or carboxyl-terminal domains of PDGFRs(11) . In
the present study, we sought to further examine the functional role of
these domains in a physiologically relevant microenvironment in an
attempt to better understand the molecular basis of biological
responses mediated by
PDGFR.
To examine the functional role of distinct structural domains
of PDGFR in ligand-dependent biological responses, two
carboxyl-terminal truncation mutants lacking amino acid residues
1025-1089 or 977-1089, designated
R
C1 and
R
C2, and a group of point mutations substituting
phenylalanine for tyrosine residues 988, 993, or 1018, designated
R(Y988F),
R(Y993F), and
R(Y1018F), were generated (Fig. 1A). In addition, we have reported previously the
construction of a
PDGFR deletion mutant lacking amino acid
residues 710-789 of the kinase insert domain, designated
R
ki-1, and point mutations within this domain converting
tyrosine 731, 742, or both to phenylalanine, designated as
R(Y731F),
R(Y742F), and
R(Y731F+Y742F),
respectively(12, 13) . Since the NIH/3T3 line is a
cell type that normally expresses both
and
PDGFRs, it
provides a physiologically relevant microenvironment for analyzing
PDGFR signal transduction. To overcome activation of endogenous
PDGFR, the cytoplasmic domain of
PDGFR mutants were linked to
the ligand-binding domain of the human CSF-1R (c-fms), which is not normally expressed in NIH/3T3
cells. Expression vectors containing the chimerae were transfected into
NIH/3T3 cells, and the ability of these chimerae to mediate
CSF-1-stimulated anchorage-independent growth, focus formation, and
chemotactic responses in NIH/3T3 cells was studied.
Figure 1:
Biochemical characterization of fms/R chimerae in NIH/3T3 cells. A, schematic
diagram of wild type
PDGFR, fms, and chimeric receptors
generated between c-fms and
PDGFRs. Coding region of the
PDGFR is represented by an openbox. Coding
region of c-fms is represented by a shadedbox. Blackboxes correspond to signal
peptide (SP), transmembrane(TM), kinase
insert (ki), and carboxyl-terminal (C) domains; B, comparison of CSF-1-induced tyrosine phosphorylation of fms/
R chimerae; C, comparison of the level of
anti-CSF-1R recoverable fms/
R protein expressed in
NIH/3T3 transfectants; D and E, comparison of
CSF-1-induced association of each chimerae with PLC
and p85 in
CSF-1-stimulated NIH/3T3 transfectants. NIH/3T3 transfectants were
either untreated(-) or treated (+) with CSF-1 (200 ng/ml)
for 5 min at 37 °C. In panelB, 2 mg of clarified
lysates were subjected to immunoprecipitation using anti-Tyr(P)
followed by SDS-PAGE and subsequent blotting with anti-Tyr(P). In panels C-E, 2 mg of clarified lysate was
immunoprecipitated with anti-CSF-1R antibody (Santa Cruz
Biotechnology). The immune complexes were then subjected to
immunoblotting with anti-
PDGFR (panelC),
anti-PLC
(panelD), or anti-p85 antibody (panelE).
We first
examined the effect of these mutations on PDGFR
autophosphorylation. NIH/3T3 cells transfected with fms/
RWT, fms/
R(Y988F), fms/
R(Y993F), fms/
R(Y1018F), fms/
R
C1, fms/
R
C2, and fms/
R
ki-1 were untreated or treated with CSF-1.
