125I-PDGF Binding Research Articles

Kinetics of 125I‐PDGF binding and down‐regulation of PDGF receptor in human arterial smooth muscle cell strains during cellular senescence in vitro

Platelet-derived growth factor (PDGF) is one of the major mitogens in serum to stimulate replication of human smooth muscle cells (SMCs) in culture. Previous studies using human fibroblasts failed to demonstrate changes in the receptor systems for growth factors during cellular senescence. We investigated the kinetics of 125I-PDGF(-BB) binding and down-regulation of the PDGF receptor in three human arterial SMC strains during cellular aging. The number of specific 125I-PDGF binding sites per cell increased slightly at a population doubling level (PDL) of 60%-80% of life span and then decreased at the PDL above 90%. The number of receptors per cell-surface area decreased with increasing in vitro age. The apparent Kd for the 125I-PDGF binding decreased with in vitro senescence. The internalization and degradation of 125I-PDGF per receptor were significantly reduced in senescent SMCs and the amount of 125I-PDGF that escaped degradation and was recycled back to the cell surface was significantly greater in senescent SMCs than young cells. Furthermore, down-regulation of the PDGF receptor was significantly greater in senescent SMCs than young cells. Immunoblot studies demonstrated that changes in beta-subunit of the PDGF receptor accounted for those in the studies using 125I-PDGF and that tyrosine phosphorylation of the PDGF receptor was significantly greater in young SMCs than aged cells. Our results suggest that age-related changes in the receptor systems for PDGF may be important contributors to the failure of DNA synthesis in senescent SMCs.

Negative feedback regulation of human platelets via autocrine activation of the platelet-derived growth factor alpha-receptor.

Human platelets contain platelet-derived growth factor (PDGF) in their alpha-granules which is released during platelet exocytosis. We show by immunoprecipitation and 125I-PDGF binding experiments that human platelets have functionally active PDGF alpha-receptors, but not beta-receptors. The PDGF alpha-receptor (PDGFR-alpha) was identified as a 170-kDa glycosylated protein-tyrosine kinase as found in other cell types. Stimulation of platelets with 0.1 unit/ml thrombin resulted in a significant increase (2-5-fold) of the tyrosine phosphorylation of the PDGFR-alpha, as determined by immunoprecipitation with phosphotyrosine antiserum as well as with PDGFR-alpha antiserum. The observed thrombin-induced autophosphorylation of the PDGFR-alpha was inhibited by the addition of a neutralizing monoclonal PDGF antibody. Thus, our results suggest that the platelet PDGFR-alpha is stimulated in an autocrine manner by PDGF secreted during platelet activation. Preincubation of platelets with PDGF inhibited thrombin-induced platelet aggregation and secretion of ATP + ADP and beta-hexosaminidase. Thrombin-induced platelet aggregation was also reversed when PDGF was added 30 s after thrombin stimulation. Inhibition of the autocrine PDGF pathway during platelet activation by the PDGF antibody led to a potentiation of thrombin-induced beta-hexosaminidase secretion. Thus, the PDGFR-alpha takes part in a negative feedback regulation during platelet activation. Our demonstration of PDGF alpha-receptors on human platelets and its inhibitory function during platelet activation identifies a new possible role of PDGF in the regulation of thrombosis.

Induction of membrane ruffling by growth factors in morphologically TPA-resistant Balb/c3T3 TR4 cells.

To investigate the biological characteristics of a Balb/c3T3 variant TR4 clone which is morphologically resistant to TPA and hypersensitive to v-src induced metastasis, we compared the responsiveness of the variant and its parent cells to growth factor-induced membrane ruffling. When the confluent cells were stimulated with PDGF, membrane ruffling was rapidly induced in TR4 but not in the parent cell cultures. In TR4 cells, membrane ruffling was observed under a phase-contrast microscope within 2 min after the addition of PDGF, reaching the maximum 5 min later and thereafter decreased gradually to the control level. There were no apparent differences in 125I-PDGF binding kinetics between TR4 and parent cells. Similar membrane ruffling was induced by other growth factors such as insulin, IGF-I, acidic or basic FGF but not by EGF or alpha- and beta-TGF, only in TR4 cells. When TR4 cells were incubated with TPA just before stimulation with these growth factors, growth factor-induced membrane ruffling was completely inhibited. Also, 5 out of 6 clones of stable fusion cells between TR4 and parent cells showed the parental type of responses to TPA and growth factors, indicating that the TR4 phenotype is recessive. These results suggest that the variant TR4 cells may acquire the genetic and recessive alteration of a cellular factor which is responsible for the regulation of growth factor-mediated membrane ruffling and that this genetic alteration occurs at a common step downstream of growth factor-mediated cascades, rather than at their receptor level.

