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J Biol Chem, Vol. 273, Issue 39, 25495-25502, September 25, 1998


Role of Immunoglobulin-like Domains 2-4 of the Platelet-derived Growth Factor alpha -Receptor in Ligand-Receptor Complex Assembly*

Keiji MiyazawaDagger , Gudrun Bäckström, Olli Leppänen, Camilla Persson, Christer Wernstedt, Ulf Hellman, Carl-Henrik Heldin, and Arne Östman§

From the Ludwig Institute for Cancer Research, S-751 24 Uppsala, Sweden

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Platelet-derived growth factor (PDGF) is a dimeric protein that exerts its effects through tyrosine kinase alpha - and beta -receptors. The extracellular part of each receptor is composed of five Ig-like domains. Recombinant forms of alpha -receptor domains 1-4 (alpha RD1-4), 1-3 (alpha RD1-3), and 1 and 2 (alpha RD1-2) were prepared after expression in Chinese hamster ovary cells and were used to study the assembly of soluble ligand-receptor complexes. When incubated with micromolar concentrations of PDGF, both alpha RD1-3 and alpha RD1-4 formed complexes of 1:2 molar composition, i.e. one dimeric PDGF molecule bound two soluble receptors. alpha RD1-3, in contrast to alpha RD1-4, formed detectable 1:1 complexes under conditions of ligand excess. alpha RD1-4 displayed an increased ability to form 1:2 complexes as compared with alpha RD1-3 under conditions of limiting concentrations of ligand. We thus conclude that Ig-like domain 4-mediated receptor-receptor interactions contribute to 1:2 PDGF·alpha RD1-4 complex formation. Since alpha RD1-4 and alpha RD1-3 were equipotent in blocking binding of subnanomolar concentrations of PDGF to cell-surface receptors, we also conclude that this effect is predominantly achieved through formation of Ig-like domain 4-independent 1:1 ligand-receptor complexes. Finally, since alpha RD1-2 bound PDGF-BB with high affinity, whereas PDGF-AA was bound only with low affinity, we conclude that Ig-like domain 3 of the PDGF alpha -receptor contains epitopes of particular importance for PDGF-AA binding and that most of the PDGF-BB-binding epitopes reside in Ig-like domains 1 and 2.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Platelet-derived growth factors (PDGFs)1 are a family of disulfide-bonded dimeric isoforms of A- and B-chains with potent mitogenic activity on connective tissue cells, glia cells, and endothelial cells (reviewed in Ref. 1). PDGF has been implicated in a number of diseases involving proliferation of PDGF-responsive cells, such as atherosclerosis, restenosis, glomerulonephritis, and certain malignancies (2).

PDGF A- and B-chains, which have ~60% identical amino acid sequences in their mature parts, form homo- and heterodimers that exert their cellular effects through two structurally related tyrosine kinase receptors, denoted alpha - and beta -receptors (3). The A-chain binds only alpha -receptors, whereas the B-chain binds both alpha - and beta -receptors.

Crystallographic analysis of PDGF-BB revealed that the two subunits are arranged in an antiparallel manner (4). Each subunit consists of a tight cystine knot motif from which two loops (loops 1 and 3) point in one direction and one loop (loop 2) points in the other direction. As a consequence of the antiparallel arrangement of the dimer, loops 1 and 3 of one subunit are juxtaposed to loop 2 of the other subunit. Mutational analysis has mapped the receptor-binding amino acid residues mainly to loops 1 and 3, but loop 2 also contributes to some extent (4-8). The dimeric PDGF molecule thus displays two receptor-binding regions, each one made up of epitopes derived from both subunits.

Both PDGF alpha - and beta -receptors consist of an extracellular part composed of five Ig-like domains, a single transmembrane region, and an intracellular split tyrosine kinase domain (9, 10). The receptors are activated by ligand-induced dimerization (11, 12). Soluble forms of the extracellular parts of the PDGF alpha - and beta -receptors undergo ligand-dependent dimerization, and functional bivalency of PDGF has been demonstrated, suggesting the formation of a 1:2 ligand-receptor complex (13-15). Ligand-binding regions of the alpha -receptor have been mapped to Ig-like domains 1-3 by analysis of deletion mutants, alpha /beta -receptor chimeras, and soluble receptor fragments (16-18). More recently, evidence has been presented suggesting that PDGF-induced receptor dimerization not only involves ligand-receptor interactions, but also receptor-receptor interactions mediated by Ig-like domain 4 (18-20).

