Synthesis and assembly of a functionally active recombinant platelet-derived growth factor AB heterodimer

A Chinese hamster ovary cell line that stably expresses transfected human platelet-derived growth factor (PDGF) A and B chain precursors was established. All three dimeric combinations of PDGF chains were produced by this cell line; their biosynthesis, assembly, and processing were followed by pulse-chase analyses. PDGF-AA, PDGF-AB, and PDGF-BB were processed to Mr values of about 30,000 and were accumulated in these forms in the medium. In addition, PDGF-BB was further processed to a 24-kDa component, which remained cell-associated. The major secreted component was PDGF-AB, which was purified and shown to have structural and functional characteristics indistinguishable from PDGF-AB purified from human platelets.     

Role of alpha beta receptor heterodimer formation in beta platelet-derived growth factor (PDGF) receptor activation by PDGF-AB.

We investigated the ability of highly purified recombinant platelet-derived growth factor (PDGF) AB to interact with the products of alpha and beta receptor genes expressed in cells independently or concurrently. Although PDGF-AB lacked any detectable ability to bind or activate beta receptors in cells expressing only this receptor, efficient beta receptor activation by this ligand was readily observed in cells coexpressing alpha platelet-derived growth factor receptors (alpha PDGFRs). beta receptor activation induced by PDGF-AB was shown to be dependent upon in vivo physical association of this receptor with alpha PDGFRs. Moreover, cross-linking analysis established the existence of PDGF-AB-induced beta PDGFR dimers in vivo. All of these findings argue that initial PDGF-AB interaction with the alpha PDGFR induces conformational changes in the ligand or receptor that facilitates efficient recruitment of beta PDGFR by this PDGF isoform.     

Circular dichroism of isolated ricin A- and B-chains

An analysis of the circular dichroism (CD) spectra of isolated ricin A- and B-chains revealed several bands not apparent in the spectrum of intact ricin. Arithmetic combination of the A- and B-chain spectra gave a composite spectrum resembling that of native ricin, indicating that the two chains did not undergo any major conformational change upon dissociation. The addition of lactose to the B-chain at pH 7.2 caused a slight perturbation of a tryptophan-derived negative CD band centred at 283 nm without change to the overall structure of the polypeptide.     

Effects of platelet-derived growth factor on bone formation in vitro

Platelet-derived growth factor (PDGF) is a polypeptide found in a variety of tissues, including bone, where it could act as an autologous regulator of skeletal remodeling. Therefore, a recombinant B chain homodimer of human PDGF was studied for its effects on bone formation in cultured rat calvariae. PDGF at 10-100 ng/ml stimulated [3H]thymidine incorporation into DNA by up to sixfold and increased the DNA content and the number of colcemid-induced metaphase arrested cells. This effect was observed in the fibroblast and precursor cell-rich periosteum. As a result of its mitogenic actions, PDGF enhanced [3H]proline incorporation into collagen, an effect that was observed primarily in the osteoblast-rich central bone. The effect of PDGF was not specific for collagen since it also increased noncollagen protein synthesis. In addition, PDGF increased bone collagen degradation. PDGF and insulin-like growth factor (IGF) I had additive effects on calvarial DNA synthesis, but PDGF opposed the stimulatory effect of IGF I on collagen synthesis and IGF I prevented the PDGF effect on collagen degradation. In conclusion, PDGF stimulates calvarial DNA synthesis which causes an increased number of collagen-synthesizing cells, but PDGF also enhances bone collagen degradation.     

Separation of ricin A- and B-chains after dithiothreitol reduction

After complete cleavage of ricin interchain disulfide bridge by 0.05 M dithiothreitol in nondenaturing conditions at 37 degrees C during 1 h 30 min, A- and B-chains were separated on a lactosaminyl-aminoethyl Biogel P-150 column at 4 degrees C, in the presence of 0.01 M dithiothreitol and 0.5 M MgCl2. A- and B-chains have been characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunology. Their specific activities have been tested by protein synthesis inhibition in a cell-free assay (rabbit reticulocyte lysate) and on whole cells (Zajdela hepatoma cells) and by hemagglutination. From these tests, the apparent cross contamination of the chains was about 0.1%.     

