THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, No. 5, Issue of February 15, pp. 3625-3631,1993 Printed in U.S.A.

Mechanism of Platelet-derived Growth Factor (PDGF) AA, AB, and BB Binding to (Y and B PDGF Receptor*

(Received for publication, July 27, 1992)

Larry J. Fretto, Andrea J. Snape, James E. Tomlinson, Joseph J. Seroogy, David L. Wolf, William J. LaRochelleS, and Neil1 A. Gieseg From COR Therapeutics Inc., South Sun Francisco, California 94080 and the $Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

The biological effects of platelet-derived growth fac- tor (PDGF) are mediated by cell surface a and j 3 PDGF receptors, which, as a result of ligand binding, undergo dimerization in a manner consistent with PDGF being bivalent. In order to directly demonstrate PDGF biv- alency and to define the binding of PDGF AB to isolated 0 receptor, we developed solid-phase binding assays using purified recombinant extracellular domain of human PDGF receptors. PDGF AA, AB, and BB were prepared from the monomeric chains expressed in Escherichia coli, and each was purified to homogene- ity; PDGF AB contained <0.5% of either homodimer. The interactions of these isoforms with immobilized PDGF receptors were examined by several ap- proaches. Scatchard analysis revealed high affinity binding (Kd = 0.5-1.0 nM) of radiolabeled PDGF AA and AB to receptor and of PDGF BB to both receptor subtypes. Contrary to previous reports, PDGF AB also bound j 3 receptor with high affinity ( K d = 0.9 nM). When a B-chain-specific monoclonal antibody that rec- ognizes the putative binding domain of PDGF BB was used for ligand detection, we found that PDGF AB binding to 6 receptor occurred exclusively through the B-chain subunit, whereas binding to a receptor oc- curred through either subunit. In addition, site-di- rected mutagenesis was used to specifically inactivate the B chain of PDGF AB, which eliminated binding to the j3 receptor without affecting a receptor binding. These results establish that PDGF is bivalent and that monovalent ligand retains high affinity receptor bind- ing.

Platelet-derived growth factor (PDGF),’ a potent mitogen for cells of mesenchymal origin, has been linked to the etiology of a number of human diseases, including atherosclerosis, glomerulonephritis, and cancer (1-6). It is a disulfide-linked dimer of two related polypeptide chains, designated A and B, which are assembled as heterodimers (PDGF AB) or homo- dimers (PDGF AA and PDGF BB) (7-9). PDGF mediates its

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

5 To whom all correspondence should be addressed COR Thera- peutics, Inc., 256 E. Grand Ave., South San Francisco, CA 94080. Tel.: 415-244-6800; Fax: 415-244-9208.

The abbreviations used are: PDGF, platelet-derived growth factor; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electropho- resis; HPLC, high performance liquid chromatography; ABTS, 2,2’- azinobis(3-ethylbenzthiazoline-6-sulfonic acid).

~~

biological activity by binding to cell surface receptors and inducing their intrinsic tyrosine kinase activity (10). Studies involving direct binding and cross-competition by PDGF iso- forms, as well as receptor down-regulation, provided indirect evidence for at least two PDGF receptor subtypes (11, 12). These observations were confirmed by molecular cloning of two related but distinct cDNAs encoding a and ,8 PDGF receptors (13-17).

The mechanism by which PDGF activates its receptors is largely unknown, but recent evidence suggests that PDGF receptor dimerization, mediated by ligand binding, is required for signal transduction (18). Based upon the ability of each PDGF isoform to bind, dimerize, and activate PDGF receptor subtypes, when expressed alone or together, a model has been proposed in which PDGF A chain binds LY receptor and PDGF B chain binds both receptors (11, 19). Furthermore, receptor dimerization is thought to occur by bivalent PDGF cross- linking two receptors such that PDGF AA induces only a/@ receptor dimers, PDGF AB induces @/a and receptor dimers, and PDGF BB induces all three receptor dimer com- binations, ala, a/@, and PIP (19-22). Although receptor bind- ing data for PDGF AA and BB are consistent with this model, there are discrepancies regarding PDGF AB binding to p receptor in the absence of a receptor (14, 15, 23, 24) and PDGF bivalency has not been directly demonstrated. To address these questions of PDGF binding specificity and valency we have developed solid-phase receptor binding as- says using pure recombinant proteins. PDGF chain-specific antibodies and ligand mutagenesis were used to prove that PDGF is bivalent but that high affinity binding can occur through a single ligand subunit.