Kinetics of 125I‐PDGF binding and down‐regulation of PDGF receptor in arterial smooth muscle cells derived from patients with moyamoya disease

Progressive stenosis or occlusion of bilateral internal carotid arteries by fibrocellular intimal thickening results in cerebral ischemia in moyamoya disease. We recently found that cultured smooth muscle cells (SMC) derived from arteries of patients with moyamoya disease responded poorly to serum mitogens, especially to platelet-derived growth factor (PDGF). In the present study, we investigated further the binding and processing of 125I-PDGF, as well as down-regulation of the PDGF receptor in arterial SMC derived from patients with moyamoya disease. The specific binding sites of 125I-PDGF were reduced significantly at both 4 degrees C and 22 degrees C on SMC from moyamoya disease compared with those from control (4.78 vs. 11.92 x 10(4)/cell at 4 degrees C), though the apparent dissociation constant (Kd) were the same. Kinetics of 125I-PDGF binding at 37 degrees C in cells from moyamoya disease showed fewer binding sites (less than 1/3 of controls) and lower degradation per cell than in those from controls, though no difference was observed in either internalization or degradation of each receptor. When SMC were exposed to lower concentrations of nonlabeled PDGF at 37 degrees C, the percentage of remaining binding sites on cells from moyamoya disease was significantly less than that from controls. This excess down-regulation of PDGF receptor in SMC from moyamoya disease may be interpreted as insufficient recycling or a decreased intracellular pool of the PDGF receptor. These results are closely correlated with the diminished proliferation responses to PDGF in SMC from moyamoya disease and provide evidence that functional alterations in vascular cells are involved in the mechanism of development of intimal thickening in moyamoya disease.

PDGF-induced mitogenic signaling is not mediated through protein kinase C and c-fos pathway in VSM cells.

This study examines the role of protein kinase C (PKC) in platelet-derived growth factor (PDGF)-induced vascular smooth muscle (VSM) cell proliferation and initial signaling events. A 24-h pretreatment of VSM cells with 200 nM phorbol 12-myristate 13-acetate (PMA) completely abolished immunologically reactive PKC activity. Depletion of PKC activity from VSM cells did not attenuate PDGF-stimulated [3H]thymidine incorporation compared with control cells. Similarly, acute activation of PKC by treatment with 200 nM PMA for 10 min had no effect on PDGF-mediated [3H]thymidine incorporation. Both PMA and PDGF increased c-fos induction to the same magnitude; however, treatment with PMA did not induce DNA synthesis in these cells. In PKC-depleted cells PDGF-mediated c-fos induction was reduced by 50-60%, while DNA synthesis in response to PDGF stimulation was not reduced. PKC depletion did not alter PDGF-stimulated increase in cytosolic calcium levels, 125I-PDGF binding, or receptor autophosphorylation. On the basis of these results, we conclude that PKC activation and c-fos induction do not play a significant role in PDGF-mediated mitogenesis in VSM cells.

PDGF receptors in the rat CNS: during late neurogenesis, PDGF alpha-receptor expression appears to be restricted to glial cells of the oligodendrocyte lineage.