A major part of platelet-derived growth factor purified from human platelets is a heterodimer of one A and one B chain

Platelet-derived growth factor, PDGF, purified from human platelets is a disulfide-bonded dimer consisting of two homologous polypeptide chains denoted A and B; it has not been known whether it is a heterodimer or a mixture of homodimers. We present here evidence that a major part of PDGF has a heterodimer structure. A highly homogeneous, 31-kDa PDGF was purified in the presence of protease inhibitors and shown to contain both chains by means of immunoprecipitations with peptide antisera specific for the A and B chains, respectively. The susceptibility of PDGF to mild acid treatment and its chromatographic behavior in reversed-phase high performance liquid chromatography and immobilized metal ion affinity chromatography, as compared to A and B chain homodimers, is consistent with a heterodimer structure. Analysis of PDGF purified according to our routine, large scale procedure revealed the major part to have a heterodimer structure. In addition, B chain homodimers were also found. With the demonstration that a major part of PDGF purified from human platelets occurs as a heterodimer, all three dimeric forms of PDGF have been identified. The following nomenclature to distinguish the various forms is suggested: PDGF-AA, a homodimer of A chains; PDGF-AB, a heterodimer; PDGF-BB, a homodimer of B chains; PDGF, any dimeric form of A or B chains.     

Similarities between fibroblast-derived growth factor and platelet-derived growth factor

Platelet-derived growth factor (PDGF), one of the most potent mitogens in serum for non-transformed cells, shares many biological and physical properties with fibroblast-derived growth factor (FDGF), a polypeptide produced by BHK cells transformed by SV40. Thus FDGF and PDGF have biological activity which is recoverable from sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, at positions indicating similar molecular weights. Further, the biological activity of both factors is heat-stable but sensitive to mercaptoethanol. FDGF and PDGF have similar abilities to induce DNA synthesis synergistically in the presence of either insulin, epidermal growth factor (EGF), vasopressin or colchicine. In contrast to other growth factors, (i) either FDGF or PDGF can induce DNA synthesis in the absence of other mitogens in 3T3 cells maintained in serum-free medium and (ii) a transient exposure of cultures to FDGF or PDGF causes a persistent stimulation of DNA synthesis. Either FDGF or PDGF enhances colony formation of non-transformed cells cultured in suspension in the presence of EGF and serum. FDGF is not PDGF adsorbed by SV40-BHK cells from serum, since SV40-BHK cells plated and grown in the absence of serum still produce FDGF. In view of the similarities between PDGF and FDGF, we suggest that they may belong to the same family of growth factors.     

The A- and B-chains of carboxypeptidase I from germinated barley originate from a single precursor polypeptide

Carboxypeptidase I from germinated barley (Hordeum vulgare) grain consists of two peptide chains linked by disulfides; the A- and B-chains contain 266 and 148 amino acid residues, respectively (Sorensen, S. B., Breddam, K., and Svendsen, I. (1986) Carlsberg Res. Commun. 51, 475-485). A cDNA library prepared from mRNA isolated from scutella of 2-day germinated barley has now been screened with a mixed oligonucleotide encoding a peptide fragment of the A-chain. Nucleotide sequence analysis of a 1443-nucleotide pair cDNA clone revealed that both chains of the enzyme are translated from a single mRNA. The coding region of the A-chain is located at the 5'-end of the cDNA and is separated from the B-chain coding region by a 165-nucleotide pair linking region. The B-chain coding region is followed by a stop codon, a 187-nucleotide pair 3'-untranslated sequence, and a short polyadenylic acid tail. The results indicate that the A- and B-chains of barley carboxypeptidase I arise by endoproteolytic excision of a 55-residue linker peptide from a single precursor polypeptide chain. The putative linker peptide is rich in proline, lysine, and arginine residues, has an apparent pI of 11.9, and appears to be excised by cleavage of peptide bonds on the COOH-terminal side of serine residues.     

Dissociation of cellular transformation from platelet-derived growth factor independence