To further study the structural basis for PDGF-induced receptor dimerization, the properties of CHO cell-derived recombinant proteins consisting of PDGF alpha -receptor Ig-like domains 1-4 (alpha RD1-4), 1-3 (alpha RD1-3), and 1 and 2 (alpha RD1-2) were compared. Using these receptor fragments, we demonstrate that alpha RD1-4 at micromolar concentrations, in contrast to alpha RD1-3, forms a 1:2 ligand-receptor complex also under conditions of ligand excess. We show that this property is a consequence of Ig-like domain 4-mediated receptor-receptor interactions. We also show that these receptor-receptor interactions do not contribute significantly to the inhibitory effect of soluble receptors on binding of subnanomolar concentrations of PDGF to cell-surface receptors. Finally, characterization of the PDGF binding properties of alpha RD1-2 demonstrates that Ig-like domain 3 of the PDGF alpha -receptor contains epitopes of particular importance for PDGF-AA binding and that most of the PDGF-BB-binding epitopes are localized within the alpha RD1-2 fragment.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Establishment of a CHO Cell Line Expressing alpha RD1-4-GST-- A polymerase chain reaction product spanning amino acids 1-419 of the PDGF alpha -receptor flanked by XhoI sites was generated. The fragment was cloned into the pCR-Script SK(+) vector (Stratagene) and sequenced by the dideoxy chain termination method. The fragment was excised by XhoI and cloned into the SalI site of the pMT2SM-GST vector (21), a gift from Dr. M. F. Gebbink (Netherlands Cancer Institute). CHO(dhfr-) cells (American Type Culture Collection) were cotransfected, using the calcium phosphate method, with pMT2SM-alpha RD1-4-GST and the G418 resistance-carrying plasmid RD2 at a 20:1 ratio. After transfection, cells were grown in Ham's F-12 medium containing 10% fetal calf serum and 0.4 mg/ml G418. G418-resistant clones were screened for secretion of alpha RD1-4-GST by an immunoprecipitation-based assay using 125I-labeled PDGF-BB and GST antiserum (22). After the first screening, the best cell line (clone 2-4) was maintained in Dulbecco's modified Eagle's medium containing 10% dialyzed fetal calf serum (Hyclone Laboratories) and increasing concentrations of methotrexate (ICN Biomedicals Inc.) to obtain clones with higher expression of the recombinant protein. Clone C5-2, which was obtained after selection under 30 µM methotrexate, was used for large-scale expression. When cultured in roller bottles with 150 ml of medium, this clone secreted ~4 µg of recombinant protein/ml/24 h.

Production and Purification of Recombinant Proteins-- CHO cells expressing alpha RD1-4-GST (clone C5-2) were expanded in 18 roller bottles in the presence of 100 µM methotrexate. After the cells reached confluency, they were cultured in RDF medium (2:1:1 RPMI 1640 medium/Dulbecco's modified minimum essential medium/Ham's F-12 medium) supplemented with 990 mg/liter glutamine, 10 mM HEPES, pH 7.4, 200 mg/liter proline, 100,000 units/liter penicillin, 100 mg/liter streptomycin, and 50 mg/liter gentamycin in the presence of 10% fetal calf serum for 2 days and then cultured in serum-free RDF medium for 3 days. Serum-free conditioned medium (3 liters) was filtered through a mesh to remove cell debris and applied to a 2.5 × 6-cm Q-Sepharose column (Amersham Pharmacia Biotech). After washing the column with 600 ml of phosphate-buffered saline, the bound protein was eluted in 0.35 M NaCl and 10 mM sodium phosphate, pH 7.4. The crude protein preparation thus obtained was incubated with 750 µl of glutathione-Sepharose (Amersham Pharmacia Biotech) for 16 h at 4 °C. The gel was then washed with 20 ml of binding buffer and further washed with 10 ml of thrombin cleavage buffer (50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, and 10 mM CaCl2).

The protein bound on the gel was digested by 100 units of bovine thrombin (Sigma) in a total volume of 2.5 ml at 37 °C for 3 h. Cleaved protein was eluted by washing with cleavage buffer; concentrated with a Centricon 50 (Amicon, Inc.) to 800 µl; and applied to a Superdex 200 (16/60) column in 0.5 M NaCl and 20 mM Tris-HCl, pH 7.5. The fraction eluted from 68 to 72 ml contained the 80-kDa protein (alpha RD1-4). The fraction eluted from 74 to 78 ml contained predominantly a 63-kDa protein (alpha RD1-3) and a small amount of the 80-kDa protein. A pure preparation of the 63-kDa protein was obtained by repeated gel permeation chromatography on the Superdex 200 column.

To prepare the lysyl endopeptidase fragment, the 80-kDa protein (30 mg) was dialyzed against 0.1 M Tris-HCl, pH 8.8, and concentrated with the Centricon 50 to 10 mg/ml. The protein was then treated with Achromobacter lysyl endopeptidase (100:1, w/w; Wako Bioproducts) at 37 °C for 16 h. The digestion was terminated by addition of 1 mM phenylmethylsulfonyl fluoride. The digest was applied to a Superdex 200 (16/60) column in 0.5 M NaCl and 20 mM Tris-HCl, pH 7.5. The gel permeation chromatography resulted in a pure fraction of a 52-55-kDa protein (alpha RD1-2) eluting from 76 to 82 ml.