Using in situ hybridization, we have visualized cells in the rat central nervous system (CNS) that contain mRNA encoding the platelet-derived growth factor alpha receptor (PDGF-alpha R). After embryonic day 16 (E16), PDGF-alpha R mRNA appears to be expressed by a subset of glial cells, but not by neurons. The temporal and spatial distribution of PDGF-alpha R+ cells, together with 125I-PDGF binding studies on subsets of glial cells in vitro, suggests that PDGF-alpha R may be expressed predominantly, or exclusively, by cells of the oligodendrocyte-type-2 astrocyte (O-2A) lineage. This conclusion is supported by the fact that the numbers of PDGF-alpha R+ cells in developing and adult optic nerves correlate well with independent estimates of the number of O-2A progenitor cells in the nerve at equivalent ages. Small numbers of PDGF-alpha R+ cells are present in the brain at E16, at which time they are found outside the subventricular germinal zones, suggesting that these cells do not express PDGF-alpha R until after, or shortly before they start to migrate away from the subventricular layer towards their final destinations. Reduced numbers of PDGF-alpha R+ cells persist in the adult CNS. PDGF-alpha R is also expressed strongly in the meningeal membranes and choroid plexus, and in the inner limiting membrane of the retina.

Receptors for platelet-derived growth factor on microvascular endothelial cells.

Endothelial cells are widely thought to be unresponsive to platelet-derived growth factor (PDGF) and devoid of PDGF receptors. However, in examining the growth factor responses of microvascular endothelial cells isolated from human omental adipose tissue, we detected PDGF-induced tyrosine phosphorylation of an 180-kD glycoprotein, subsequently identified as the cellular receptor for PDGF by specific immunoprecipitation. Scatchard analysis of 125I-PDGF binding to human microvascular endothelial cells revealed 30,000 PDGF receptors/cell with a kD of 0.14 nM. PDGF stimulated tyrosine phosphorylation of PDGF and other cellular proteins in a dose- and time-dependent manner, with half-maximal receptor phosphorylation occurring at 0.3 nM recombinant human PDGF-BB within 1 min of PDGF exposure. Normal cellular consequences of receptor activation were also observed, including tyrosine phosphorylation of a 42-kD protein and serine phosphorylation of ribosomal protein S6. Furthermore, PDGF was mitogenic for these cells. The finding of functional PDGF receptors on human microvascular endothelial cells suggests an important direct role for PDGF in neovascularization.

Regulation of proliferation and differentiation of myoblasts derived from adult mouse skeletal muscle by specific isoforms of PDGF.

The expression of receptors and the mitogenic response to PDGF by C2 myoblasts, derived from adult mouse skeletal muscle, was investigated. Employing 125I-PDGF binding assays, we showed that the cells exhibit high level binding of PDGF-BB (approximately 165 x 10(3) molecules/cell at saturation) and much lower binding of the PDGF-AA and PDGF-AB (6-12 x 10(3) molecules/cell at saturation). This indicates that the C2 myoblasts express high levels of PDGF receptor beta-subunits and low levels of alpha-subunits. PDGF-BB enhances the proliferation of C2 cells maintained in 2% FCS by about fivefold. PDGF-AB had a moderate effect on cell proliferation (less than twofold) and PDGF-AA had no effect. Inverse effects of PDGF isoforms on the frequency of differentiated myoblasts were observed; the frequency of myosin-positive cells was reduced in the presence of PDGF-BB while PDGF-AA and PDGF-AB had no effect. PDGF may thus act to increase the number of myoblasts that participate in muscle regeneration following muscle trauma by stimulating the proliferation and by inhibiting the differentiation of myogenic cells.

Autoregulation of MeWo metastatic melanoma cell growth: characterization of intracellular (FGF, MGSA) and secreted (PDGF) growth factors.