ST2-3T3, a spontaneously transformed BALB/c-3T3 cell line which does not require platelet-derived growth factor (PDGF) for growth, was fused to THO2, a PDGF-responsive non-transformed BALB/c-3T3 cell line, in order to learn whether transformation is expressed coordinately with PDGF independence. Hybrid cells were selected and grown in medium containing both HAT (hypoxanthine-aminopterin-thymidine) and ouabain; unfused cells of each parental type were killed in HAT-ouabain medium. Five independently isolated ST2-3T3xTHO2 hybrid cell lines were established and characterized for both transformation and PDGF responsiveness. All five were transformed, having a disorganized growth pattern and achieving a final cell density similar to that of ST2-3T3 cells. Two of these lines did not respond to a brief treatment with PDGF: the mitogen neither induced the synthesis of a PDGF-modulated lysosomal protein (termed MEP), nor stimulated the cells to enter the S phase; one line responded to PDGF by synthesizing both MEP and DNA, whereas two others synthesized MEP but not DNA. In contrast, four independently isolated cell lines obtained by fusing PDGF-responsive non-transformed BALB/c-3TC cells to the THO2 line were all PDGF-responsive for both MEP and DNA synthesis and were not transformed. It appears that PDGF independence is not required for the transformation of BALB/c-3T3 cells.     

Specificity of alkaline elastase Bacillus on the oxidized insulin A- and B-chains

The substrate specificity of alkaline elastase Bacillus from alkalophilic Bacillus sp. Ya-B was investigated using oxidized insulin A- and B-chains. Under time-limited cleavage, the initial cleavage site of the enzyme on the oxidized insulin A-chain and B-chain was at the leucine13-tyrosine14 bond and the leucine15-tyrosine16 bond, respectively. When the cleavage was completed, three major cleavage sites and three minor cleavage sites on the A-chain, and five major cleavage sites and four minor cleavage sites on the B-chain were found. However, most of the peptides produced after complete hydrolysis of the A- or B-chain by the enzyme were composed of four to six amino acid residues. The results suggest that this enzyme cleaves the oxidized insulin A- and B-chains in a block-cutting manner.     

Interactions between the receptors for platelet-derived growth factor and epidermal growth factor

Preincubation of Swiss 3T3 cells or human fibroblasts with purified platelet-derived growth factor (PDGF) at 4 degrees C or 37 degrees C rapidly inhibits subsequent binding of 125I-epidermal growth factor (125I-EGF). The effect does not result from competition by PDGF for binding to the EGF receptor since (a) very low concentrations of PDGF are effective, (b) cells with EGF receptors but no PDGF receptors are not affected, and (c) the inhibition persists even if the bound PDGF is eluted before incubating the cells with 125I-EGF. PDGF does not affect 125I-insulin binding nor does EGF affect 125I-PDGF binding under these conditions. Endothelial cell-derived growth factor also competes for binding to PDGF receptors and inhibits 125I-EGF binding. The inhibition demonstrated by PDGF seems to result from an increase in the Kd for 125I-EGF binding with no change in the number of EGF receptors.     

The molecular biology of platelet-derived growth factor

PDGF is a connective tissue mitogen that has been associated with clotted blood serum for at least 300 million years. It regulates the expression of cell cycle "early genes" in normal fibroblasts. Induction of early genes is preceded by stimulation of a tyrosine-specific kinase. The putative structural gene for PDGF has been acquired by an acutely transforming retrovirus and is expressed in many connective tissue tumors. Further work is needed to determine whether (i) production of PDGF by tumor cells confers a proliferative advantage on these cells, (ii) tyrosine-specific phosphorylations mediate the induction of cell cycle early genes by PDGF, and (iii) products of cell cycle early genes play any functional role in the 10-12 hr chain of events that culminates in replicative DNA synthesis and cell division. In the meantime, these very issues represent candidate functions for other viral oncogenes and their cellular homologs. Some of these genes could act at the onset of the mitogenic cascade by causing the production of automitogenic growth factors. Others may function in the interior of the cascade by promoting tyrosine-specific phosphorylations. Still others may be mutated or rearranged homologs of cell cycle early genes whose expression is normally modulated by extracellular growth factors.     

Expression of epidermal growth factor and platelet-derived growth factor receptors during cervical carcinogenesis

Summary Altered expression of epidermal growth factor receptor (EGFR) is common in a variety of epithelial malignancies, including cervical cancer. However, the prognostic significance of EGFR expression is controversial for cervical cancer. Platelet-derived growth factor receptor (PDGFR) expression status is unknown in cervical cancer. Our results demonstrated that expression of EGFR and PDGFR was greatly enhanced in vivo and in organotypic cultures of low-grade cervical dysplastic tissues, but levels were decreased in high-grade lesions. To our knowledge, this is the first report identifying the expression of PDGFR in human epithelium. When low-grade dysplastic organotypic culture tissues were induced to differentiate more completely, EGFR expression, but not PDGFR expression, was relocalized to the basal layer as seen in normal tissues. Differentiation also induced phosphorylation of EGFR but not PDGFR. Our results suggest a role for EGFR and PDGFR during the early stages of cervical carcinogensis, and demonstrate the facility of organotypic cultures to study the role of these growth factors in the development of cervical cancer.     