Purity of the proteins was assessed by SDS gel electrophoresis. The concentration of the purified proteins was determined by amino acid analysis. Iodination of alpha RD1-3 and alpha RD-14 was performed using the chloramine-T method (24).

Identification of the 80-, 63-, and 55-kDa Proteins as alpha RD1-4, alpha RD1-3, and alpha RD1-2, Respectively-- The 80- and 63-kDa proteins were subjected to amino-terminal amino acid sequencing on an Applied Biosystems Model 494A peptide sequencer after SDS gel electrophoresis and transfer to polyvinylidene difluoride membrane. In both cases, amino termini were blocked, suggesting that the amino terminus of each peptide is common, most likely located at Gln-24, which is the predicted amino terminus of the natural PDGF alpha -receptor protein.

To locate the carboxyl termini of the 80- and 63-kDa proteins, tryptic peptide mapping on a narrow-bore µRPC C2/C18 SC 2.1/10 column was performed as described previously (23). After comparison of peptide maps, three peptides unique for the 80-kDa protein were selected for sequencing. One peptide was of the sequence YLVPR, corresponding to the expected carboxyl terminus of alpha RD1-4 after cleavage of alpha RD1-4-GST at the thrombin cleavage site in the linker between alpha RD1-4 and GST (see Fig. 1). No peptides corresponding to GST sequences were found. The second unique alpha RD1-4 peptide had a sequence of ISWLK (amino acids 347-351), and the third localized to the region between the two other peptides. Inspection of the amino acid sequence of the PDGF alpha -receptor revealed the presence of sequence PPPRIS at positions 343-348, which fits with the optimum sequence for thrombin cleavage (40). Together, these observations suggest that the 63-kDa protein was generated by internal cleavage of alpha RD1-4 at position 346 and thus lacks most of Ig-like domain 4 (see Fig. 1). This notion was further supported by the amino acid composition of the 63-kDa protein (data not shown) as well as by the size differences after enzymatic deglycosylation between the 80- and 63-kDa proteins, as estimated by SDS gel electrophoresis (data not shown). The identification of the amino and carboxyl termini of the 55-kDa protein to positions 24 and 194, respectively, was based on the amino acid composition of the protein (data not shown) together with the fact that there are no lysine residues located between positions 37 and 194.

Gel Chromatography of PDGF-BB·alpha RD1-4 Complexes-- PDGF-BB was mixed with 170 µM alpha RD1-4 at molar ratios of 0:1, 0.25:1, 0.5:1, and 1:1 in 0.5 M NaCl and 20 mM Tris-HCl, pH 7.5, and incubated at room temperature for 2 h. The mixtures were then analyzed on a Superdex 200 PC (3.2/30) column operated at room temperature in the SMART system (Amersham Pharmacia Biotech). The following proteins (obtained from Bio-Rad) were used as standards to calibrate the column: thyroglobulin (670 kDa), bovine gamma -globulin (158 kDa), and ovalbumin (44 kDa).

Complex Formation Assays and Native Gel Electrophoresis-- alpha RD1-4, alpha RD1-3, or alpha RD1-2 at concentrations of 12.5-50 µM was mixed with various concentrations of PDGF-AA or PDGF-BB in 10 µl of 0.5 M NaCl and 20 mM Tris-HCl, pH 7.4. Electrophoresis of proteins on 4% agarose gels was performed in 10 mM PIPES, 4 mM sodium acetate, pH 6.8, and 0.1% Triton X-100 at 10 V/cm for 20 min. Native polyacrylamide gel electrophoresis was performed using 7.5% acrylamide gels at room temperature as described (14).

Dynamic Light Scattering-- Hydrodynamic radii of proteins were derived from dynamic light scattering measurements on DynaPro-801 (Protein Solutions, Inc.) at protein concentrations ranging between 12.5 and 70 µM.

PDGF alpha -Receptor Binding Assay-- PDGF-AA or PDGF-BB was 125I-labeled by the chloramine-T (24) and Bolton and Hunter (25) methods, respectively. Binding assays were performed as described previously (26) using porcine aortic endothelial cells stably expressing the PDGF alpha -receptor (27). 125I-Labeled PDGF-AA or PDGF-BB at a concentration of 0.16 nM was preincubated with various concentrations (3-3000 nM) of alpha RD1-4, alpha RD1-3, or alpha RD1-2 in binding buffer for 15-30 min before addition to cell cultures.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression and Purification of Soluble Forms of PDGF alpha -Receptor Ig-like Domains 1-4, 1-3, and 1 and 2-- An expression vector encoding a fusion protein of PDGF alpha -receptor Ig-like domains 1-4 and GST (alpha RD1-4-GST) was generated in which the part encoding the PDGF alpha -receptor was connected with the GST-encoding part by a thrombin cleavage recognition sequence (Fig. 1). Transient transfection in COS cells followed by metabolic labeling and immunoprecipitations with a GST antiserum revealed a secreted component of 110 kDa upon analysis by SDS gel electrophoresis (data not shown).