EXPERIMENTAL PROCEDURES

Materials-Sis 1 monoclonal antibody prepared against recombi- nant human PDGF BB and anti-PDGF, a goat polyclonal antibody raised against human platelet PDGF, have been previously described (25). The monoclonal antibody designated anti-KT3 was raised against the peptide Thr-Pro-Pro-Pro-Glu-Pro-Glu-Thr as described previously (26). The human cDNAs encoding PDGF A and B chains used in this study have been characterized previously (27, 28). The human cDNAs encoding p PDGF receptor (29) and 01 PDGF receptor were gifts from Dr. Lewis T. Williams and Dr. Jaime Escobedo.

Expression and Purification of the PDGF Receptor Extracellular Domain-The 01 and p PDGF receptor cDNAs were isolated from a human placenta X GTlO cDNA library (29). Standard mutagenesis techniques were used to introduce a translation termination signal a t codon 525 and 530 for the cy and (3 receptor cDNAs, respectively (30). Following mutagenesis each cDNA was sequenced by the dideoxy- nucleotide method to confirm that the coding region was the same as previously published (17, 29). Each cDNA was inserted into the mammalian expression vector pBJ-1 (31) and co-transfected into CHO cells with the selectable marker plasmid pSV2neo (32). After

3625

3626 Mechanism of PDGF Receptor Binding G418 selection, cell clones overexpressing each protein were isolated and cultured in a hollow-fiber bioreactor (Unisyn Fibertec Corp., San Diego, CA). Conditioned media containing the extracellular domains of the PDGF receptors (10 mg/liter) was applied to Tresyl-agarose (Schleicher & Schuell) coupled to monoclonal antibodies raised against the PDGF receptor extracellular domains. After immunoaf- finity chromatography, the receptor proteins were further purified on Sephacryl S200 HR (Pharmacia LKB Biotechnology Inc.); this step also removed any traces of monoclonal antibody that would interfere with immunological detection of receptor-bound PDGF in the assays described below.

Construction of PDGF Expression Vectors-PDGF A-chain cDNA was mutagenized by standard techniques (30) to introduce an NcoI site with an in-frame methionine codon followed by a glycine and six histidine codons fused to the wild-type sequence at codon 89. The cDNA sequence encoding the carboxyl terminus of the protein was modified by fusion of wild-type codon 187 to sequences encoding the KT3 epitope (TPPPEPET) followed by a translation stop codon and a HindIII restriction site. The PDGF B-chain cDNA was mutagenized to introduce an NcoI site that generated a methionine codon at position 83. Additionally, wild-type codon 182 was fused to five histidine codons followed by a translation stop codon and a HindIII restriction site. The DNA sequence of each construct was verified by the dideoxynucleotide method (33). Each PDGF coding sequence contained within the NcoI to HindIII restriction fragment was in- serted into the pQE-6 expression vector (Qiagen Inc., Chatsworth, CAI.

Expression and Purification of PDGF Ligands-The expression constructs described above were introduced into the M15 strain of Escherichia coli and PDGF expression was induced with 1 mM iso- propyl-j3-D-thioglactopyranoside for 5-6 h at 37 "C. Cells were solu- bilized in 6 M guanidine HC1, and thiol groups were protected by S- sulfonation as previously described (34). PDGF A or PDGF B mon- omeric proteins were adsorbed to nickel-nitriloacetic acid-agarose (Qiagen Inc.) at pH 8.0 and were eluted with 8 M urea at pH 4.5 or 5.9, respectively. To facilitate dimer formation, the PDGF concentra- tion was adjusted to 0.4 mg/ml in 2 M urea at pH 7.6 containing 5 mM reduced glutathione and 0.5 mM oxidized glutathione followed by incubation at room temperature for 48 h (34). PDGF homodimers were purified by ion-exchange chromatography as previously de- scribed (34,35). The yield of each purified homodimer from l liter of bacterial culture was approximately 30 mg. The same conditions were used to prepare PDGF AB, except that 0.2 mg/ml each PDGF A and B chain were present in the renaturation reaction. Following ion- exchange chromatography, PDGF AB contained slight amounts of PDGF AA and BB. To remove these contaminants, PDGF AB was applied to an HPLC reverse phase column (0.46 X 25 cm, C4, Vydac) in 0.7 M acetic acid, pH 3.6, and eluted at 0.5 ml/min in 0.1% trifluoroacetic acid with a 5-37% acetonitrile gradient (120 min). Ligand concentrations were determined using the extinction coeffi- cients, E$;?& of purified recombinant PDGF AA, AB, and BB calculated from the predicted amino acid composition as 0.59, 0.51, and 0.46, respectively.