MeWo melanoma cells (clone LC1) secrete a potent mitogenic activity susceptible to reinitiate DNA replication in quiescent rodent fibroblasts (CCL39, NRK-49F, NIH-3T3) but not in BHK-21 kidney cells. This activity appears to be closely related to platelet-derived growth factor (PDGF) based on 1) its cationic nature, heat and acid resistance, but sensitivity to reducing agents; 2) its apparent molecular weight (33 kDaltons) as estimated by Biogel filtration, once dissociated from binding proteins by mild acidic treatment; 3) its weak affinity for heparin; and 4) its ability to compete with 125I-PDGF for binding to human and rodent fibroblasts, and to be recognized by anti-PDGF antibodies. Although MeWo cells coexpress the PDGF-A and PDGF-B (c-sis) chain gene transcripts, the secreted product shows reactivity on CCL39 fibroblasts more compatible with the PDGF-BB than with the PDGF-AB isoform. MeWo cell lysates contain activities that bind moderately and strongly to heparin-Sepharose, being eluted with 1.0 and 2.0 M NaCl, respectively. The latter may correspond to basic fibroblast growth factor (basic FGF), consistent with the expression of basic FGF gene mRNAs. The former has not been fully characterized and is probably not the product of the acidic FGF gene. In addition, MeWo cells react positively with the FB2 AH7 antibody, thus indicating that they elaborate material similar to melanoma growth-stimulating activity (MGSA). MeWo cells proliferate in serum-free medium in a cell-density-dependent fashion, both in liquid and semisolid cultures. Their division is modestly enhanced by basic FGF and by human and porcine PDGF but not by the factors that they release. However, the absence of demonstrable 125I-PDGF binding sites on MeWo cells, in conjunction with their lack of sensitivity to suramin growth inhibition, suggests that the secreted PDGF does not act as an autocrine factor. Instead, the autonomous proliferation of MeWo melanoma cells may result from the concerted action of basic FGF and MGSA, which are mostly cell-associated.

Prognostic significance of mRNA‐encoding estrogen receptor and epithelial growth factor receptor in breast carcinoma progression into lymph nodes: 1. Estrogen receptor encoding mRNA

The main objective of this study was to differentiate between lymph nodes infiltrated by estrogen receptor-positive (ER+) and estrogen receptor-negative (ER-) breast carcinoma. Lymph nodes were obtained from 40 postmenopausal cancer patients, 10 from each disease stage. Six patients from each group had estrogen receptor-positive (BCaER+) and four estrogen receptor-negative (BCaER-) tumors. Both tumor-containing (T) and uninvolved (N) lymph nodes from the same patient were examined by the following parameters: magnitude of lymph node nucleic acid hybridization with cDNA probes from breast cancer MCF-7ER+ and MCF-7ER- cells; and binding capacity of 3H-estradiol, 125I-EGF, and 125I-PDGF binding and protein kinase C activities of the lymph nodes. Concomitant with the appearance of transformed cells, several events occur: Tumor cells induce stimulation of mononuclear cells and macrophages and evoke T- and B-cell proliferation, leading to the synthesis of tumor cell membrane-associated antibodies. In estrogen receptor-positive (ER+) breast carcinoma, estrogens and host hormonal modulatory mechanisms stimulate production and release of epithelial growth (EGF) and platelet-derived growth factors (PDGF). These factors are characterized by protein kinase C activities. There is infiltration of tumor cells into the lymph node and infiltration of leukocytes into the tumor site. In the lymph node, tumor progression depends on tumor cell proliferation rate and metastatic aggressiveness. The experiments described in this study document the changes that occur in lymph nodes, with differences between nodes infiltrated with BCaER+ and BCaER- breast carcinomas. Hybridization of 32P-cDNA from MCF-7ER+ cells with cellular RNA from BCaER+ involved (T) lymph nodes is greater than with cellular RNA from uninvolved (N) lymph nodes. The magnitude of hybridization correlated (P less than 0.005) with the disease stage.

Mesangial cells express PDGF mRNAs and proliferate in response to PDGF.

Platelet-derived growth factor (PDGF) is a potent mitogen for cells of mesenchymal origin and is released and/or synthesized by platelets, macrophages, endothelial cells, and rat mesangial cells. In the present investigation, we found that human glomerular mesangial cells in culture release a PDGF-like protein which competes for 125I-PDGF binding to human foreskin fibroblasts and is mitogenic for these fibroblasts. The competing and to a lesser extent the mitogenic activities present in the conditioned medium are partially recognized by an anti-PDGF antibody. Northern blot analysis of poly(A)+ RNA from human mesangial cells demonstrates the expression of both PDGF A- and B-chain mRNAs. PDGF also binds to mesangial cells in a specific manner and stimulates DNA synthesis and cell proliferation. These data suggest that a PDGF-like protein secreted by mesangial cells or released from platelets, monocytes, or endothelial cells during glomerular inflammation may function as an autocrine or a paracrine growth factor for these cells. The biological role of PDGF in mediating proliferative and other inflammatory events in the glomerulus remains to be identified.

Ligand activation causes a phosphorylation-dependent change in platelet-derived growth factor receptor conformation.