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  		Fig. 1.   Structure of recombinant forms of different parts of the PDGF alpha -receptor extracellular domain. The expression plasmid pMT2SM-alpha RD1-4-GST directs the expression of a secreted fusion protein consisting of PDGF alpha -receptor sequences corresponding to Ig-like domains 1-4 (amino acid residues 24-419), a linker sequence containing a thrombin recognition sequence (ls), and GST. Borders of the different Ig-like domains and positions of cysteine residues are indicated. The amino acid sequence of the linker region and the thrombin and lysyl endopeptidase recognition sites are shown. Cleavage sites are indicated by arrows. After cleavage with thrombin and purification by gel permeation chromatography, pure fractions of alpha RD1-4 and alpha RD1-3 were obtained. alpha RD1-2 was generated and purified by lysyl endopeptidase cleavage of alpha RD1-4 and subsequent gel permeation chromatography.

To obtain a stable source for the production of the PDGF alpha -receptor extracellular domain, the plasmid was transfected into CHO(dhfr-) cells, and after selection in increasing concentrations of methotrexate, a clone that secreted ~4 µg of alpha RD1-4-GST/ml/24 h was identified. The clone was expanded and grown in cycles consisting of 2 days of culture in the presence of serum followed by 3 days of culture in serum-free medium. Serum-free conditioned medium was used for purification of recombinant proteins.

Purification was performed by ion-exchange chromatography on Q-Sepharose followed by affinity purification on glutathione-Sepharose. The immobilized fusion protein was then cleaved with thrombin, and the released protein was subjected to gel chromatography on a Superdex 200 column. Analysis by SDS gel electrophoresis of the broad peak from the gel permeation chromatography showed a fraction of two components with molecular masses of 80 and 63 kDa (data not shown). Pure fractions of the two forms were obtained by pooling the early and late eluting peak fractions separately and subjecting them to rechromatography (Fig. 2A). Sequencing of internal peptides and analysis of amino acid composition were used to structurally characterize the two proteins (see "Material and Methods" for details). From this analysis, it was concluded that the 80-kDa form was generated through cleavage at the linker sequence between the PDGF alpha -receptor and GST and that the 63-kDa component was formed through thrombin cleavage at Arg-346 (Fig. 1). We thus conclude that the 80-kDa protein corresponds to Ig-like domains 1-4 of the PDGF alpha -receptor and that the 63-kDa form is a carboxyl-terminally truncated form that is lacking most of Ig-like domain 4. The two proteins will hereafter be referred to as alpha RD1-4 and alpha RD1-3, respectively.


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  		Fig. 2.   Purification of alpha RD1-4, alpha RD1-3, and alpha RD1-2. A, analysis of alpha RD1-4 (lane 1) and alpha RD1-3 (lane 2) by SDS-7.5% polyacrylamide gel electrophoresis under reducing conditions; B, analysis of alpha RD1-2 by SDS-10% polyacrylamide gel electrophoresis under reducing conditions. Proteins were visualized by Coomassie staining, and positions of marker proteins are indicated to the left.

To obtain a fragment corresponding to Ig-like domains 1 and 2, we took advantage of the absence of any lysine residues between positions 37 and 194 in the PDGF alpha -receptor. alpha RD1-4 was subjected to cleavage by Achromobacter lysyl endopeptidase, and after gel permeation chromatography, a fraction of a 52-55-kDa protein was obtained (Fig. 2B). Since amino acid analysis indicated the presence of 1.5-2 mol of lysine/mol of protein, we concluded that the preparation was composed predominantly of a protein encompassing amino acid residues 24-194 with some contribution of a protein cleaved at Lys-36 and thus encompassing amino acid residues 37-194. Amino acid residues 24-194 correspond approximately to Ig-like domains 1 and 2 of the PDGF alpha -receptor, and the preparation will hereafter be referred to as alpha RD1-2 (Fig. 1).

alpha RD1-4 Forms a 1:2 Complex with PDGF Also under Conditions of Ligand Excess-- The complete extracellular region of the PDGF alpha -receptor forms ligand-dependent dimers (14). To investigate if this was also a property of alpha RD1-4, the protein was subjected to analyses, in the absence or presence of PDGF-BB, by dynamic light scattering, gel chromatography, and native gel electrophoresis.

As shown in Table I, addition of ligand to alpha RD1-4 shifted the apparent molecular mass from 110 to 240 kDa, as analyzed by dynamic light scattering. When analyzed by gel permeation chromatography, alpha RD1-4 eluted as a 130-kDa protein in the absence of PDGF-BB. In the presence of PDGF-BB, the peak shifted to an elution position corresponding to 260 kDa (Fig. 3A). SDS gel electrophoresis of the proteins of the 260-kDa peak confirmed the presence of PDGF-BB in this complex (Fig. 3B). Thus, we conclude that Ig-like domain 5 is not required for the formation of a stable ligand-receptor complex.