PDGF Solid-phase Binding Assays-Affinity-purified j3 PDGF receptor extracellular domain protein was immobilized in 96-well microtiter plates (Dynatech Products Co.) and incubated with PDGF as previously described (36). The same procedures were followed for (Y PDGF receptor except that, following its immobilization, wells were blocked with 25 mM HEPES, 100 mM NaCI, 0.2% Tween 20, pH 7.6. Colorimetric detection of receptor-bound PDGF was achieved using Sis 1, anti-KT3, or anti-PDGF antibodies, incubated at 0.5 pg/ml in binding buffer (0.3% gelatin, 25 mM HEPES, pH 7.6, 100 mM NaCI, 0.01% Tween 20) for 2 h a t 24 "C. Wells were washed with binding buffer and incubated with peroxidase-conjugated anti-mouse I g G or anti-goat IgG (Boehringer Mannheim). Wells were washed again, and peroxidase substrate (ABTS") was added; product formation was monitored at 650 nm using a plate reader (Molecular Devices).

Scatchard Analysis-Binding of IZ5I-PDGF (0.1-10 nM) to immo- bilized cr and j3 PDGF receptor extracellular domains was performed for 4 h a t 24 "C in microtiter wells as described above. After washing the wells three times with binding buffer (see above), bound PDGF was solubilized in 1% SDS, 0.5% bovine serum albumin for y count- ing. Nonspecific binding was determined in duplicate a t each concen- tration of '"I-PDGF using a 150-fold excess of the appropriate unlabeled PDGF isoform and was 5-1576 of the total 9 - P D G F binding, which was determined in triplicate. Data were analyzed by

the method of Scatchard (37). PDGF BB was labeled with diiodo- Bolton Hunter reagent (Du Pont-New England Nuclear; 1 mCi/5 pg PDGF) to give a specific activity of 50,000-90,000 cpm/ng (38). The same specific activity was achieved for PDGF AA radiolabeled using chloramine T (39) and for PDGF AB using either labeling method. Both Iz5I-PDGF AB preparations had the same binding affinities, and the results shown were for PDGF AB labeled by the chloramine T procedure.

RESULTS

Expression and Purification of PDGF Isoforms-In order to prepare dimeric PDGF isoforms, PDGF A and B chains were individually expressed in E. coli, purified, and folded in uitro. The added histidine residues encoded at the amino terminus of the A chain and at the carboxyl terminus of the B chain served as an affinity tag for purification by immobilized nickel ion chromatography. The expression levels attained for PDGF A and B chain were 15 and 30% of total bacterial protein, respectively, as determined by SDS-PAGE (data not shown). The sulfonated PDGF A and B chains eluted from the nickel- nitriloacetic acid-agarose were 90-95% pure, and their respec- tive yields were 60 and 100 mg/liter bacterial culture. Homo- dimer forms of PDGF were generated in high yields (50-60%) after 48 h of renaturation and, as shown in Fig. lA, were greater than 95% pure following cation exchange chromatog- raphy. Heterodimeric PDGF AB was the major isoform gen- erated when equal amounts of A chain and B chain were mixed prior to renaturation, as shown previously (35). Reverse phase C4 HPLC of the partially purified heterodimer prepa- ration resolved all three isoforms; PDGF AA, AB, and BB were eluted with 21.0, 23.5, and 26.0% acetonitrile, respec- tively (data not shown).

Fig. 1 shows the subunit structure and purity of each of our recombinant wild-type ligands as assessed by SDS-PAGE (40) followed by silver-staining (panel A ) and immunoblotting (panels B-D). Under nonreducing conditions each PDGF isoform migrated as a single band with a distinctive mobility corresponding to 33 kDa for PDGF AA, 30 kDa for PDGF AB, and 28 kDa for PDGF BB. As expected, PDGF AB had a mobility intermediate to that of the homodimers. SDS- PAGE analysis of reduced isoforms confirmed that they were