Total cell lysates were immunoprecipitated with anti-phosphotyrosine
antibody (anti-Tyr(P)). The proteins in immune complexes were
electrophoretically separated, transferred to a membrane, and
immunoblotted with anti-Tyr(P). As shown in Fig. 1B,
while fms/
R
C2 showed approximately 50% reduction in
tyrosine phosphorylation, the other chimerae exhibited levels of
tyrosine phosphorylation that were comparable to that of fms/
RWT. Consistent with previous findings, we did not
detect tyrosine phosphorylation of endogenous
PDGFR upon CSF-1
triggering (Fig. 1B, lanes1 and 2). These data suggest that amino acids 977-1024 within the
carboxyl-terminal domain of
PDGFR may contain relevant tyrosine
autophosphorylation sites. Alternatively, the reduction in the level of fms/
R
C2 tyrosine phosphorylation may be due to
conformational changes induced by deletion of the carboxyl-terminal
domain of
PDGFR. To examine the expression levels of chimeric
receptors in the NIH/3T3 transfectants, immunoprecipitates were
prepared from equivalent amount of cell lysates using antiserum
directed against the extracellular domain of c-Fms (anti-CSF-1R). The immune complexes were then electrophoretically
separated and immunoblotted with anti-
PDGFR antiserum raised
against a synthetic peptide corresponding to amino acids 959-973
of the receptor. As shown in Fig. 1C, the levels of fms/
R chimeric proteins expressed were comparable among
each NIH/3T3 transfectant. Under the same conditions, endogenous
PDGFR protein could not be detected in the anti-CSF-1R immune
complex prepared from control NIH/3T3 cells (Fig. 1C, lanes 1). Cell surface expression levels of the chimeric
proteins were also determined to be very similar by
fluorescence-activated cell sorting analysis using a monoclonal
antibody directed against the extracellular domain of the CSF-1R (data
not shown). Together, these results suggest that the stoichiometry of
CSF-1-induced tyrosine autophosphorylation of all mutant receptors,
except fms/
R
C2, was comparable to that of fms/
RWT.
We next examined the effect of these
mutations on receptor association with its known substrates. The
ectopic expression and biochemical analyses of R
C1 and
R
C2 in 32D hematopoietic cells indicated that amino acid
residues 977-1024 contain the major determinants necessary for PLC
association and activation (data not shown). Therefore, the ability of
mutant fms/
R chimerae to coimmunoprecipitate with
PLC
was compared using anti-CSF-1R antibody. As shown in Fig. 1D, this antibody coimmunoprecipitated comparable
levels of PLC
from NIH/3T3 cells expressing fms/
RWT
and fms/
R
C1. In contrast, the level of anti-CSF-1R-
recoverable PLC
was either reduced by about 90% in NIH/3T3 cells
expressing fms/
R(Y988F), fms/
R(Y993F), or
it was completely abolished in fms/
R
C2 or fms/
R(Y1018F) transfectants after CSF-1 stimulation.
Consistent with these results, the level of anti-Tyr(P)-recoverable
PLC
protein was also reduced (data not shown). As shown in Fig. 1E, each chimera associated to a similar extent
with the 85 kDa regulatory subunit of PI 3-kinase (p85). Thus, our
findings indicate that amino acid residues 977-1024 within the carboxyl
terminus of
PDGFR contain the major binding site for PLC
but
not p85. These results are consistent with previous data demonstrating
that tyrosine 1021 of the
PDGFR is required for association with
PLC
, since the amino acid residues surrounding tyrosine 1018 of
the
PDGFR are very similar to those surrounding tyrosine 1021 of
the
PDGFR (16) . Under the same conditions, fms/
R
ki-1, fms/
R(Y731F), and fms/
R(Y731F+Y742F) failed to coprecipitate p85, even
though the ability of these mutants to associate with PLC
was not
impaired (data not shown).
To examine the effect of these mutations
on ligand-stimulated biological responses, we next tested the ability
of each transfectants to form colonies in semisolid medium. As shown in Fig. 2, NIH/3T3 expressing fms/RWT formed colonies
of sizes greater than 30 µm in diameter with an efficiency of
around 25% (panelsC and D). Moreover, each
transfectant exhibited a similar ability to form progressively growing
colonies in the presence of CSF-1 except NIH/3T3 cells transfected with
the vector alone (panelsA and B). These
data suggest that ligand-stimulated association of PLC
with
PDGFR is not required for colony formation in soft agar, since fms/
R(Y1018F), which failed to bind PLC
completely,
exhibited CSF-1-induced colony forming efficiency comparable to that of
the fms/
RWT transfectant. In addition, neither deletion
of 80 amino acids from the kinase insert nor truncation of 112 amino
acids from the carboxyl terminus of
PDGFR detectably impaired the
ability of CSF-1 to mediate colony formation. Taken together, our
findings suggest that either the kinase insert or carboxyl-terminal
domain is dispensable for anchorage-independent cellular growth.