                              
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Table I
Analysis by light scattering of alpha RD1-4 with and without ligand
Hydrodynamic radii (Rh) of alpha RD1-4 and PDGF-BB were measured by dynamic light scattering at the indicated concentrations in 0.15 M NaCl and 20 mM Tris-HCl, pH 7.5, at 37 °C. The estimated molecular masses were calculated from the Rh values and the sample temperature using a standard curve for globular proteins. The measurement for PDGF-BB was performed at a higher concentration because of the sensitivity limit of detection.


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  		Fig. 3.   Analysis of alpha RD1-4 and PDGF-BB·alpha RD1-4 complexes by gel chromatography. A, gel chromatography profiles of mixtures of alpha RD1-4 and PDGF-BB on a Superdex 200 PC (3.2/30) column. The elution positions of marker proteins are indicated by arrows. B, analysis of peak fractions by SDS-10% polyacrylamide gel electrophoresis under reducing conditions. Lane 1, 130-kDa peak from chromatogram 1 in A; lane 2, 260-kDa peak from chromatogram 3; lane 3, control PDGF-BB. Proteins were visualized by Coomassie staining, and positions of marker proteins are indicated to the left.

To determine the stoichiometry of the PDGF-BB·alpha RD1-4 complex, titration experiments were performed with a fixed receptor concentration of 50 µM and ligand/receptor ratios ranging between 0.06:1 and 4:1. Complex formation was assayed by a shift in mobility during electrophoresis under native conditions on agarose (Fig. 4A) and polyacrylamide (Fig. 4B) gels. At a 0.5:1 molar ratio of PDGF-BB and receptor, all ligand and receptor occurred in complex, confirming that the complex is composed of one PDGF dimer and two receptors. We also analyzed the complex formation under conditions of ligand excess. Interestingly, neither in the electrophoresis assays (Fig. 4) nor during gel chromatography (Fig. 3A) was there any evidence for 1:1 complexes under conditions of ligand excess, indicating that formation of 1:2 complexes is favored under these experimental conditions.


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  		Fig. 4.   Determination of the stoichiometry of the PDGF-BB·alpha RD1-4 complex. alpha RD1-4 (50 µM) was mixed with various concentrations of PDGF-BB and analyzed by native electrophoresis on 4% agarose (A) and 7.5% polyacrylamide (B) gels. Ligand/receptor ratios are indicated at the bottom of each panel. Proteins were visualized by Coomassie staining.

The size of alpha RD1-4 was estimated to 80 kDa by SDS gel electrophoresis. The predicted size of a complex composed of one PDGF molecule (30 kDa) and two receptors would thus be 190 kDa, which is somewhat lower than the sizes determined by both dynamic light scattering and gel chromatography. However, also the monomeric free receptor gave higher estimated values in the gel chromatography and dynamic light scattering assays than in SDS gel electrophoresis, most likely reflecting an extended structure of alpha RD1-4.

Ig-like Domain 4 Interactions Mediate Stability of 1:2 Complexes under Conditions of Ligand Excess-- We have recently demonstrated that Ig-like domain 4-mediated receptor-receptor interactions contribute to PDGF-induced dimerization of receptors in intact cells (20). To investigate how Ig-like domain 4 is involved in this process, we compared alpha RD1-4 and alpha RD1-3 with regard to formation of ligand-receptor complexes.

In the complex forming assay using electrophoresis on agarose gels, alpha RD1-3 was found to form a complex of slower mobility after ligand addition (Fig. 5A, lanes 5-7). The slower migrating component was deduced to be a 1:2 PDGF-BB·alpha RD1-3 complex since all PDGF-BB and alpha RD1-3 were complexed at a 0.5:1 ligand/receptor molar ratio.


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  		Fig. 5.   Comparison of 1:2 PDGF·alpha RD1-4 and PDGF·alpha RD1-3 complex stability at ligand excess. alpha RD1-4 or alpha RD1-3 at a concentration of 50 µM was mixed with PDGF-BB and incubated at room temperature for 2 h in the absence (A) or presence (B) of tracer amounts of 125I-labeled alpha RD1-4 or alpha RD1-3. Complex formation was analyzed by native electrophoresis on 4% agarose gels. Ligand/receptor ratios are indicated at the bottom of each panel. Proteins were visualized by Coomassie staining (A) or with a phosphoimager (B).

Interestingly, in parallel to a decrease in the amount of the 1:2 complex, a novel slowly migrating component appeared with a mobility in between those of the 1:2 complex and the free ligand, most likely representing a 1:1 ligand-receptor complex at conditions of ligand excess (Fig. 5A, lane 8). To confirm that the slowly migrating complex represented an alpha RD1-3-containing species rather than tailing PDGF, an experiment was performed using tracer amounts of 125I-labeled receptors. Free receptor and receptor complexes were detected by a phosphoimager (Fig. 5B). As in Fig. 5A, the addition of excess ligand did not lead to the appearance of any novel receptor-containing species in the case of alpha RD1-4 (Fig. 5B, lane 4); however, a more slowly migrating receptor-containing species was seen when PDGF and alpha RD1-3 were mixed at a 4:1 molar ratio (Fig. 5B, lane 8). We thus conclude that the preferential formation of 1:2 ligand-receptor complexes also under conditions of ligand excess is favored by the presence of Ig-like domain 4.