1 2 3 4 5 6 1 2 3 A kDa 6

- 45

- 31 C

-21 D

- 14

FIG. 1. Subunit structure, purity, and immunoreactivity of recombinant PDGF isoforms. 12.8% SDS-PAGE analysis (40) of purified PDGF AA (lanes I and 4 ) , PDGF AB (lanes 2 and 5 ) . and PDGF BB (lanes 3 and 6 ) . A, silver-staining of nonreduced PDGF (lanes 1-3) and reduced PDGF (lanes 4-6); 1 pg of PDGF was loaded in each lane except lane 5 (2 pg of PDGF AB). Reduced samples contained 5% 2-mercaptoethanol. Electrophoretic mobilities of pro- tein standards are indicated at right. B-D, Western blot analyses of nonreduced PDGF using polyclonal and chain-specific monoclonal antibodies, which were detected with peroxidase-coupled secondary antibodies and luminol (enhanced chemiluminescence, Amersham). B, anti-PDGF detection of 1.5 ng of AA, 2.5 ng of AB, and 1.0 ng of BB. C, anti-KT3 detection of 1.0 ng of AA, 30 ng of AB, and 10 ng of BB. D, Sis 1 detection of 10 ng of AA, 5 ng of AB, and 2 ng of BB.

Mechanism of PDGF Receptor Binding 3627

A 1 2 3 4 5 6

AB-, OOO-O -00- c A A

B 1 2 3 4 5 6

AB+ c B B

FIG. 2. Isoform purity of recombinant PDGF AB. HPLC- purified PDGF AB was subjected to 12.8% SDS-PAGE and Western blot analysis in the presence or absence of added PDGF homodimers. A, Anti-KT3 detection of PDGF AB (40 ng/lane) and PDGF AA not added in lane I or added a t 0.3% (lane 2), 1.0% (lane 3 ) , 2.0% (lane 4 ) , and 4.0% (lane -5). PDGF AA (0.4 ng) alone (lane 6). R, Sis 1 detection of PDGF AB (40 ng/lane) and PDGF BB not added in lane I or added at 0.3% (lane 2) , 1.0% (lane 3 ) , 2.0% (lane 4 ) , and 4.0% (lane 5); PDCF BB (4 ng) alone (lane 6 ) .

disulfide-linked dimers and that the HPLC-purified PDGF AB preparation comprised equal amounts of PDGF A and B chain (Fig. lA, lune 5 ) . The electrophoretic mobilities of the reduced PDGF chains corresponded to 16 kDa for A chain and 14 kDa for B chain, which are in reasonable agreement with the masses predicted from amino acid sequence of 13 and 12 kDa, respectively. Immunoblot analysis of the nonre- duced isoforms showed that anti-PDGF antibody detected all three recombinant dimers as single bands with the expected mobilities (Fig. 1B). In addition, anti-KT3 antibody specifi- cally detected our KT3-flagged A chain in PDGF AA and AB, although with very different sensitivities (Fig. IC). Sis 1 specifically detected B-chain epitopes in both PDGF AB and BB (Fig. 1D). These data clearly establish the identity of our PDGF AB preparation and show that residual contamination by PDGF AA or BB was not evident. To determine the lowest levels of homodimer contamination that could be detected, PDGF AB was mixed with decreasing quantities of either PDGF AA or BB prior to SDS-PAGE and immunoblot analy- sis. As shown in Fig. 2.4, levels of PDGF AA equivalent to 0.3% (w/w) contamination were readily detected by anti-KT3 antibody, which was 25-fold more sensitive for detecting the homodimer (Fig. IC). In the case of added PDGF BB (Fig. 2B) , as little as 1.0% was detected by Sis 1, which was &fold more sensitive for detecting PDGF BB (Fig. 1D). In the unadulterated PDGF AB preparation, neither PDGF AA nor BB were detected (Fig. 2, lune 1 ), thereby allowing for reliable characterization of heterodimer binding.

These structural analyses demonstrated that the renatured PDGF chains formed disulfide-linked dimers that were rec- ognized by Sis 1 and anti-PDGF antibodies, which are known to be specific for a biologically active conformation (25, 41). T o directly assess the biological activity of our recombinant PDGF isoforms, mitogenic activity was assayed on NIH/3T3 cells; no difference was observed, as compared to PDGF with native sequence.* Therefore, the addition of a histidine stretch and/or the KT3 epitope did not functionally alter these PDGF ligands.

