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(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 (PDGF) ( 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 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 The expression of reciprocal chimeric Construction of Expression Vectors-The DNA representing
the human
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
Figure 2:
ECD 2-3 (
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 PDGF
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
PDGF
Figure 5:
Binding of PDGF-AA or -BB to ECD 2-3
(
Figure 6:
Scatchard analysis of
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
(
In this report we describe bacterial expression,
purification, and refolding of the Previously, it
was shown that an We have also examined the physical
characteristics of the 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 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
Volume 270,
Number 46,
Issue of November 17, 1995 pp. 27595-27600
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Platelet-derived Growth Factor
(PDGF) Receptor for PDGF-AA and PDGF-BB Binding (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
)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) . (PLC
),
Src, Grb2, Nck, and the tyrosine phosphatase Syp (for a review, see (11) ), which activate a number of inter-linked downstream
signaling pathways.
- (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./-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.
-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.Protein Expression, Refolding, and Purification
Protein
expression and nickel-affinity purification was performed as described
previously(19) . The three domain types (2-3, 1-3,
and 1-5) were refolded by dialyzing into 2 M urea buffer
(100 mM phosphate), pH 7.4, containing 1 mM EDTA, 150
mM NaCl, and 0.1% -mercaptoethanol. Subsequently, the
proteins were dialyzed into refolding buffer consisting of 100
mM phosphate, 150 mM NaCl, 2 mM reduced
glutathione, 1 mM oxidized glutathione, 1 mM EDTA at
pH 7.4. The refolded proteins were then dialyzed into 100 mM phosphate buffer, pH 7.4, to remove the reduced and oxidized
glutathione. The refolded domains were further purified by FPLC (Mono
Q) (Pharmacia) and analyzed by 4-20% SDS-PAGE (Bio-Rad) under
reducing and nonreducing conditions.Solid-phase Binding Assays
Direct immobilization
of the -PDGFR extracellular domains (ECDs) 2-3, 1-3,
and 1-5 were performed by overnight incubation in immunosorbent
96-well plates (Falcon 3912) at room temperature using 80 ng of
purified protein in 50 µl of PBS in 0.02% sodium azide. Binding
studies were performed with the monoclonal antibody to ECD 2 of the
-PDGFR (mAb R1), PDGF-AA and -BB. The bound PDGF-AA or -BB
were detected by anti-PDGF antibodies to the COOH-terminal
region(20) . The NH-terminal SH2 domain of
GTPase-activating protein was also directly immobilized and used as
controls. For competition experiments, suramin was added at increasing
concentrations in the presence of PDGF-AA (10 nM) or -BB (10
nM). Immunodetection was followed as described
previously(21) .Ligand Binding Assay
ECD 1-3 or ECD
2-3 was labeled by the chloramine-T method (specific activity 8
10
cpm/ng). PDGF-AA or PDGF-BB (40 nM)
were immobilized by overnight incubation in immunosorbent 96-well
plates (Falcon 3912) at room temperature in 50 µl of PBS in 0.02%
sodium azide. Immobilized PDGF ligands were washed by ice-cold HEPES
binding buffer (25 mM HEPES, 150 mM NaCl, 5
mM KCl, 1.2 mM MgSO, and 0.1% bovine
serum albumin, pH 7.5) once and incubated in the presence of 5 ng/ml
labeled ECD preincubated in the presence of increasing concentrations
of unlabeled ECD, PDGF-AA, or PDGF-BB for 2 h at 16 °C. Free I-ECD was removed by washing wells three times with
ice-cold HEPES binding buffer and bound
I-ECD was
quantitated and analyzed by the method of Scatchard.
Far UV Circular Dichorism Spectroscopy
The
-PDGFR ECD 2-3, 1-3, and 1-5, for circular
dichroism spectroscopy were prepared in PBS at a protein concentration
of 0.15 mg/ml. The protein concentrations of the stock solutions, used
in CD measurements, were determined spectrophotometrically (23) and the spectra scaled according to standard CD spectra
for immunoglobulins. The spectra were recorded using a 1-mm demountable
``strain free'' quartz cuvette on a JASCO J-500C
spectropolarimeter in the wavelength range of 260-195 nm at room
temperature. The following settings were used: bandwidth, 1 nm; time
constant, 2.0 s; step resolution, 0.1 nm; scan speed, 10
millidegree/min; sensitivity, 1 millidegree/cm. Each spectrum
represents an average of 4 scans with the baseline subtracted.