The effect of ligand binding on platelet-derived growth factor (PDGF) receptor conformation was examined using peptide antibodies directed against specific receptor domains. Antiserum 83, which was directed to the receptor's carboxyl terminus (residues 934-951), preferentially immunoprecipitated the ligand-activated form of the PDGF receptor from 35S-labeled BALB/c 3T3 cells. By contrast, two antisera directed against other receptor sequences precipitated unactivated and activated receptors equally well. Denatured receptors were recognized equally by all antisera, even 83. Thus, ligand activation caused a change in PDGF receptor conformation that enhanced accessibility of the antibody to the carboxyl terminus. The activated receptor conformation was induced by three different forms of PDGF (AA and BB homodimers and AB heterodimers) and was reversed by suramin, a polyanionic compound that dissociates PDGF from the receptor. The inhibitory effect of suramin on receptor conformation was abolished by the phosphatase inhibitor, sodium orthovanadate, suggesting that receptor phosphorylation mediated the conformational change. In a cell-free assay, the change in receptor conformation was induced by PDGF only in the presence of ATP and was inhibited by adenyl-5'-yl imidodiphosphate, a nonhydrolyzable analog of ATP. The functional significance of receptor conformation was examined in Chinese hamster ovary fibroblasts transfected with wild-type or mutated forms of the PDGF receptor. When receptor tyrosine kinase activity was abolished by a mutation of the ATP binding site the receptor no longer underwent PDGF-induced conformational change and did not mediate PDGF-induced mitogenesis even though 125I-PDGF binding was normal. These findings show that ligand binding elicits a phosphorylation-dependent change in PDGF receptor conformation that may be important for receptor function.

Dissociation of the ligand and dephosphorylation of the platelet-derived growth factor receptor

The ligand-induced phosphorylation of the platelet-derived growth factor (PDGF) receptor was followed at 37°C by a rapid dephosphorylation which was roughly parallel to the down regulation of the 125I-PDGF binding sites. At 4°C, when the ligand-receptor complexes remain associated with the cell surface, the phosphorylated form of the receptor was more stable. However if the ligand was dissociated from the receptor by means of a mild acid wash or a treatment with suramin, the dephosphorylation of the receptor also occurred at a low temperature. These data suggest that, due to the dissociation of the ligand, the kinase activity of the receptor is switched off so that the phosphotyrosine-containing receptors remain exposed to the action of phosphatases that rapidly dephosphorylate them.

Synthesis, phosphorylation, and degradation of multiple forms of the platelet-derived growth factor receptor studied using a monoclonal antibody.

We have developed a monoclonal antibody, designated PR7212 (IgG1), which specifically recognizes the platelet-derived growth factor receptor (PDGFR) of primate cells. The antibody recognizes an extracellular epitope of the receptor, demonstrated by its ability to bind to intact cells. Using this antibody, we have detected three forms of PDGFR of approximately 180, 164, and 130 kDa. All three of the forms were detected by Western blot analysis of human dermal fibroblasts. Immunoprecipitates of 32P-labeled membrane extracts of human dermal fibroblasts demonstrate that phosphorylation of all three forms of the receptor is stimulated by PDGF. In addition, several smaller molecules were detected, ranging in size from 113 to 49 kDa, which are also phosphorylated in response to PDGF addition. These smaller molecules may be either PDGFR kinase substrates or partially degraded PDGFR. Only the 180- and the 164-kDa forms of the receptor are detectable from immunoprecipitates of soluble extracts of 35S-metabolically labeled cells. Pulse-chase experiments demonstrate that the 164-kDa form is a precursor of the 180-kDa molecule. After PDGF binding at 37 degrees C, the 180-kDa form disappears from the cell surface in parallel with a decrease in 125I-PDGF binding, providing evidence that occupation results in internalization of PDGFR rather than inactivation.

Regulation of glycogen synthase activity by growth factors. Relationship between synthase activation and receptor occupancy.