PDGF Isoform Receptor Binding-Solid-phase binding as- says were developed to characterize the receptor binding

'Human PDGF used to evaluate the effects of adding flag se- quences to our ligands was PDGF BB from Amgen (now from R&D Systems, Minneapolis, MN) and PDGF AA from Upstate Biotech- nology, Inc. (Lake Placid, NY).

properties of the PDGF isoforms. Duan et ul. (36) have demonstrated that the immobilized extracellular domain of mouse (3 PDGF receptor retained binding properties similar to those of the full-length receptor. Using this strategy, a secreted extracellular domain form of each PDGF receptor was expressed in CHO cells and isolated from conditioned media. SDS-PAGE analysis of immunoaffinity-purified LY and (3 receptor proteins after reduction revealed single silver- stained bands with electrophoretic mobilities corresponding to 110 and 100 kDa, respectively (Fig. 3A). The differences between these apparent masses and the predicted protein masses of 58 kDa, as well as the observed size heterogeneity, are probably due to variable levels of glycosylation, as previ- ously described for (3 PDGF receptor (36). The identity of these proteins was confirmed by immunoblot analysis using polyclonal antibodies raised against synthetic peptides (Fig. 3, B and C).

When PDGF receptor extracellular domain was immobi- lized in microtiter wells (see "Experimental Procedures") and incubated with increasing concentrations of "'1-PDGF AA, AB, or BB in the presence or absence of excess unlabeled ligand, specific binding was saturable and was >80% of total binding for each of the ligand concentrations tested. Such binding experiments were performed for each receptor/ligand interaction, and Scatchard plots of the data that best repre- sent the relative affinities of each interaction are shown in Fig. 4. In all cases, a single line fit the data best with corre- lation coefficients > 0.95 indicating a single-affinity class of binding sites. LY receptor bound all PDGF isoforms with high affinity (Kd = 0.5-0.7 nM). As expected, p receptor did not bind PDGF AA but did bind PDGF BB with high affinity ( K d

= 0.5 nM). Surprisingly, PDGF AB bound (3 receptor with an affinity ( K d = 0.9 nM) comparable to that of PDGF BB. Consistent with these results, immunologic detection of un- labeled PDGF binding to immobilized receptor was saturable, 90% specific, and half-maximal a t approximately 1 nM ligand (see below). Although the data shown in Fig. 4 were from a single experiment, a total of 20 Scatchard analyses have been performed, and the average & and B,,, values for each receptor/ligand pair were approximately 1.0 nM and 400 pM, respectively.

Human PDGF homodimers with native sequence were ra- diolabeled and analyzed in the solid-phase binding assay under the same conditions as described above. The binding parameters obtained from Scatchard plots of these data (not

A B C 1 2 1 2 1 2

kDa -.

200 - -!

92- , ,"

69 -

46 -

I I

FIG. 3. Purity and immunologic identity of recombinant PDGF receptor extracellular domains. 7% SDS-PAGE analyses of immunoaffinity-purified (Y (lanes 1 ) and @ (lanes 2 ) PDGF receptor extracellular domains. A, silver staining of 750 ng of each receptor and of protein standards (150 ng each, in the far left lane). R and C, Western blot analyses of each receptor protein (70 ng/lane) using polyclonal antibodies directed against synthetic peptides correspond- ing to sequences from (Y receptor ( R ) or @ receptor ( C ) .

3628 Mechan i sm of PDGF Receptor Binding

c m

0 2 0 0 4 0 0 600 AA Bound (pM)

, KdsO.7nM

0 2 0 0 4 0 0 6 0 0 800 AB Bound (pM)

0.1 2 .

5 0.06-

Kd=O.SnM

0.00 0 2 0 4 0 6 0

BE Bound (pM)

0.20.

5 c m 0.3- m 0.10-

Kd=O.SnM Kd=O.SnM

0 200 400 6 0 0 0 2 0 4 0 6 0 8 0 0.00 1 - .

AB Bound (pM) BE Bound (pM)

FIG. 4. Scatchard analyses of “‘1-PDGF isoform binding to immobilized PDGF receptor extracellular domain. 01 PDGF receptor binding by PDGF AA (panel A ) , PDGF AB (panel B ) , and PDGF BB (panel C) and f i PDGF receptor binding by PDGF AB (panel D ) and PDGF BB (panel E ) was measured in microtiter wells as described under “Experimental Procedures.” B/F, the ratio of specifically hound ligand to free ligand after 4 h a t 24 “C. The dissociation constants ( K d ) for each binding interaction are shown.

shown) were the same as those reported above for our flagged recombinant PDGF, demonstrating that our sequence modi- fications did not measurably affect receptor binding.

