(Received for publication, February 10, 1995; and in revised form, August 31, 1995)
From the
The purpose of this study was to bacterially express, purify,
and refold combinations of the extracellular immunoglobulin (Ig)-like
domains (2-3, 1-3, and 1-5) of the human
-platelet-derived growth factor receptor (
PDGFR) to
characterize molecular interactions with its ligand, platelet-derived
growth factor (PDGF). The far UV circular dichroism spectroscopy of the
-PDGFR extracellular domains (ECDs) revealed a predominantly
-sheet protein, with a structure consistent with folded Ig-like
domains. The addition of PDGF-BB to these ECD types changed the
conformation of all three types with a decrease in mean residue
ellipticity in the following rank order: 1-5 = 1-3
> 2-3. In striking contrast, addition of PDGF-AA to these ECD
types markedly changed the conformation of ECD 2-3, by an
increased mean residue ellipticity but no changes were observed for
ECDs 1-3 and 1-5. PDGF-AA bound to the immobilized ECD
types 2-3, 1-3, and 1-5 at concentrations of 20, 11,
and 7.5 nM, respectively. In contrast, PDGF-BB bound the ECD
types 2-3, 1-3, and 1-5 at concentrations of 3, 3,
and 2.2 nM, respectively. Scatchard analysis of binding
studies using labeled ECDs indicated that PDGF-BB bound ECD 1-3
and ECD 2-3 with K
values of 74 and
72 nM, respectively. While, PDGF-AA bound ECD 1-3 and
ECD 2-3 with K
values of 33 and 87
nM, respectively. Therefore, our results indicated that the
loss of ECD 1 impaired the binding affinity of
PDGFR ECD 1-3
toward PDGF-AA without having a similar effect on PDGF-BB binding.
Together all of our data suggest that ECD 1 is differentially required
for proper orientation of PDGF-AA but not PDGF-BB binding determinant
within ECDs 2 and 3.
Platelet-derived growth factor (PDGF) ()is a potent
serum mitogen, promoting the growth of mesenchymal cells (for a review,
see (1) ). PDGF exists as a disulfide-linked dimer (M
28,000), composed of two homologous polypeptide
chains designated A and B(2, 3, 4) . Both
homodimers (AA and BB) and a heterodimer (AB) have been isolated from
serum (5) and bind with high affinities to either or both cell
surface-glycosylated receptors designated as
(6) and
(7) of 180 and 185 kDa, respectively. Although both
receptors are highly homologous, the three PDGF isoforms bind the
-PDGFR (8) while the
-PDGFR primarily binds the
PDGF-BB form(4) .
The PDGFRs are members of the receptor
tyrosine kinases, which possesses five immunoglobulin-like
extracellular domains, a single transmembrane spanning motif, and a
split intracellular tyrosine kinase domain(7) . Ligand binding
leads to receptor dimerization, which activates the tyrosine kinase
leading to trans-autophosphorylation of the
receptor(9, 10) . The tyrosine-phosphorylated receptor
now becomes a target for binding Src homology region 2 (SH2) domains of
a number of signaling molecules, which include phosphatidylinositol-3
kinase, GTPase-activating protein, phospholipase C (PLC
),
Src, Grb2, Nck, and the tyrosine phosphatase Syp (for a review, see (11) ), which activate a number of inter-linked downstream
signaling pathways.
A number of other growth factor receptors have
been shown to dimerize in the the presence of ligand. These include the
soluble extracellular domain of the epidermal growth factor
receptor(12) , the human growth hormone receptor(13) ,
and the - (14) and
-PDGFR(15) . For the
PDGFR, both groups expressed all five immunoglobulin-like domains in
Chinese hamster ovary cells (15) and in baculovirus-infected
insect cells(14) . These glycosylated receptor extracellular
domains were shown to exist as dimers but when deglycosylated appeared
as monomers (15) . These soluble receptors dimerized in the
presence of added PDGF (14, 15) , confirming the
conclusion that receptor dimerization is essential for tyrosine kinase
activation.