Expression, Refolding, and Purification of the
In order to
characterize the molecular interactions between the -PDGFR ECDs 2-3, 1-3, and 1-5-PDGFR
extracellular domains 2-3, 1-3, and 1-5 and PDGF-AA
or -BB, the above domain combinations were cloned and expressed in
bacteria (Fig. 1A). Each ECD combination was purified
to about 90% by Ni chelate affinity chromatography
and analyzed by 4-20% SDS-PAGE under reducing conditions. As
shown in Fig. 1B, the apparent molecular masses for
domains 2-3, 1-3, and 1-5 are 32, 46, and 70 kDa,
respectively, predicting that they exist as soluble monomers under
reducing and denaturing conditions. However, under nonreducing but
denaturing conditions, domains 2-3 exist as a monomer while
domains 1-3 and 1-5 exist as oligomers (data not shown).
This suggests that domain 1 possesses the determinants that may mediate
the receptor oligomerization of ECD 1-3 and ECD 1-5 by
interdisulfide bonds.
-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.
A
neutralizing mAb -PDGFR ECDs 2-3, 1-3, and 1-5 Are
Recognized by Receptor-specific Monoclonal AntibodyR1 directed against the -PDGFR, bound the
native receptor with high affinity(25) . The epitope recognized
by the mAb R1 was shown to be denatured under reducing
conditions(18) . Furthermore, the major PDGFR epitope
binding to mAb R1 was localized to Ig-like domain 2 (18) .
These findings indicated that high affinity binding of mAb R1 to
PDGFR requires proper folding of determinants within Ig-like
domain 2. Therefore, bacterially expressed domains 2-3,
1-3, and 1-5 were analyzed for proper folding by solid
phase binding assays with the monoclonal antibody of the -PDGFR
(mAb R1). Results shown in Fig. 2indicated binding to the
ECD 1-5, ECD 1-3 with a half-maximal value of around 0.4
nM while ECD 2-3 showed half-maximal binding at
concentration of about 0.6 nM. This confirmed that all three
receptor ECD types were correctly refolded, however, the maximal
binding of ECDs 1-3 and 1-5 was estimated to be
approximately 2-fold higher than that of ECD 2-3. Therefore, our
results suggest that ECD 1 may be indirectly involved in binding of mAb
R1.
), 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.
CD Spectroscopic Studies of PDGF-AA and -BB-induced
Conformational Changes in the Three ECD Types
Far-UV CD
spectroscopy was performed to investigate the folding of the three ECD
types, to determine whether they possessed Ig-like structures and to
study ligand-induced conformational changes. The CD spectra of the
purified ECD 2-3, 1-3, and 1-5 indicated domains
composed essentially of -sheet (trough at 215 nm) similar to that
observed for Ig-like domains and also confirmed that they were
correctly folded. The calculated secondary structure for the 3 ECD
types are 45-56% -sheet and 44-56% turns (Table 1)(28) . Fig. 3, A-C, show the
far-UV CD spectra of the the ECDs and their recorded spectra of a 1:1
mixture with the indicated PDGF ligands. Moreover, a calculated spectra
for a 1:1 mixture of ECD and PDGF ligand is indicated. Fig. 3D, shows the spectrum of PDGF-AA or -BB which has
a calculated -sheet content of 60%. The addition of PDGF-AA at an
equimolar ratio to the three ECD types showed a significant
conformational change for domains 2-3 (increased mean residue
ellipticity) in which the -sheet content increased from 56 to 100%
but not for domains 1-3 and 1-5 (Table 1). However,
the addition of EGF to these ECD types did not show conformational
changes, eliminating the possibility of nonspecific binding and
conformational changes (data not shown).
ECD complexes
calculated (dotted lines).
ECD complexes calculated (dotted
line).
Binding of PDGF-AA and -BB to ECDs 2-3, 1-3,
and 1-5
The wild type receptor as well as the eukaryotic
expression of all five Ig-like domains have shown high affinity binding
to both PDGF-AA and
-BB(14, 15, 26, 27) . To investigate
binding of PDGF-AA and -BB to each ECD type, we developed a solid phase
binding assay in which the ECDs were immobilized. As shown in Fig. 5A, the immobilized ECD 1-3 and ECD 1-5 each
bound PDGF -AA at a concentration of 2-20 nM. However,
the binding capacity of ECD 2-3 was 50% less as compared to ECD
1-3 and ECD 1-5. This is very similar to the differential
observed for mAb R1 binding to these ECDs. PDGF-BB bound to all
three ECD types at concentrations between 2.0 and 3.0 nM (Fig. 5B), indicating that ECD 2-3 possess
the major determinants for PDGF-BB binding. We next quantitated the
affinity of ECD 1-3 and ECD 2-3 for each PDGF ligand.