Purification of the receptor for platelet-derived growth factor from porcine uterus

The receptor for platelet-derived growth factor has been purified to homogeneity on a large scale from porcine uterus. The purification procedure utilizes solubilization of uterus membranes by Triton X-100, followed by sequential chromatographies on wheat germ agglutinin-Sepharose, fast protein liquid chromatography Mono-Q, and anti-phosphotyrosine-Sepharose. About 160 micrograms of homogeneous and functionally active 170-kDa receptor could be purified from 5 kg of uterus tissue. The pure receptor responded to platelet-derived growth factor stimulation by autophosphorylation, indicating that the receptor has a kinase domain as an integral part of the molecule. A rabbit antiserum was produced against the pure receptor, which specifically recognizes the intact 170-kDa receptor.     

Chemical and biological properties of a growth factor from human-cultured osteosarcoma cells: Resemblance with platelet-derived growth factor

A human osteosarcoma cell line, U-2 OS, cultured under serumfree conditions, was shown to produce a growth factor (osteosarcoma-derived growth factor, ODGF) for human-cultured glial cells, fibroblasts, and other cells. ODGF, collected from the spent medium of 2 OS cultures, was purified by a sequence involving heparin-Sepharose chromatography, hydrophobic chromatography, gel chromatography, and preparative gel electrophoresis in SDS. Purified ODGF, at a concentration of 3 ng/ml, elicited a mitogenic response in human glial cells equivalent to 50% of that afforded by human serum at a final concentration of 1%. The preparation was estimated to be > 50% pure. The biological activity of ODGF resided in a cationic, relatively heat-resistant, reduction-susceptible protein with a molecular weight of 30,000 (by gel chromatography and SDS-gel electrophoresis). The electrophoretic behaviour of radioiodinated ODGF suggested that the protein was composed of two different polypeptide chains (about 13,000-14,000 and 16,000-17,000 daltons, respectively) linked via disulphide bonds. The molecular makeup of ODGF was thus similar to that of platelet-derived growth factor. ¹²⁵I-ODGF could be precipitated by an antibody to platelet-derived growth factor, indicating that the two factors were immunologically related. Resemblance with platelet-derived growth factor was also indicated by the finding that the latter (but not, e.g., fibroblast growth factor or epidermal growth factor) competed with ¹²⁵I-ODGF for binding to human-cultured glial cells.     

Human platelet-derived growth factor: structure and function

Human platelet-derived growth factor (PDGF) is a heat-stable, cationic polypeptide transported in blood in the alpha granules of platelets. It is released from platelets during blood clotting. PDGF has been resolved into at least two closely related active polypeptides, PDGF-I and PDGF-II, each consisting of two inactive chains linked together by disulfide bonds. PDGF stimulates the growth of normal cells in culture, including fibroblasts, arterial smooth muscle cells, and glial cells. In addition, PDGF has been shown to stimulate cell migration and many diverse metabolic functions such as amino acid transport, protein synthesis, cholesterol ester synthesis, phospholipid turnover, and prostacyclin synthesis. It modulates receptor binding of other active components such as epidermal growth factor, luteinizing hormone, low-density lipoprotein, and somatomedin C. Specific cell membrane receptors for PDGF have been demonstrated in arterial smooth muscle cells and fibroblasts.     

Binding domains and epitopes in platelet-derived growth factor

Trypsin treatment of recombinant PDGF-BB from Escherichia coli leads to the liberation of a small carboxy-terminal fragment and two internal segments without dissociating the molecule. The remaining core of 21 kDa retained a considerable binding affinity of 8.4 nM. By use of various peptide fragments obtained from monomeric recombinant PDGF-B, a receptor binding domain was assigned to one of these internal trypsin-sensitive segments. This segment is enriched in charged residues, suggesting mainly hydrophilic interactions with the receptor. Circular dichroism measurements of recombinant PDGF-BB showed a high content of random structure and only a small percentage (less than 10%) of alpha-helical structures. This structure was very rigid since the addition of 70% trifluoroethanol or 1% SDS did not change the circular dichroism spectrum. On the basis of these results, a tentative structure was generated by computer modeling.