The expression of reciprocal chimeric /
-PDGFRs
in an interleukin-3-dependent cell line (32D), showed that Ig-like
domains 2-3 of the
-PDGFR must contain the major high
affinity determinants for PDGF-AA
binding(16, 17, 18) . Furthermore, deletion
within the second Ig-like loop of the
-PDGFR resulted in a marked
decrease in binding PDGF-AA but not PDGF-BB(17) . To further
evaluate the structural roles of Ig-like domains 2 and 3 in PDGF
binding, we expressed and purified combinations of the
-PDGFR ECDs
(2-3, 1-3, and 1-5) in quantities suitable for
structure-function studies.
Construction of Expression Vectors-The DNA representing
the human -PDGFR extracellular domains 2-3 (ECD
2-3
), 1-3 (ECD
1-3
), and 1-5 (ECD
1-5
) (6) were synthesized by the
polymerase chain reaction method with a BamHI and HindIII site at the 5` and 3` ends, respectively. The
polymerase chain reaction products were cloned into the pQE9 plasmid
(type 4) possessing an NH
-terminal six-histidine tag
(Qiagen). DNA sequencing confirmed the authenticity of the cloned
inserts. The recombinant vectors were transfected into competent M15 Escherichia coli cells carrying the plasmid pREP4.
To each of the domain types, recombinant human PDGF-AA, PDGF-BB, and EGF (UBI) at a concentration of 0.15 mg/ml was added and the spectra were recorded as described above. The spectra for the growth factors alone were also measured as described above. In order to measure conformational changes assuming 1:1 stoichiometry, the combined spectrum of the ligand and receptor, added together, digitally, was compared to the spectrum of the complex as described by Timm et al.(24) .
Figure 1:
A, a schematic
representation of the -PDGFR extracellular domain showing the
signal peptide (SP), the five Ig-like domains and the
transmembrane region (TM), and the ECD 1-5, 1-3,
and 2-3 constructs designed for this study. B, the
electrophoretic separation of the expressed, purified, and refolded ECD
1-5 (lane 2), 1-3 (lane 3), and 2-3 (lane 4) using 4-20% SDS-PAGE under reducing conditions
and stained with Coomassie Blue. The apparent molecular masses for
domains 1-5, 1-3, and 2-3 are 70, 46, and 32 kDa,
respectively.
Figure 2:
ECD 2-3 (), 1-3
(
), and 1-5 (
) folding assessed by a monoclonal
antibody to
-PDGFR ECD 2 (mAb
R1). The three ECD types were
immobilized and probed with increasing concentrations of mAb
R1.
The signals were detected with goat anti-mouse Fc conjugated to
alkaline phosphatase. The results are represented as absorbance at 405
nm against mAb
R1 concentration.
Figure 3:
The far-UV CD of the uncomplexed (A) ECD 2-3 (thin line) (B) ECD
1-3 (thin line) (C) ECD 1-5 (thin
line), and (D) PDGF (AA or BB) (thin line)
recorded at a protein concentration of 0.15 mg/ml in PBS. A-C also shows the spectrum of PDGF-AA complexed ECD types (dash
lines) and the combined spectrum of the PDGFECD complexes
calculated (dotted lines).
Interestingly, the addition of PDGF-BB to ECD 2-3, 1-3, and 1-5 markedly changed their conformation in the following order: 1-5 = 1-3 > 2-3 (decreased mean residue ellipticity) (Fig. 4, A-C). These conformational changes are in the opposite direction to that observed when PDGF-AA was added to ECD 2-3 (Fig. 3, A-C).
Figure 4:
The far-UV CD of the uncomplexed (A) ECD 2-3 (thin line) (B) ECD
1-3 (thin line), and (C) ECD 1-5 (thin line) recorded at a protein concentration of 0.15 mg/ml
in PBS. A-C also shows the spectrum of PDGF-BB complexed ECD
types (dash lines) and the combined spectrum of the
PDGFECD complexes calculated (dotted
line).
