(Received for publication, February 28, 1996)
From the
We have reported previously that a chimeric platelet-derived
growth factor receptor (PDGFR) possessing the ligand binding domain of
the PDGFR and the intracellular domain of the
PDGFR
(
R) was markedly more efficient
than the wild type
PDGFR (
RWT) in its ability to enhance
PDGF-A transforming activity in NIH/3T3 fibroblasts. To determine the
region within the cytoplasmic domain of
PDGFR that confers this
higher transforming activity, we generated several additional
/
PDGFR chimerae. When a chimeric PDGFR possessing the first
933 amino-terminal amino acids from the
PDGFR and the final 165
amino acids from the carboxyl-terminal of the
PDGFR
(
R) was cotransfected with the
PDGF-A gene into NIH/3T3 cells, it showed a similar high efficiency to
enhance PDGF-A chain transforming activity as
R. However, when chimeric PDGFRs
in which either the kinase insert domain (
RKI) or the last 79
amino acids from the carboxyl-terminal end of the
PDGFR
(
R) were substituted into
PDGFR sequences were cotransfected with PDGF-A, they showed
similar low efficiencies in enhancing transforming activity as the
RWT. These results predicted that the 86 amino acids following the
tyrosine kinase 2 domain of
PDGFR (amino acid residues 942-1027)
were responsible for the higher transforming activity of
PDGFR. To
confirm this finding, we next constructed a chimera in which amino acid
residues 942-1028 of the
PDGFR
(
R) were substituted for those in
the
PDGFR. Cotransfection experiments indicated that
R increased transforming activity of
PDGF-A to similar extent as the
R
or
R. Therefore, our findings
define a critical domain within the noncatalytic region of
PDGFR
intracellular domain that confers the higher focus forming activity
mediated by the
PDGFR.
Platelet-derived growth factor (PDGF) ()is a potent
mitogen for connective tissue cells(1) . This growth factor is
composed of dimers of A and B chains encoded by distinct genes. All
three PDGF isoforms (PDGF-AA, PDGF-BB, and PDGF-AB) have been
identified(2) . They bind with different affinities to two
related receptor molecules, designated
PDGFR and
PDGFR, which
are also encoded by two distinct
genes(3, 4, 5) . Accumulating evidence
indicates that PDGF-induced receptor activation involves recruitment of
receptor dimers(6, 7, 8, 9) . In
fibroblasts where both PDGFRs are expressed, PDGF-BB binds
,
, or
PDGFR dimers, PDGF-AA binds only
PDGFR homodimers, and PDGF-AB binds
or
PDGFR
dimers, but does not bind
PDGFR
dimers(1, 7, 10) .
Activation of the PDGFR
tyrosine kinase domain leads to physical association and tyrosine
phosphorylation of many substrates, including Src, the 85-kDa subunit
of phosphatidylinositol 3-kinase (p85), Nck, Ras GTPase-activating
protein (RasGAP), phospholipase C- (PLC
), and
Syp(11, 12, 13, 14, 15) .
The specific tyrosine residues interacting with each of these
substrates have been identified for the
PDGFR. Src binds to
tyrosines 579 and 581 within the juxtamembrane domain(16) , and
p85 binds to tyrosines 740 and 751 within the kinase insert domain of
PDGFR(17, 18, 19) . Tyrosine 751 is also
the association site for Nck (20) . RasGAP binds to tyrosine
772 within the kinase insert domain of
PDGFR(18, 19) . Syp and PLC
bind to tyrosine
1009 and 1021, respectively, within the carboxyl-terminal domain of
PDGFR(21, 22) . In addition, Shc and protein
kinase C-
have been shown recently to be phosphorylated on
tyrosine following PDGF-BB stimulation(23, 24) . Among
these substrates, both activated
PDGFR and
PDGFR
tyrosine-phosphorylate p85 and PLC
with similar
stoichiometry(25) . However, the
PDGFR has been shown to
associate more efficiently than the
PDGFR with the PLC
in
vivo(25, 26) . Moreover, RasGAP and Syp have been
shown to be poor substrates for the
PDGFR in comparison to the
PDGFR(26, 27) .
Although both PDGF-AA and
PDGF-BB are mitogenic as well as chemotactic for cells possessing
appropriate PDGFRs(28) , we and others have shown previously
that PDGF-BB is 10-100-fold more efficient than PDGF-AA at
inducing transformation of NIH/3T3 cells(29, 30) . It
is known that PDGF-BB stimulates - as well as
PDGFRs, while
PDGF-AA binds only to the
PDGFR. Thus, the greater transforming
efficiency of PDGF-B could be due to a quantitative increase in the
level of activated PDGFRs and/or differences in substrate specificity
of
versus
PDGFRs. We have observed previously that
cotransfection of the PDGF-A chain gene with wild type
PDGFR
(
RWT) increased PDGF-A chain transforming activity by
approximately 2-fold. In contrast, cotransfection of a chimeric
receptor possessing
PDGFR PDGF-A ligand binding domain and the
remaining sequences, including the catalytic domain from
PDGFR
(
R), resulted in a 17-fold
enhancement of PDGF-A chain transforming activity(25) . Thus,
our previous findings indicated that the increased transforming
activity of PDGF-B in comparison with PDGF-A could be due to distinct
substrate specificities of the two PDGFRs. In the present manuscript,
we have identified a specific region within the carboxyl-terminal of
the
PDGFR that is responsible for mediating the enhanced focus
formation of PDGF-B.