Ig-like Domain 4 Contributes to Complex-forming Ability at Micromolar Concentrations, but Not to PDGF-neutralizing Activity at Nanomolar Concentrations-- To investigate if Ig-like domain 4 quantitatively contributes to the ability to form 1:2 ligand-receptor complexes, alpha RD1-4 and alpha RD1-3 were compared with regard to their abilities to form 1:2 ligand-receptor complexes under conditions of limiting amounts of ligand and using lower concentrations of receptor than in previous experiments (Fig. 6, A and B). In native acrylamide electrophoresis, the migratory positions of the 1:2 PDGF-BB·alpha RD1-4 complexes (Fig. 6A, lane 2) were well separated from the migratory positions of the 1:2 PDGF-BB·alpha RD1-3 complexes (lane 4). When alpha RD1-4 and alpha RD1-3, at concentrations of 12.5 µM, were mixed with limiting concentrations of PDGF-BB, complexes between PDGF and alpha RD1-4 were preferentially formed at the expense of PDGF·alpha RD1-3 complexes (Fig. 6A, lanes 6-8). Similar results were obtained when the formation of PDGF-AA·receptor complexes was analyzed (data not shown). Further evidence for an involvement of Ig-like domain 4 in the formation of soluble 1:2 ligand-receptor complexes was obtained when the fraction of free and complexed receptors was analyzed after incubation of 3 µM alpha RD1-3 and alpha RD1-4, with trace amounts of 125I-labeled receptor, together with various concentrations of PDGF-BB (Fig. 6B). At 0.37 µM PDGF, 17% of alpha RD1-4 occurred as a ligand complex (Fig. 6B, lane 2), whereas <5% of alpha RD1-3 was present in complex with PDGF under the same conditions (lane 6). The reason that complete complex formation is not observed at a 1:2 ligand/receptor ratio in this experiment, in contrast to what was observed in Fig. 4, 5 and 6A, is most likely that lower receptor concentrations were used.


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  		Fig. 6.   Comparison of alpha RD1-4 and alpha RD1-3 in the formation of 1:2 ligand-receptor complexes. alpha RD1-4 and alpha RD1-3 at concentrations of 12.5 (A) and 3 (B) µM were incubated with PDGF-BB at various ligand/receptor ratios as indicated and analyzed by native electrophoresis on 7.5% polyacrylamide gels. Positions of PDGF·alpha RD1-4 and PDGF·alpha RD1-3 complexes are indicated by closed and open arrows, respectively. Proteins were visualized by Coomassie staining (A) or with a phosphoimager (B).

alpha RD1-4 and alpha RD1-3 were also analyzed for their abilities to neutralize the binding of subnanomolar concentrations of 125I-PDGF-AA to cell-surface receptors (Fig. 7). When mixed with 0.16 nM 125I-PDGF-AA, both proteins reduced, in a dose-dependent manner, the binding of 125I-PDGF-AA to cell-surface receptors. Half-maximal competition was observed at 30 and 100 nM for alpha RD1-3 and alpha RD1-4, respectively. Similar results were obtained with 125I-PDGF-BB (data not shown). Thus, Ig-like domain 4-mediated receptor-receptor interactions do not contribute significantly to the ability to sequester ligand in this concentration range.


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  		Fig. 7.   Neutralization by alpha RD1-4 and alpha RD1-3 of 125I-PDGF-AA binding to cell-surface PDGF alpha -receptors. Porcine aortic endothelial cells expressing the PDGF alpha -receptor were incubated with 5 ng/ml 125I-PDGF-AA together with various concentrations of alpha RD1-4 (open circles) and alpha RD1-3 (closed circles). Unlabeled PDGF-BB at 160 ng/ml reduced the binding of 125I-PDGF-AA to 600 cpm.

alpha RD1-2 Binds PDGF-BB, but Not PDGF-AA-- To further localize the region(s) within alpha RD1-3 that mediates ligand binding, we investigated the properties of alpha RD1-2 in complex forming assays and in cell-surface receptor binding inhibition assays (Figs. 8 and 9). The receptor fragment was mixed with PDGF-AA and PDGF-BB at a 1:2 ligand/receptor molar ratio, and the presence of complex was analyzed by native polyacrylamide gel electrophoresis (Fig. 8). When mixed with PDGF-BB, almost all of alpha RD1-2 appeared in complex with ligand (lane 6). In contrast, after mixing alpha RD1-2 with PDGF-AA, most of the receptor remained as free receptor (lane 5).