Figure 5:
Binding of PDGF-AA or -BB to ECD 2-3
(), 1-3 (
), and 1-5 (
) using an
immunosorbent assay. The ECD types were immobilized, PDGF added at
increasing concentrations and probed with antibodies to the
COOH-terminal region of the ligand. The signals were detected with goat
anti-mouse Fc conjugated to alkaline phosphatase. Absorbance at 405 nm
are plotted against (A) PDGF-AA and (B) PDGF-BB
concentrations. The N-SH2 domain of GTPase-activating protein (GAP) (
) was used as a
control.
Figure 6:
Scatchard analysis of I-ECD
binding to PDGF. PDGF-BB (A and B) or PDGF-AA (C and D) was immobilized on immunosorbent 96-well plates as
described under ``Experimental Procedures.'' Immobilized PDGF
ligand was incubated in the presence of 5 ng/ml of indicated
I-ECD preincubated in the presence of increasing
concentrations of appropriate unlabeled ECDs. Data was then subjected
to Scatchard analysis(32) .
To further demonstrate the specificity of the binding interaction, we utilized suramin as a potent PDGF antagonist. Competition studies demonstrated that suramin inhibited PDGF-AA or -BB binding to the ECD types with half-maximal values in the range 1.25-2.5 µM (Fig. 7, A and B). This assay provides means to test potential agonists or antagonists as described previously(21, 22) .
Figure 7:
Binding of PDGF-AA or -BB to ECD 2-3
(), 1-3 (
), and 1-5 (
) is inhibited by
suramin. The ECD types were immobilized, PDGF added at a constant
concentration with increasing concentrations of suramin and probed with
antibodies to the COOH-terminal region of the ligand. The signals were
detected with goat anti-mouse Fc conjugated to alkaline phosphatase. %
of maximal binding is plotted against suramin concentration in the
presence of PDGF-AA (A) and PDGF-BB (B).
In this report we describe bacterial expression,
purification, and refolding of the -PDGFR ECDs 2-3,
1-3, and 1-5 to evaluate structural features that
contribute to PDGF binding. The nativeness of ECDs was confirmed by 1)
CD spectroscopy and 2) a monoclonal antibody (mAb
R1) which
recognizes a nonlinear epitope within ECD 2. In vitro binding
studies using an immunoadsorbant assay indicated that immobilized ECD
types 2-3, 1-3, and 1-5 each bound to PDGF-BB at very
similar concentrations (2-3 nM). However, ECD 2-3,
1-3, and 1-5 bound PDGF-AA at concentrations of 20, 11, and
7.5 nM, respectively. Scatchard analyses of binding studies
using labeled ECD 1-3 and ECD 2-3 cross-competed with
unlabeled ECD types or PDGF ligands, indicated that deletion of ECD 1
from ECD 1-3 impaired PDGF-AA binding by more than 2-fold without
affecting PDGF-BB binding. Therefore, our results suggest that
PDGFR extracellular domain 1 is differentially required for high
affinity interaction with PDGF-AA but not PDGF-BB.
Previously, it
was shown that an /
chimeric PDGFR, in which domain 1 of the
-PDGFR was substituted for domain 1 of the
-PDGFR, bound to
PDGF-AA with high affinity. Another
/
chimeric PDGFR in which
ECD 1-3 of the
-PDGFR was substituted for the ECD 1-3
of the
-PDGFR also bound to PDGF-AA with high affinity (16) . In addition, a carboxyl-terminal deletion mutants
encoding the first two Ig-like domains (
R
)
and an internal deletion mutants lacking Ig-like loop 3
(
R
) could not bind to
PDGF-AA(18) . Moreover, an internal deletion mutant lacking
Ig-like loop 2 (
R
) did not affect PDGF-BB
binding but affected PDGF-AA binding(17) . Taken together these
results suggest that ECD 1 of the
-PDGFR is not directly involved
in PDGF-AA binding. Instead ECD 1 is likely to be required for
correctly orienting the other domains with respect to each other, so
that the high affinity binding determinants are positioned close to
each other. In contrast, for PDGF-BB, it appears that the determinants
on ECD 2-3 are in the correct orientation, as indicated by high
affinity binding to PDGF-BB.