Platelet-derived growth factor (PDGF) was found to stimulate the activity of glycogen synthase, an enzyme subjected to regulation by phosphorylation-dephosphorylation reactions. In Swiss mouse 3T3 cells, the time course of enzyme activation by PDGF is very similar to that of epidermal growth factor (EGF) and insulin. A 3-fold maximal stimulation was observed by 30 min, and the enzyme activity ratio returned to basal levels by 100 min. The PDGF effect was maximal at 1 ng/ml (30 pM) and half-maximal stimulation was observed at 0.2 ng/ml (6 pM). Parallel measurements of 125I-PDGF binding indicate that binding was maximal by 10 min and thus should not be rate-limiting for enzyme activation. In addition, presaturation of the receptors with PDGF at 4 degrees C did not expedite subsequent enzyme activation at 37 degrees C. Removal of PDGF in the media after the 4 degrees C pretreatment did not affect the enzyme activation response, indicating that the continued presence of PDGF in the medium is not necessary after receptor binding is saturated. Results of sequential addition of PDGF, EGF, and insulin indicated a refractory period in the response system. This property is evident even when the second addition involved a different growth factor and is independent of the sequence of addition of the factors. There was little additivity in the actions of the three growth factors in effecting enzyme activation and suggests a common intermediate element in the three signalling pathways.

Tumor promoter enhances mitogenesis by PDGF with little effect on PDGF binding.

Preincubation of Swiss 3T3 cells with the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) at 37 degrees C is observed to cause only a small (approximately 10%) decrease in maximal binding of 125I-platelet-derived growth factor (125I-PDGF), and does not affect the affinity of 125I-PDGF binding to these cells. Under the same conditions, the affinity of the epidermal growth factor receptor is greatly reduced, possibly resulting from phosphorylation by protein kinase C. TPA is also shown to have no effect on the kinetics of internalization or degradation of bound 125I-PDGF. Although TPA has little or no effect on these properties of the PDGF receptor, it was found to act in a synergistic fashion with low, but not high, concentrations of PDGF to increase DNA synthesis by 3T3 cells. Since TPA has previously been shown to activate protein kinase C, these findings suggest that protein kinase C does not regulate the ligand-binding properties of the PDGF receptor, and that the observed synergism between TPA and PDGF in stimulating mitogenesis reflects effects of TPA on other processes in the mitogenic pathway.

Suramin binds to platelet-derived growth factor and inhibits its biological activity.

The polyanion suramin was recently found to inhibit binding of 125I-PDGF (platelet-derived growth factor) to Balb/c 3T3 cell membranes. Cultured Swiss 3T3 cells were used to investigate the mode of action of suramin and to monitor its effect on the biological activity of PDGF. Evidence is presented that suramin inhibits cellular binding of PDGF by binding to PDGF itself, thereby preventing it from binding to its cell surface receptor: First, while suramin inhibited 125I-PDGF binding with a half maximum inhibition concentration of approximately 60 microM or 90 micrograms/ml in a simultaneous competition assay, it was inactive in a sequential radioreceptor assay, in which an inhibitor is expected to be active if it interacts with the receptor (even with relatively low affinity) but to be inactive if it interacts with PDGF. Second, suramin prevented immunoprecipitation of 125I-PDGF in a dose-dependent manner, with a half maximum effective concentration of approximately 50 microM. Furthermore, suramin efficiently dissociated 125I-PDGF bound to its cell surface receptor, whereas unlabeled PDGF even in large excess was virtually inactive. This is also in line with the proposed direct interaction between PDGF and suramin, since such an interaction can be envisaged to induce a conformational change in the PDGF-receptor complex, resulting in an increased off-rate of the complex. Reduced 125I-PDGF binding in the presence of suramin correlated directly with a suramin dose-dependent inhibition of PDGF-induced incorporation of 3H-thymidine into quiescent Swiss 3T3 cells and of the proliferation of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

マクロファージ由来増殖因子の生化学的研究

Activated macrophages have been known to produce mitogens for fibroblasts and vascular smooth muscle cells (Macrophage Derived Growth factor, MDGF) and these factors could play an important role in development of atherosclerosis.MDGF secreted by human alveolar and peritoneal macrophages was studied to further characterize this factor. Concentrated macrophage conditioned medium supported the growth of vascular smooth muscle cells for more than 14 days and was mitogenic for quiescent Swiss 3T3 cells in 3H-thymudine incorporation assay. When condition medium was fractionated by Sephadex G100 gel filtration chromatography, two active peaks were detected apart from major protein peak. The first peak had apparent molecular weight of 30, 000 and most of its activity was blocked by monospecific anti-PDGF IgG. This fraction also compete for 125I-PDGF binding to fibroblasts. The second peak eluted between MW 10, 000-20, 000, and its activity was blocked by anti-PDGF IgG. Metabolic study using 35S-cystein revealed that these proteins were actually synthesized by macrophages.These data shows that macrophages synthesized and secrete PDGF like mitogens, which is major part of MDGF.