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  		Fig. 8.   Comparison of alpha RD1-4 and alpha RD1-2 in complex forming assay. alpha RD1-4 or alpha RD1-2 at a concentration of 25 µM was incubated with or without 12.5 µM PDGF-AA or PDGF-BB, as indicated. Complex formation was analyzed by native electrophoresis on 7.5% polyacrylamide gels. Proteins were visualized by Coomassie staining.

In cell-surface binding inhibition assays, inhibition of 125I-PDGF-BB binding to cell-surface PDGF alpha -receptors was observed using 3-10-fold higher concentration of alpha RD1-2 as compared with alpha RD1-3 and alpha RD1-4 (Fig. 9, right panel). In contrast, when alpha RD1-2 was assayed for inhibition of 125I-PDGF-AA binding to PDGF alpha -receptors, ~100-fold higher concentrations of alpha RD1-2 than of alpha RD1-3 or alpha RD1-4 were required for similar inhibitory effects (Fig. 9, left panel). Together, these findings demonstrate that alpha RD1-2 binds PDGF-BB, but not PDGF-AA, with rather high affinity and that Ig-like domain 3 of the PDGF alpha -receptor contains epitopes of particular importance for PDGF-AA binding.


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  		Fig. 9.   Neutralization by alpha RD1-2 of 125I-PDGF-AA and 125I-PDGF-BB binding to cell-surface PDGF alpha -receptors. Porcine aortic endothelial cells expressing the PDGF alpha -receptor were incubated with 5 ng/ml 125I-PDGF-AA (left panel) or 125I-PDGF-BB (right panel) with various concentrations of alpha RD1-4 (open squares), alpha RD1-3 (closed circles), and alpha RD1-2 (open circles). Unlabeled PDGF-BB at 160 ng/ml reduced the binding of 125I-PDGF-AA and 125I-PDGF-BB to 50 and 18%, respectively.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this study, we have used three forms of the PDGF alpha -receptor extracellular part, alpha RD1-4, alpha RD1-3, and alpha RD1-2, to explore the mechanism and structural basis of ligand binding and receptor dimerization. Through the use of gel permeation chromatography and two different types of native gel electrophoresis, we confirmed, in agreement with previous studies (13, 14), that soluble alpha RD1-4 forms a complex at a 1:2 ligand/receptor ratio (Figs. 3 and 4). Using the same assay system, we further demonstrated that a 1:2 complex also forms under conditions of ligand excess, suggesting that the complex is stabilized by additional types of interactions.

The finding that the 1:2 PDGF·alpha RD1-3 complex, in contrast to the 1:2 PDGF·alpha RD1-4 complex, was partially disrupted under conditions of ligand excess (Fig. 5, A and B) localizes the interactions mediating stability under conditions of ligand excess to Ig-like domain 4. Receptor-receptor interactions involving Ig-like domain 4 of the PDGF alpha -receptor have recently been shown to occur and also to be required for activation of PDGF receptors (20). The experiments of the present study provide direct evidence that Ig-like domain 4-mediated receptor-receptor interactions contribute to the stability of the ligand-receptor complex. They also suggest a mechanism whereby these interactions contribute to ligand activation. Since dimerization of receptors is the activating event, our findings suggest that Ig-like domain 4 interactions contribute to receptor activation by promoting the formation of 1:2 complexes at the expense of 1:1 complexes over a wide range of ligand concentrations.

Further support for a role of the PDGF receptor Ig-like domain 4 in receptor dimerization, but not in direct ligand binding, has been provided through the recent characterization of monoclonal antibodies against Ig-like domain 4 of the PDGF beta -receptor that block receptor signaling without affecting ligand binding (18, 19). Ig-like domain 4 of the stem cell factor receptor has also been shown to be involved in receptor-receptor interactions (28). Moreover, a recent study comparing the properties of soluble forms of the vascular endothelial cell growth factor receptor Flt-1 indicated that at micromolar concentrations of soluble receptor domains, cross-linked 1:2 ligand-receptor complexes were detected only in receptors containing both the ligand-binding Ig-like domains 1-3 and Ig-like domain 4 (29). Together, these findings suggest that Ig-like domain 4 in several tyrosine kinase receptors is involved in receptor-receptor interactions. Interestingly, in all these cases, an anomalous Ig-like domain lacking two conserved cysteine residues is involved. This structural feature is also conserved in other receptors structurally related to the PDGF receptors, such as the colony-stimulating factor-1 receptor (30) and Flk2 (31), as well as in Flt-1-related receptors, such as KDR (32) and Flt-4 (33).

alpha RD1-4 and alpha RD1-3 were further compared with regard to their relative affinity for PDGF (Figs. 6 and 7). Whereas a difference between the two forms was detected in the assay performed at micromolar concentrations of receptor (Fig. 6), no difference was observed in the binding inhibition assay that scored for neutralization of subnanomolar concentrations of PDGF (Fig. 7). We therefore conclude that the neutralizing effect is achieved predominantly through the formation of 1:1 ligand-receptor complexes without involvement of receptor-receptor interactions. Thus, Ig-like domain 4 is important for ligand binding and complex formation on cell-surface receptors at subnanomolar concentrations of ligand, but not when soluble receptor domains are used; this difference is most likely due to the high local receptor concentration at the cell surface, which promotes receptor-receptor interactions.