Bivalency of PDGF Demonstrated Using PDGF Antibody- Sis 1, a PDGF B-chain-specific monoclonal antibody, was used to examine the mechanism of PDGF receptor binding to PDGF AB and BB. Both of these isoforms were detected by Sis 1 on Western blots (Fig. lD), demonstrating that a single B chain contains the epitopes required for antibody recogni- tion (25). Since Sis 1 blocks PDGF BB binding to its receptor (25) , we reasoned that the converse might be true, namely receptor would block binding of Sis 1 to PDGF BB, if both subunits were engaged in receptor binding. On the other hand, PDGF BB might bind receptor through one subunit only, leaving the other subunit available for Sis 1 detection. To distinguish between these two alternatives, Sis 1 was incu- bated with receptor-bound PDGF, as shown in Fig. 5. As expected, Sis 1 did not detect PDGF AA bound to N receptor, but it readily detected PDGF BB bound to either a or @ PDGF receptor. PDGF AB was also detected by Sis 1 when bound t o N receptor (Fig. 5 A ) but, unlike PDGF BB, was not detected by Sis 1 when bound to p receptor (Fig. 5 B ) . That PDGF AB was indeed bound to @ receptor under these conditions was confirmed using the anti-KT3 antibody for detection (Fig. 5B). Therefore, PDGF AB is bivalent for binding to N receptor but functionally monovalent for binding to receptor, because this interaction occurs exclusively through the B-chain sub- unit.

PDGF B-chain Mutant Used to Generate Monovalent Het- erodimer-As a separate approach to determine PDGF val- ency, site-directed mutagenesis was used to specifically inac- tivate the B-chain subunit of PDGF AB. Codons 108-109 were deleted from the PDGF B-chain cDNA and the encoded mutant B-chain protein was renatured in the presence or absence of wild-type A chain to give mutant heterodimer,

‘09. These proteins were purified by the same methods as described for the wild-type isoforms. As compared to wild-

PDGF ~ ~ ~ 1 0 8 - 1 0 9 , or mutant homodimer, PDGF BA10*-109 B

E C 0 v) (0

n 0

0 1 2 3

PDGF (nM)

E C 0 v) (D

0 n

0.6 -

0.4 -

0.2 -

0.0 0 1 2 3 4

PDGF (nM)

FIG. 5. Determination of PDGF valency using a B-chain- specific monoclonal antibody. Receptor-bound PDGF AB or BB was detected with antibodies specific for A chain (anti-KT3) or B chain (Sis l), using the solid-phase binding assay described under “Experimental Procedures.” A , Sis 1 detection of PDGF AB (W), PDGF BB (O), or PDGF AA (0) bound to immobilized 01 PDGF receptor. B, Sis 1 detection of PDGF AB (W) or PDGF BB (0). and anti-KT3 detection of PDGF AB (A) bound to immobilized f i PDGF receptor.

Mechanism of PDGF Receptor Binding 3629

type PDGF BB, mutant homodimer had >100-fold reduced ability to compete for 12'I-PDGF BB binding to immobilized (Y or /3 receptor (Fig. 6), proving that the deletion had effec- tively blocked B-chain binding.

The binding activity of PDGF ABA'"lW could be accurately evaluated only if wild-type PDGF AA had been effectively removed by C4 HPLC, whereas contamination by the mutant PDGF BB homodimer would have no effect. By mixing PDGF

with trace amounts of PDGF AA its level of purity could be quantified. As shown in Fig. 7, immunoblot analysis of PDGF ABA'oR"W using anti-KT3 antibody readily detected PDGF AA added a t 0.2%, but none was detected without addition ( l a n e 1 ), indicating that contamination by PDGF AA was insignificant. When analyzed on immobilized CY recep- tor, PDGF AB*'Os"W showed saturable binding that was in- distinguishable from that of wild-type PDGF AB (Fig. 8A), demonstrating that active B chain was not required for effi- cient binding to CY receptor. In contrast, interaction of the mutant heterodimer with /3 receptor was greatly impaired as evidenced by a 100-fold reduction in its ability to compete for wild-type l2'1-PDGF AB binding (Fig. 8B). Therefore, site- directed mutagenesis can be effectively used to generate mon- ovalent PDGF ligands that retain a high level of binding activity.