We have also examined the physical
characteristics of the -PDGFR ECD types in the absence and
presence of PDGF. Our data indicate that PDGF induces distinct
conformational changes in the three ECD types. PDGF-AA significantly
changed the conformation of ECD 2-3 by increasing the mean
residue ellipticity but did not change the conformation of ECDs
1-3 or 1-5. It is postulated that the orientation of ECD
2-3, with respect to each other by domain 1, may be essential for
correct exposure of the binding determinants. This observation is
reflected in the direct binding assay where the efficiency of ECD
2-3 for PDGF-AA binding is enhanced in the presence of ECD 1. In
contrast, PDGF-BB binding changed the conformation all three domain
types with the following rank order: 1-5 = 1-3 >
2-3 by decreasing the mean residue ellipticity. Since direct
binding of the three ECD types to PDGF-BB are very similar, the
conformational change observed here is likely to be due to an induced
fit mechanism.
The crystal structure and mutagenesis studies
performed on PDGF-BB has allowed the identification of three surface
loops, two at one end of the molecule (loops 1 and 3) and the third
(loop 2) at the opposite end of the elongated twisted -sheet
monomer. The dyad axis relating the two monomers brings loops 1 and 3
of one monomer close to loop 2 of the symmetry related
monomer(29, 30, 31) . Since, PDGF-BB and -AA
have a high degree of sequence similarity they are predicted to possess
similar three-dimensional structures. The sequences within the same
three loops in PDGF-AA, when compared with those identified for
PDGF-BB, have changed from basic to hydroxy amino acids. These major
sequence differences between PDGF-AA and -BB loop regions may
contribute to the different affinities and mode of interaction with the
-PDGFR observed in this study.
Taken together, all of our findings are consistent with the proposed model depicted in Fig. 8, A and B, demonstrating the molecular interaction of PDGF-AA and PDGF-BB with the ECD 1-3 and ECD 2-3. According to this model, during the refolding process, the determinants within ECD 1 induce conformational changes within ECD 2 required for a tight interaction with PDGF-AA but not with PDGF-BB. As shown in Fig. 8A the requirement of ECD 1-induced changes in ECD 2 and 3 for PDGF-AA binding is further necessitated by the distinct physiochemical properties of binding determinants within PDGF-AA in comparison to PDGF-BB (see discussion above). Fig. 8B also illustrates how the loss of determinants within ECD 1 leads to lack of conformational change within ECD 2 and 3 necessary for tight PDGF-AA bindings. The physical consequence of this effect is PDGF-AA-induced conformational changes in ECD 2 and 3 (measured by CD and reflected by the reduced affinity) to allow such binding. In contrast the binding of PDGF-BB to ECD 2-3 is not affected since the binding site for this ligand is in the correct orientation.
Figure 8: Schematic model illustrating the differential molecular interaction of PDGF-AA and PDGF-BB with extracellular domains 2-3 and 1-3. A, molecular interaction of PDGF-AA and PDGF-BB with extracellular domains 1-3. B, molecular interaction of PDGF-A and PDGF-B with ECD 2-3. I, II, and III denote extracellular domains 1, 2, and 3. PDGF-AA is depicted by as a closed circle; PDGF-BB is depicted by a hatched oval. The non-identity of PDGF isoforms is based on changes from basic to hydroxy amino acids within the three surface loop. Induced fit is designated by subtle changes in the structure of ECD 2-3 and 1-3. The conformational changes induced by PDGF-AA in extracellular domains 2 and 3 is depicted by re-orientation of the IgG-like domains.
In conclusion, we show that for PDGF-AA binding, ECD 2
and 3 are necessary and that ECD 1 orients these domains with respect
to each other so that binding determinants are correctly positioned in
three-dimensional space. However, for PDGF-BB, ECD 2-3 are
correctly oriented for high affinity binding. The availability of large
amounts of purified -PDGFR ECDs will allow us to define the
molecular interactions with PDGF in detail using x-ray crystallography.
Furthermore, since PDGF and its receptor are implicated in a number of
disease states including cancer, the immunosorbent assay system
developed will help screen and identify potential agonists and
antagonists of the
-PDGFR that could be of therapeutic value.