For in vitro kinase assays, total cell lysates
(about 2 mg) were subjected to immunoprecipitation using mAb-R1.
The immune complexes were extensively washed and incubated in 20 mM Tris (pH = 7.5), 10 mM MgCl
, 5 mM MnCl
, 10 µg/ml aprotinin, and 50 µCi of
[
-
P]ATP (Amersham Corp.) for 15 min at room
temperature. The immune complex reactions were then washed and
electrophoretically separated on 8% SDS-polyacrylamide gel
electrophoresis. The gels were dried and autoradiogrammed. The levels
of PDGFRs and the kinase activities of each
/
chimerae in the
various transfectants were quantitated using scanning densitometer
(PDI, Inc.) and expressed relative to the signal derived from the
RWT. Immunoblot and immunoprecipitation analyses, NIH/3T3
transfection and cotransfection assays were performed as described
previously(34) .
Figure 1:
Schematic
diagram of RWT,
RWT, and the
/
PDGFRs chimerae. The
coding region of
PDGFR is represented by an open box. The
coding region of the
PDGFR is represented by a shaded
box. The black box corresponds to the signal peptide (SP) and transmembrane (TM) domain. Tyrosine kinase
domain (TK1 and TK2), kinase insert (KI),
and carboxyl-terminal (CT) domains are
indicated.
Cotransfection of PDGF-A chain with RKI
resulted in production of similar numbers of foci as those induced by
cotransfection of PDGF-A with
RWT. In contrast, cotransfection of
R with PDGF-A resulted in marked
increase in transforming activity similar to that induced by
cotransfection of PDGF-A with
R.
Moreover,
R exhibited a similar
ability to enhance the transforming activity of PDGF-A as that of
R. These results suggest that
substitution of KI domain of the
PDGFR with the KI domain of the
PDGFR does not enhance transforming activity. However, the
replacement of the carboxyl-terminal domain of the
PDGFR with that
of
PDGFR is sufficient to confer the higher transforming activity.
Finally, we also showed that cotransfection of PDGF-A with
R did not result in enhanced
transformation, suggesting that the most distal 79
PDGFR amino
acids are not responsible for conferring the high transforming activity
mediated by the
PDGFR. Since the expression vectors containing
PDGF-A and
/
PDGFR cDNAs each contain different drug-resistant
markers(29, 33) , the number of cells expressing both
cDNAs was also determined by analyzing the number of colonies that
survived in the presence of both HAT/mycophenolic acid and geneticin.
As shown in Table 1, each cotransfection resulted in similar
numbers of double marker-selected colonies, suggesting similar levels
of plasmid DNA were transfected into the cells. Together, these results
predicted that the 86 amino acids (amino acids 942-1028) following
tyrosine kinase 2 domain of the
PDGFR may be responsible for its
transforming activity.
Figure 2:
Comparison of the ability of RWT and
chimeric
/
PDGFRs to enhance PDGF-A transforming function in
NIH/3T3. NIH/3T3 cells were transfected with expression vectors
containing PDGF-A (A) or cotransfected with PDGF-A and LTR-gpt (B),
RWT (C),
R (D),
R (E), or
R (F). Transformed foci
were detected 3 weeks after transfection, and cells were fixed with 10%
buffered formalin phosphate and stained with Giemsa
stain.
Since the level of
receptor expression and their kinase activities have been documented to
affect transforming activity of receptor tyrosine kinases expressed in
NIH/3T3 cells(35, 36) , we next sought to examine the
level of human chimeric /
PDGFR protein and its kinase
activity in each transfectant. Total cell lysates prepared from NIH/3T3
cells transfected with
RWT,
R,
R,
R, or vector alone were
immunoprecipitated using a monoclonal antibody, mAb-
R1, which
recognizes human
PDGFR, but not endogenous murine
PDGFRs
present in NIH/3T3(31) . The immune complexes were then
subjected to either immunoblot analysis using anti-
PDGFR serum or
an in vitro kinase assay. Since the anti-
PDGFR is
directed against the extracellular domain of the
RWT, it was
possible to compare the expression levels of each chimera and the wild
type
PDGFR using this antibody. As shown in Fig. 3A, anti-
PDGFR specifically detected proteins
with molecular masses of approximately 190 kDa in lysates from cells
transfected with the human
RWT or the
/
chimerae. Note
that under these experimental conditions, the murine
PDGFR
naturally expressed in NIH/3T3 cells, was not observed (Fig. 3A, lane 1). Quantitation of the amounts of the
human receptor detected from each transfectant revealed that NIH/3T3
cells transfected with
R,
R, and
R expressed about 1.7-, 0.7-, and
1.0-fold that of
RWT, respectively. Thus, the higher transforming
activity of
R was not due to higher
protein expression, since the steady state level of this chimera was
found to be nearly identical to
RWT level in the transfectants.