Ganglioside-mediated modulation of cell growth, growth factor binding, and receptor phosphorylation.

Glycosphingolipids added exogenously in cell culture are slowly incorporated into plasma membranes, inhibit cell growth, and modify growth behavior ( Laine , R. A., and Hakomori, S. (1973) Biochem. Biophys. Res. Commun. 54, 1039-1045; Keenan , T. W., Schmid, E., Franke , W. W., and Wiegandt , H. (1975) Exp. Cell Res. 92, 259-270). With the availability of purified growth factors and serum-free culture conditions in recent years, we have been able to examine this phenomenon in mouse Swiss 3T3 cells in greater detail with the following results. 1) Cell growth (cell number increase) in serum-free medium was specifically inhibited by the presence of GM1 and to a lesser extent by GM3, but not by NeuAcnLc4 , although the gangliosides were incorporated equally well into cell membranes. GM3 inhibited both platelet-derived growth factor (PDGF)- and epidermal growth factor-stimulated mitogenesis determined by thymidine incorporation, while GM1 could only inhibit PDGF-stimulated mitogenesis. NeuAcnLc4 had no effect on mitogen-stimulated thymidine incorporation. 2) The concentration-dependent binding of 125I-PDGF binding to cells indicated that cells whose growth was inhibited by GM1 or GM3 showed an increased affinity for PDGF as compared to cells grown without addition of ganglioside, while the total number of receptors stayed the same. Addition of ganglioside did not affect the binding of 125I-EGF. 3) No direct interaction was observed between gangliosides and growth factors as evidenced by the lack of competition by ganglioside-containing liposomes for cellular binding of 125I growth factors. 4) GM1 and GM3, but neither NeuAcnLc4 nor Gb4 , inhibited the PDGF-stimulated tyrosine phosphorylation by membrane preparations of a 170,000 molecular weight protein, which is probably the PDGF receptor. Thus, the level of gangliosides GM1 and GM3 in membranes may modulate PDGF receptor function by affecting the degree of tyrosine phosphorylation and may alter the affinity of the receptor for PDGF.

Platelet-derived growth factor receptors form a high affinity state in membrane preparations. Kinetics and affinity cross-linking studies.

The specific binding of 125I-PDGF (platelet-derived growth factor) to intact fibroblasts becomes relatively nondissociable during incubation at 37 degrees C. To characterize the interaction of PDGF with its receptors under conditions in which there is no receptor internalization, we have studied the binding of 125I-PDGF to membrane preparations derived from mouse 3T3 cells and rat liver. The binding sites had the affinity and specificity characteristics expected of PDGF receptors. At 37 degrees C (but not at 4 degrees C) the specific binding of 125I-PDGF to membranes gradually became nondissociable as assessed by either dilution or by addition of excess unlabeled PDGF. This tight binding was not due to a covalent interaction since the polyanionic compound suramin readily dissociated specifically bound 125I-PDGF. This property of suramin was used to expose rat liver PDGF receptors which were occupied by endogenous PDGF. Affinity cross-linking studies demonstrated that the formation of the nondissociable state of 125I-PDGF binding was associated with the binding of 125I-PDGF to a 160,000-dalton protein and to a 110,000-dalton species. The cross-linked binding sites could be adsorbed to wheat germ agglutinin and to anion exchange resins. The isoelectric point of both cross-linked species determined by two-dimensional gel electrophoresis was approximately 4.7. These data demonstrate that in membrane preparations, PDGF binds to an anionic 160,000-dalton glycoprotein which is likely to be the receptor. A high affinity state of PDGF binding, which is formed rapidly at 37 degrees C, can be dissociated by suramin.