A partial amino acid sequence of human platelet-derived growth factor, the major mitogen in serum for cells of mesenchymal origin, has been determined. A region of 104 contiguous amino acids shows virtual identity with the predicted sequence of p28sis, the putative transforming protein of simian sarcoma virus (SSV). This similarity suggests a mechanism for transformation by SSV and other agents, involving expression of growth factors.

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A cellular receptor for platelet-derived growth factor (PDGF) was demonstrated by incubation of "MI-labeled PDGF with human foreskin fibroblast cultures followed by liberation of cell-bound radioactivity with Triton X-100. The cellular binding of labeled PDGF in the presence of increasing amounts ofunlabeled PDGF showed saturation; Scatchard analysis ofbinding data indicated a single class of receptors having kd = 1 x 10-9 M. The number of PDGF binding sites was -3 X 105/cell. Labeled PDGF binding reached an apparent equilibrium after 3 hr at 4TC. At 370C, it passed a maximum after 30 min and then decreased with time due to degradation of the tracer. A large excess of unlabeled PDGF reduced labeled PDGF binding by more than 90% whereas similar doses of epidermal growth factor, fibroblast growth factor, or insulin had no effect. It was concluded that PDGF did not share receptors with these factors. PDGF receptors were found on skin fibroblasts, normal and malignant glial cells, smooth muscle cells, and 3T3 cells but not on epithelial-derived cells, neuroblastoma cells, endothelial cells, or peripheral lymphocytes. As only the receptor-positive cells-i.e., the connective tissue-and glia-derived cells-are responsive to stimulation with PDGF, these findings imply a functional significance of the PDGF receptor.The human platelet-derived growth factor (PDGF) is a Mr 30,000 cationic protein, probably composed of two polypeptide chains linked by disulfide bonds (1, 2). PDGF is stored in the a-granules of platelets (3,4) and released during the plateletrelease reaction (3-5). The factor stimulates the proliferation of various cultured cells, such as human glial cells (1, 6, 7), and a number of connective tissue-derived cells, e.g., arterial smooth muscle cells (8, 9) human fibroblasts (10, 11), and mouse 3T3 cells (12)(13)(14)(15). For these cell types, PDGF actually constitutes the major part of the growth-promoting activity of serum.The access to pure "2I-labeled PDGF (1) has made it possible to study the interaction between PDGF and its target cells. In this paper, we describe a specific binding of "2I-labeled PDGF to cultured cells. A preliminary report has been presented elsewhere (16). MATERIALS AND METHODSGrowth Factors. PDGF was purified from fresh platelets as described (2). The final product was >90% pure as estimated from analytical NaDodSO4polyacrylamide gel electrophoresis.Epidermal growth factor (EGF) and fibroblast growth factor (FGF) were purchased from Collaborative Research (Waltham, MA). Insulin was obtained from Vitrum (Stockholm, Sweden).Radiolabeing of PDGF. PDGF was labeled with "5I according to Hunter and Greenwood (17) as described (1). Five micrograms of PDGF was labeled with 0.5 mCi (1 Ci = 3.7 X 101" becquerels) of '25I. 125I-Labeled PDGF was separated from unbound radioactivity on a Sephadex G-25 column eluting with 1 M acetic acid containing serum albumin at 1 mg/ml (1). The specific activity was 16,000 cpm/ng, corresponding to -0.2 atoms of iodine per molecule of PDGF. The present ...

A cationic protein that stimulates DNA synthesis in human cultured cells was isolated from human platelets by ion exchange chromatography, hydrophobic chromatography, gel chromatography, and gel electrophoresis in sodium dodecyl sulfate. The electrophoretic behavior of biologically active or radioiodinated an reduced growth factor indicated that the native protein (Q30,000 daltons) was composed of two different polypeptides (z:13,000-14,000 and 16,000-17,000 daltons, respectively) linked via reduction-susceptible bonds. The stimulatory activity on human glial cells of the purified product at a concentration of t4 ng/ml (0.13 nM) was equal to that of 1% human serum.The major multiplication-stimulating activity of serum for cultured cells originates from the blood platelets (1-3). Although partial purification of the platelet-derived growth factor (PDGF) has been described (4-7) it has not yet been purified to homogeneity, and the characterization of the factor is still incomplete. PDGF is a protein or proteins resistant to heat and to treatment with various dissociating agents such as 4 M guanidine-HCl, 6 M urea, or 2% sodium dodecyl sulfate (NaDod-S04) but susceptible to reducing agents. The physiological function of PDGF is not known. PDGF is of interest both in relation to platelet function in vivo and as a representative of the group of potent growth-promoting substances for cultured cells (for a review, see ref. 8).The present communication deals with the-chemical properties of PDGF and the utilization of these properties in a purification protocol for PDGF leading to an electrophoretically pure product. Previous studies indicated that the growth-promoting activity of human platelets resides in at least two components, one anionic and the other cationic, that may be separated by ion exchange chromatography on CM-Sephadex (4, 5). The cationic PDGF served as starting point for the present experiments. A preliminary report has been presented elsewhere (9). MATERIALS AND METHODSAssay for multiplication-stimulating activity Multiplication-stimulating activity was estimated by using the incorporation of [3H]thymidine into trichloroacetic acid-precipitable material of serum-deprived, sparse cultures of a normal human glial cell line, U-787 CG (10), as described (5). Multiplication-stimulating activity was expressed relative to the activity given by medium containing 1% of a reference serum derived from a pool of healthy human subjects. The reference assay resulted in 10-18 X 103 cpm per dish, corresponding to 55-80% labeled nuclei. Controls, receiving no multiplication-stimulating activity, incorporated 1-3 X 103 cpm per dish (3-10% labeled nuclei). Purification of PDGFGeneral. Protein was determined as described (11). All operations were performed at 40C unless otherwise specified. Plastic containers or siliconized glassware was used to minimize losses of multiplication-stimulating activity on vessel walls.Ion Exchange Chromatography. Platelet lysate was prepared from outdated human platelets essentially a...