Soluble forms of the intact extracellular domain of the PDGF beta -receptors have previously been shown to inhibit PDGF action through sequestration of the ligand (15). Similarly, beta -receptor Ig-like domains 1-4 have also been shown to block PDGF action (34). In both cases, IC50 values between 20 and 100 nM were reported, which are similar to the values found in our study. Our data predict that one way to convert soluble extracellular domains of receptors to more potent antagonists would be to engineer them to adopt dimeric forms. In agreement with this prediction, a disulfide-linked dimeric form of Ig-like domains 1-3 of the PDGF beta -receptor fused to the second and third constant Ig-like domains of the heavy chain of IgG was reported to block PDGF at significantly lower concentrations than the monomeric forms (35).

To further map the ligand-binding region(s) within Ig-like domains 1-3, we compared alpha RD1-3 and alpha RD1-2 with regard to PDGF-AA and PDGF-BB binding (Figs. 8 and 9). We found that Ig-like domain 3 is of great importance for PDGF-AA binding, but contributes less to PDGF-BB binding. The observation that Ig-like domain 3 is of great importance for PDGF-AA binding to PDGF alpha -receptors is in good agreement with a recent study assaying PDGF-AA binding to immobilized PDGF alpha -receptor fragments (18). However, our demonstration that Ig-like domain 3 of the PDGF alpha -receptor is of lesser importance for binding of PDGF-BB than PDGF-AA represents a novel finding.

The observation that the binding regions of PDGF-AA and PDGF-BB are not structurally coincident is also in agreement with previous studies; PDGF-AA binds with higher affinity than PDGF-BB to Ig-like domains 2 and 3 (36), and neomycin selectively inhibits PDGF-AA binding to PDGF alpha -receptors (37). Studies using a PDGF alpha -receptor mutant lacking the major part of Ig-like domain 2 (amino acid residues 150-189) suggested the presence of a PDGF-AA-specific epitope within that region (38). This is to some extent in contrast to our results, and the reason remains unclear at present. However, it is possible that the PDGF-BB epitope(s) in Ig-like domain 2 lies outside that region and that the loss of PDGF-AA binding of the deletion mutant is caused by indirect effects on the structure of Ig-like domain 3.

The demonstration that PDGF-BB binds alpha RD1-2, together with previous observations demonstrating the lack of importance of Ig-like domain 1 for PDGF binding (18, 36), identifies Ig-like domain 2 of the PDGF alpha -receptor as the structure containing the major binding epitope(s) for PDGF-BB binding. Interestingly, it was recently demonstrated by crystallographic analysis that the structurally related vascular endothelial cell growth factor binds predominantly to Ig-like domain 2 of the vascular endothelial cell growth factor receptor Flt-1 (39).

Our present investigation thus confirms and extends previous studies characterizing the structural basis and mechanism of PDGF-induced receptor activation. We conclude that PDGF-AA binding occurs predominantly through interactions with Ig-like domains 2 and 3, whereas PDGF-BB binding occurs predominantly via Ig-like domain 2. Furthermore, Ig-like domain 4-mediated receptor-receptor interactions contribute to complex formation and promote the formation of 1:2 ligand-receptor complexes at micromolar concentrations of soluble receptor and at physiologic levels of cell-surface receptors. However, these interactions are not significantly involved in the neutralizing activity of subnanomolar concentrations of PDGF by soluble receptors. The availability of large amounts of well characterized soluble PDGF alpha -receptor, as described in this paper, will allow future studies aiming at an improved understanding of the structural basis for ligand-receptor interaction, such as crystallographic analysis, as well as the setup of solid-phase assays to be used in screens for PDGF receptor antagonists interfering with ligand-receptor or receptor-receptor interactions.

    ACKNOWLEDGEMENTS

We thank Takeshi Shimomura for advice on cell cultures, Mats Sandgren for assistance with protein purification, and Sherry Mowbray for helpful comments and suggestions.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported in part by the Uehara Memorial Life Science Foundation. Present address: Dept. of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Inst. of Technology, 4259 Nagatsuta, Midori-Ku, Yokohama 226, Japan.

§ To whom correspondence should be addressed: Ludwig Inst. for Cancer Research, P. O. Box 595, S-751 24 Uppsala, Sweden. E-mail: Arne.Ostman{at}LICR.uu.se.

The abbreviations used are: PDGFs, platelet-derived growth factors; CHO, Chinese hamster ovary; alpha RD1-4, alpha RD1-3, and alpha RD1-2, PDGF alpha -receptor Ig-like domains 1-4, 1-3, and 1 and 2, respectively; GST, glutathione S-transferasePIPES, 1,4-piperazinediethanesulfonic acid.
    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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