ABAIOs-lW

DISCUSSION

This study directly demonstrates that PDGF is bivalent and defines the specific binding interactions between PDGF isoforms and receptor subtypes. This undertaking was greatly facilitated by having homogeneous ligand and receptor pro- teins (Figs. 1-3) with which to establish a defined system for analyzing ligand binding. The observed high affinity binding ( K d = 0.5-1.0 nM) of PDGF AA and AB to immobilized N

l o o w I '0 60 0

4 0 1 20

.01 .1 1 1 0 1 0 0

PDGF (nM)

FIG. 6. Receptor binding analysis of PDGF BA'o"'08 homo- dimer. Increasing amounts of PDGF BA'oR-lm homodimer (0, .) or wild-type PDGF BB (0,U) were coincubated with '*'I-PDGF BR (10 ng/ml) on immobilized (Y (circles) or p (squares) PDGF receptor extracellular domain. Relative amount of receptor-bound '251-PDGF B B is expressed as percent of control binding (6,850 cpm/well for (Y

receptor and 12,000 cpm/well for p receptor) in the absence of competitor. Determinations were performed in triplicate.

1 2 3 4 5 6 - " " ..

ABAl08-109+ t A A

FIG. 7. Analysis of mutant PDGF AB for contamination by wild-type PDGF AA. 12.8% SDS-PAGE and Western blot analysis with anti-KT3 antibody of HPLC-purified PDGF (40 ng/ lane) in the absence (lane I ) or presence of PDGF AA added at 0.2% (lane 2) , 0.8% (lane 3 ) , 2.0% (lane 4 ) , or 4.0% (lane 5) by weight. PDGF AA (2 ng) alone (lane 6).

A

0.0 0 3 6 9 1 2

PDGF (nM)

10

PDGF AB. A, saturation binding of PDGF ABA'OR"09 (m) or wild- FIG. 8. Receptor binding analysis of monovalent mutant

type PDGF AB (0) to immobilized a PDGF receptor extracellular domain was detected with anti-PDGF antibody as described under "Experimental Procedures." B, increasing amounts of PDGF AB""

(.) or wild-type PDGF AB (0) were coincubated with "'I-PDGF AB (10 ng/ml) on immobilized @ PDGF receptor extracellular domain. Relative amount of receptor-bound I2'I-PDGF AB is expressed as percent of control binding in the absence of competitor (18,000 cpm/ well). Values are averages of triplicate determinations.

receptor and of PDGF BB to both immobilized receptor subtypes (Fig. 4) reflects the established PDGF isoform bind- ing specificities characteristic of native full-length PDGF receptors and therefore validated our assay system.

There are conflicting reports in the literature concerning the ability of PDGF AB to bind /3 PDGF receptor. A compar- ison of PDGF AB binding to CY and /3 PDGF receptors was first made in human fibroblasts that express both receptor subtypes. Heldin and co-workers (11,42) found that, following removal of a receptor by down-regulation, PDGF AB did bind /3 receptor but with a weaker affinity than that of PDGF BB. Under similar conditions, Hart et al. (12) were unable to detect PDGF AB binding to p receptor. In a more direct approach, /3 PDGF receptor was expressed in CHO cells or baby hamster kidney cells, which lack endogenous PDGF receptors. PDGF AB bound to /3 receptor on these cells, albeit with a reduced affinity relative to PDGF BB (14, 15, 23, 43). In contrast, Heidaran et dl. (24) could not detect binding of highly purified PDGF AB to 32D cell transfectants expressing /3 PDGF receptors and suggested that binding in previous studies may have been due to PDGF BB in the PDGF AB preparations. Alternatively, a low /3 receptor number on 32D transfectants may limit the detection of bound PDGF AB, especially if its affinity was less than that of PDGF BB. Under the conditions of our assay, PDGF AB not only bound the /3 receptor but did so with an affinity comparable to PDGF

3630 Mechanism of PDGF Receptor Binding

BB (Fig. 4). This result is not easily attributable to PDGF BB in our PDGF AB preparation because by immunoblot analysis there was clearly less than 1.0% contamination by either homodimer (Fig. 2). Moreover, saturable binding of PDGF AB to preceptor (half-maximal at 1.5 nM) was detected by an antibody that recognizes PDGF AB but not PDGF BB (Fig. 5B).

Our observation that PDGF AB binds both receptors is consistent with a bivalent PDGF model proposed by Heldin, Bowen-Pope, and others (11, 19,42) in which PDGF A-chain subunits bind a receptor and PDGF B-chain subunits bind either receptor subtype. Receptor dimerization induced by PDGF has been demonstrated by measuring the physical association of receptors using sucrose gradient analysis ( X ) , chemical cross-linking (20, 21, 22, 24), nondenaturing gel electrophoresis (21), and coimmunoprecipitaton of receptor subtypes (19, 20, 22, 24), and evidence that it is required for signal transduction has recently been provided by Ueno et al. (18) using recessive dominant receptor mutants. The bivalent model proposes that PDGF receptor dimerization occurs by one ligand molecule cross-linking two receptors. Such a mech- anism has been identified for human growth hormone in which co-crystallization of the ligand-receptor complex led to the surprising observation that two receptor binding sites exist on one ligand and each site binds a receptor causing dimerization (44). Although this is also an attractive mecha- nism for PDGF-induced receptor dimerization, PDGF biva- lency has not heretofore been proven.