Figure 2:
CSF-1-stimulated cell growth of NIH/3T3
transfectants in semisolid media. NIH/3T3 cells (1
10
) transfected with indicated DNAs were suspended in DMEM
supplemented with 10% calf serum and 0.4%
Seaplaque-agarose(15) . Cells were then fed with DMEM
containing 10% calf serum in the presence or absence of CSF-1 (100
ng/ml) once per week. Photographs were taken by using light microscope
after 2 weeks. Results are representative of at least three independent
experiments.
CSF-1 has been shown to induce efficient focus formation of NIH/3T3
cells expressing fms/RWT, fms/
R(Y731F), and fms/
R(Y731F+Y742F)(15) . Therefore, we
sought to examine the ability of individual mutant chimera to mediate
CSF-1-dependent focus formation of NIH/3T3 cells. As shown in Fig. 3, CSF-1 treatment induced approximately 140 foci in
NIH/3T3 cells transfected with 1 µg of the fms/
RWT
expression vector (panelsC and D).
Truncation of 64 amino acid residues from the carboxyl terminus (fms/
R
C1) or 80 amino acid residues from the kinase
insert domain (fms/
R
ki-1) did not affect the level
of ligand-stimulated focus formation, respectively (Fig. 3, panels E and F and panels O and P).
In striking contrast, truncation of an additional 48 amino acid
residues (fms/
R
C2) resulted in 10-fold less focus
forming activity in response to CSF-1 treatment (Fig. 3, G and H). These data suggest that amino acid residues
977-1024 within the carboxyl-terminal domain of the
PDGFR are
important for mediating focus formation. Transfection of chimeric
receptor containing mutation of tyrosine 988, 993, or 1018 within this
domain each reduced CSF-1-induced focus formation by about
5-10-fold (Fig. 3, panels I and J, K and L, and M and N). A similar number
of marker-selected colonies were observed in parallel plates,
suggesting that equal levels of DNA were transfected into these cells
(data not shown). Since the CSF-1-dependent focus formation of NIH/3T3
cells by various constructs correlated well with the ability of mutant
receptors to bind PLC
, our results suggest that PLC
is
probably involved in the focus forming function mediated by the
PDGFR. However, it is still possible that other putative
substrates of
PDGFR that interact with this region may also be
involved in mediating this response. In contrast to the mutations
within the carboxyl-terminal domain, none of the mutations within the
kinase insert domain of
PDGFR reduced ligand-induced
focus-formation (data not shown and (15) ). These data suggest
that substrates that bind to phosphotyrosine residues within amino acid
residues 710-789 of the kinase insert domain of the
PDGFR,
including p85 subunit of PI 3-kinase, are not required for efficient
focus formation in NIH/3T3 cells.
Figure 3:
CSF-1-stimulated focus formation of
NIH/3T3 transfectants. NIH/3T3 cells were transfected by the calcium
phosphate precipitation method, using 1 µg of indicated fms/PDGFR chimeric receptor cDNA expression vectors and
40 µg of calf thymus DNA as carrier. Transfected cells were
maintained in medium containing 5% calf serum and switched to medium
containing 2% calf serum with or without 100 ng/ml human CSF-1 5 days
post-transfection. Focus formation was scored 2-3 weeks after
transfection. Parallel plates were also stained and photographed.
Similar results have been obtained in two other independent
experiments.
Ligand-stimulated PDGFR is
able to mediate chemotactic signaling in 32D hematopoietic and NIH/3T3
cells(9, 12, 17) . To compare the ability of
the various fms/
R mutants to mediate chemotactic
responses in NIH/3T3 cells, we examined the ability of each
transfectant to migrate through a membrane toward the CSF-1 ligand (14) . As shown in Fig. 4, while deletions of amino acid
residues 1025-1089 (fms/
R
C1) or mutation of
residues Tyr-988 or Tyr-993 did not impair CSF-1 dependent chemotactic
response, deletion of amino acid residues 977-1089 (fms/
R
C2) or mutation of Tyr-1018 inhibited
chemotaxis by less than 50%. Therefore, our results suggest that
mutation within the carboxyl terminus of
PDGFR did not impair
chemotaxis to the same extent as focus formation. In contrast, deletion
of 80 amino acids within the kinase insert domain or mutations of PI
3-kinase association sites reduced ligand-dependent chemotactic
responses in NIH/3T3 cells by greater than 80%. Thus, our findings
suggest that substrates interacting with the kinase insert domain of
PDGFR are required for efficient chemotactic responses in NIH/3T3
fibroblasts.