Figure 3:
Comparison of the protein levels of the
RWT and each chimera and their kinase activity in the NIH/3T3
transfectants. The levels of
/
PDGFR chimera expressed in each
transfectant and their kinase activity is shown. Cell lysates from
marker-selected cultures were subjected to immunoprecipitation using
monoclonal mAb-
R1 antibody that recognizes human but not the
murine
PDGFR. Following SDS-polyacrylamide gel electrophoresis,
proteins were transferred to Immobilon-P and immunoblotted with
anti-
PDGFR antiserum (A). Similar amount of cell lysates
was also subjected to immunoprecipitation using mAb-
R1 followed by
an in vitro kinase assay as described under
``Experimental Procedures'' (B).
The level of ligand-induced receptor autophosphorylation is a good
measurement for receptor tyrosine kinase activity(37) .
Therefore, we next subjected immune complexes prepared using
mAb-R1 to an in vitro kinase assay. Quantitation of data
presented in Fig. 3B demonstrated that the level of
receptor autophosphorylation of
R,
R, and
R was 0.5-, 1.1-, and 0.5-fold that
of
RWT, respectively. Since the
R exhibited lower autokinase
activity as compared with that shown by
RWT, our findings suggest
that the higher transforming activity of this chimera is not due to the
higher level of receptor tyrosine kinase activity.
In previous
studies we showed that the higher focus forming activity of PDGF-B was
due to distinct biochemical properties of the PDGFR intracellular
domain(25) . In the present report, we have utilized a PDGF-A
focus formation enhancement assay to localize a region within the
PDGFR carboxyl terminus that is responsible for this effect. This
region designated as the minimal transforming domain of the
PDGFR
is 86 amino acids in length and most likely represents an independent
functional domain, since attempts to further dissect this domain led to
a reduction in transforming activity (data not shown). The minimal
transforming domain of
PDGFR has minimal effect on expression
level or kinase activity of the receptors, suggesting that the
difference in transforming activity mediated by
PDGFR and
PDGFR may be due to their different substrate specificity.
Activation of the
PDGFR has been reported to induce significantly
higher levels of tyrosine phosphorylation of RasGAP and Nck than
PDGFR(25, 27) . However, the binding sites within
PDGFR for both Nck and RasGAP are within its kinase insert
domain(13, 18, 19, 20) . Since
substitution of
PDGFR KI domain with that of
PDGFR did not
confer the high transforming activity to the
PDGFR, our findings
suggest that Nck or RasGAP are not likely to be important for
PDGF-induced transformation. Furthermore, we have found that Shc, which
is involved in mediating Ras
activation(24, 38, 39) , is
tyrosine-phosphorylated to a similar extent by activated
and
PDGFR and each
/
PDGFR chimerae expressed in 32D
transfectants. (
)Thus, the possibility that Shc is the major
downstream signaling molecule that regulates the higher transforming
activity of
PDGFR versus
PDGFR is
unlikely(40) .
The minimal transforming domain of the
PDGFR contains four tyrosine residues. Among these tyrosines 966
and 970 are not phosphorylated in vivo.(
)In
contrast, tyrosine residues 1009 and 1021 have been shown to undergo
ligand-dependent tyrosine phosphorylation and association with Syp and
PLC
,
respectively(11, 12, 13, 14, 15) .
The
PDGFR has been shown previously to interact with PLC
and
Syp with a lower efficiency(25, 27) . In addition we
have shown that the low transforming activity of PDGF-A mediated by the
PDGFR can be abolished by mutation of the PLC
binding
site(41) . Therefore, our data indicate that PLC
and/or
Syp may be involved in the higher transforming activity of PDGF-B
mediated by the
PDGFR. Interestingly, it has been reported
recently that Tyr
and Tyr
are also
required for efficient ligand-induced ubiquitination of the activated
PDGFR leading to proper degradation of the ligand-activated
receptor (42) . In addition, the minimal transforming domain of
the
PDGFR contains a hydrophobic region that has been shown
previously to be essential for proper ligand-induced internalization
and down-regulation of activated
PDGFRs(43) . Thus, it
would be remiss not to consider the possibility that the higher
transforming activity of the
PDGFR may also be due to the
difference in the ligand-induced post-translational regulation of the
PDGFR. Further studies to dissect the role of this region in the
ligand-induced receptor processing and substrate phosphorylation will
potentially help us to elucidate the exact molecular basis for
PDGF-induced transformation of NIH/3T3 cells by the
PDGFR.