In the first of two separate approaches to determine PDGF valency, we studied ligand binding to immobilized receptor extracellular domain, for which receptor dimerization would be unlikely. If PDGF is bivalent, these conditions should favor a monovalent binding interaction in which one subunit of the ligand would be receptor-bound and the other would have its receptor binding domain exposed, which, under phys- iological conditions, would bind a second receptor molecule. This hypothesis was tested using the PDGF B-chain-specific monoclonal antibody, Sis 1, which recognizes epitopes within the putative receptor binding domain (25, 45). PDGF AB bound to p receptor was not detected by Sis 1, whereas PDGF BB was, indicating that PDGF AB bound ,6 receptor entirely through the B-chain subunit, thereby blocking the Sis 1 antibody epitopes (Fig. 5B). In contrast, PDGF AB bound to a receptor was readily detected, consistent with it being able t o bind through the A-chain subunit, leaving the B-chain subunit exposed for Sis 1 recognition (Fig. 5A). These results demonstrate that PDGF is bivalent and each subunit binds independently in a manner consistent with the homodimer binding specificities. Therefore high affinity ( K d = 0.5 nM) binding can occur through monovalent interactions in the absence of receptor dimerization involving binding of both ligand subunits. Scatchard analysis of PDGF BB binding to full-length native receptor showed a 5-10-fold higher affinity than that to immobilized re~eptor .~ This suggests that recep- tor dimerization may increase ligand binding affinity due to bivalent binding. If so, that could explain why PDGF BB binds p receptor on cells with a higher affinity than does PDGF AB and yet these isoforms bind with similar affinity in our assay. Other factors such as receptor truncation or immobilization could account for the reduced affinity of PDGF BB but if this were the case, the difference between the affinities of PDGF AB and BB observed on whole cells would be expected to persist in the solid-phase binding assay.

The second strategy to demonstrate PDGF bivalency relied

’ L. J. Fretto, unpublished observations.

on our previous study that used deletion scanning mutagenesis to map a small domain (13 residues) within the v-sis/PDGF B chain critical for receptor activation (45). We chose a deletion in this region that inactivated PDGF B but did not prevent its disulfide-linked dimer formation, thereby allowing folding of wild-type and inactive mutant chains to form novel dimers. In this way we were able to specifically inactivate the B chain of PDGF AB and evaluate the effects on receptor binding. Our finding that p receptor binding was blocked whereas binding to a receptor was unaffected (Fig. 8) confirms that PDGF AB is bivalent and that p receptor binding is mediated solely through the B-chain subunit. More impor- tantly, monovalent PDGF showed saturable binding over the same concentration range as wild-type ligand (Fig. 8A) and therefore may act as a receptor antagonist if bivalency is required for receptor activation. This prediction is supported by a study by Hammacher et al. (42), which demonstrated that PDGF BB effects were blocked by PDGF AB on cells containing only p receptors. In addition, several studies have shown that PDGF AB can activate p receptor only if a receptor is present, suggesting that monovalent binding does not cause receptor activation (19, 22, 24, 42). A recent study that analyzed PDGF mutants lacking covalent linkage of subunits suggested that monomeric PDGF has agonist activity (46), but it is just as likely that the active species was a noncovalently linked dimer. Therefore, additional character- ization of monovalent PDGF could provide a direction for antagonist development and additional insight into the mech- anism of PDGF receptor activation.

Acknowledgments-We thank Dr. Lewis T. Williams and Dr. Jaime Escobedo for the generous gift of PDGF receptor cDNAs and for helpful discussions throughout the course of these studies. We also thank Dr. Stuart Aaronson and Dr. Keith Robbins for Sis 1 mono- clonal antibody, anti-PDGF antibody, and PDGF B cDNA. The PDGF A cDNA was kindly provided by Drs. Christer Betsholtz and Carl-Henrik Heldin. Antibodies used to purify PDGF receptor were provided by Maria-Amelia Escobedo and Dr. Vanitha Ramakrishnan. We are grateful to Dr. David Phillips for continued support and helpful discussions.

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