Accordingly, the level of I-ECD 1-3 or
I-ECD 2-3 binding to the immobilized PDGF-AA or
PDGF-BB was determined in the presence of increasing concentrations of
unlabeled homologous ECDs. The results were then analyzed by the method
of Scatchard using a one ligand/one class of binding site model. As
shown in Fig. 6A, Scatchard analysis of
I-ECD 1-3 binding to PDGF-BB measured high affinity
binding sites with a K
of 74 nM. A
similar analysis of I-ECD 2-3 binding to PDGF-BB
measured a similar number of binding sites with a K
value of 72 nM (Fig. 6B). Furthermore,
as shown in Fig. 6C, Scatchard analyses of I-ECD 1-3 binding to PDGF-AA estimated high
affinity binding sites with a K
value of 33
nM. In striking contrast, I-ECD 2-3
reproducibly showed a similar number of binding sites with a K
value of 87 nM (Fig. 6D). Thus deletion of ECD 1 impaired the
affinity of ECD 2-3 for PDGF-AA binding by more than 2-fold,
without having a comparable effect on PDGF-BB binding. Under these
conditions, the binding of I-ECD 1-3 or 2-3
was also competed by increasing concentrations of unlabeled PDGF-AA or
PDGF-BB with similar kinetics as shown with unlabeled ECDs (data not
shown). These results are consistent with the finding that the soluble
-PDGFR receptors have K values of
30-100 nM, but when immobilized provided a value of 0.4
nM(27) . Furthermore, -PDGFR ECD 1-3 was
expressed in NIH 3T3 cells as a Fc fusion protein bound to PDGF-BB with
an affinity of 1.5 nM. The -PDGFR ECD 1-3 competed
with the -PDGFR ECD 1-3 for PDGF-BB binding with a
half-maximal value of 10 nM(21) , thus confirming that
the recombinant -PDGFR ECD types shown here compare well with
those expressed in eukaryotic systems. Furthermore, our findings
suggest that the extent of complex formation between PDGF and ECDs is
at least 78% in an equimolar mixture since the K value of ECD for PDGF-AA or PDGF-BB is not greater than 80
nM. Thus, our data indicates that most ECDs exist in complexes
with PDGF under the experimental conditions used for CD spectroscopy.
), 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.
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) .
), 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).
-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./ 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.-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.-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.
-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.
)-PDGFR, -platelet derived growth
factor receptor; ECD, extracellular domain; PAGE, polyacrylamide gel
electrophoresis; PBS, phosphate-buffered saline; mAb, monoclonal
antibody.
We thank Alex DeSeabra for excellent technical
support, Amal Salama for reading this manuscript, and Drs. Jacalyn
Pierce and Steve Tronick for support and encouragement.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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K. Miyazawa, G. Backstrom, O. Leppanen, C. Persson, C. Wernstedt, U. Hellman, C.-H. Heldin, and A. Ostman Role of Immunoglobulin-like Domains 2-4 of the Platelet-derived Growth Factor alpha -Receptor in Ligand-Receptor Complex Assembly J. Biol. Chem., September 25, 1998; 273(39): 25495 - 25502. [Abstract] [Full Text] [PDF]
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G. Fuh, B. Li, C. Crowley, B. Cunningham, and J. A. Wells Requirements for Binding and Signaling of the Kinase Domain Receptor for Vascular Endothelial Growth Factor J. Biol. Chem., May 1, 1998; 273(18): 11197 - 11204. [Abstract] [Full Text] [PDF]
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N. A. Lokker, J. P. O'Hare, A. Barsoumian, J. E. Tomlinson, V. Ramakrishnan, L. J. Fretto, and N. A. Giese Functional Importance of Platelet-derived Growth Factor (PDGF) Receptor Extracellular Immunoglobulin-like Domains. IDENTIFICATION OF PDGF BINDING SITE AND NEUTRALIZING MONOCLONAL ANTIBODIES J. Biol. Chem., December 26, 1997; 272(52): 33037 - 33044. [Abstract] [Full Text] [PDF]
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A. Uren, J. Jakus, J. F. de Mora, A. Yeudall, E. Santos, S. Gutkind, and M. A. Heidaran Carboxyl-terminal Domain of p27Kip1 Activates CDC2 J. Biol. Chem., August 29, 1997; 272(35): 21669 - 21672. [Abstract] [Full Text] [PDF]
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B. Barleon, F. Totzke, C. Herzog, S. Blanke, E. Kremmer, G. Siemeister, D. Marme, and G. Martiny-Baron Mapping of the Sites for Ligand Binding and Receptor Dimerization at the Extracellular Domain of the Vascular Endothelial Growth Factor Receptor FLT-1 J. Biol. Chem., April 18, 1997; 272(16): 10382 - 10388. [Abstract] [Full Text] [PDF]
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T. Hosoda, K. Monzen, Y. Hiroi, T. Oka, E. Takimoto, Y. Yazaki, R. Nagai, and I. Komuro A Novel Myocyte-specific Gene Midori Promotes the Differentiation of P19CL6 Cells into Cardiomyocytes J. Biol. Chem., September 14, 2001; 276(38): 35978 - 35989. [Abstract] [Full Text] [PDF]
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