Figure 4:
CSF-1-stimulated chemotactic response of
NIH/3T3 transfectants requires PDGFR association with PI 3-kinase
activity. Cell migration was assayed by a modified Boyden chamber
technique using a Transwell cell culture insert (Costar), which
contained a filter with a pore size of 8 µm(14) . Briefly,
100 ng/ml CSF-1 ligand diluted in DMEM was first added to the bottom
chamber. 1
10
NIH/3T3 cells were then added to the
top chamber, and cells that had crossed the filter after 24 h of
incubation at 37 °C were counted. Results are representative of at
least three independent experiments.
Mutation of the PLC association site of the
PDGFR or fibroblast growth factor receptors has been reported to
have no effect on ligand-stimulated mitogenic
responses(16, 18) . Consistent with these findings,
ectopic expression of
R
C1 or
R
C2 in 32D
hematopoietic cells indicates that amino acid residues 977-1089
encompassing the PLC
binding site are not necessary for
PDGF-induced mitogenic responses (data not shown). Moreover, our data
indicate that fms/
R transfectants containing a mutation
of the PLC
binding site or even a larger truncation of amino acid
residues 977-1089 of the carboxyl-terminal domain of
PDGFR did not
reduce the ability of the receptor to mediate formation of colonies in
the presence of the CSF-1. In contrast, analyses of carboxyl-terminal
truncation mutants to mediate CSF-1-induced focus formation revealed
that the presence of amino acids 977-1024 of the
PDGFR was
necessary for this effect (see Fig. 3). Interestingly, mutation
of Tyr-988, Tyr-993, or Tyr-1018 within this domain each reduced not
only the CSF-1-induced focus formation but also receptor association
with the PLC
. These findings suggest that activation of PLC
is likely to be involved in focus formation but not in
anchorage-independent cell growth. We have observed that the inhibitory
effect of Y1018F mutation on the CSF-1-induced focus formation or
receptor association with PLC
was more pronounced than that shown
by mutation of either Y988F or Y993F. Although the biological
significance of the difference observed remains to be established, our
data suggest that Tyr-1018 may be differentially involved in receptor
association with PLC
and mediating the focus forming activity of
PDGFR. PDGF-induced focus formation of NIH/3T3 cells is
characterized by the loss of contact inhibition and morphological
alterations, which are considered to be the hallmark of neoplastic
diseases. Interestingly, diacylglyceride and inositol trisphosphate,
the enzymatic reaction products of PLC
activation, have also been
implicated in stimulating redistribution of cytoskeletal components in
a variety of transformed cells(19, 20, 21) .
In addition, a recent report suggests that protein kinase C-
is
involved in transformation of NIH/3T3 cells(22) . Together,
these findings suggest that PLC
and its associated proteins may
mediate focus forming activity of PDGF by stimulating protein kinase
C-dependent changes in the cytoskeletal architecture of NIH/3T3 cells.
In contrast to the mutations within the carboxyl-terminal domain, a
double mutation within the kinase insert domain of PDGFR that
completely abrogated receptor-associated PI 3-kinase fully inhibited
ligand-stimulated cell movement but not focus formation or colony
formation of NIH/3T3 transfectants. This result is consistent with
previous findings showing that PDGF-induced PI 3-kinase association
with the
PDGFR is required for chemotactic response(23) .
Thus our findings indicate that the region of
PDGFR involved in
ligand-dependent chemotactic signaling is distinct from that required
for focus formation and anchorage-independent growth functions mediated
by the
PDGFR. Finally, we would be remiss not to mention the
possibility that differences between the signaling mechanisms uncovered
in this study may potentially reflect the inherent property of
CSF-1R/
PDGFR chimera utilized.