Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.     

Differential expression of platelet-derived growth factor receptors in human malignant glioma cell lines.

Glioma cells in culture express platelet-derived growth factor (PDGF) A- and B-chains and secrete PDGF-like activity that is mainly PDGF-AA. In this work, we show that the PDGF alpha- and beta-receptors are independently expressed in human malignant glioma cells. We also define three different receptor phenotypes that are related to the morphology of glioma cells: cells with only alpha-receptors, only beta-receptors, or with both types of receptors. By the help of Northern blot analyses, 125I-PDGF-binding experiments, and immunoprecipitations the receptors are shown to be structurally normal PDGF receptors, except for minor variations in size that probably are due to differences in glycosylation. PDGF-BB induces DNA synthesis in cells of all three receptor phenotypes, whereas PDGF-AA or PDGF-AB has this effect only on cells with alpha- or with alpha- and beta-receptors. 125I-PDGF-AB binds with high affinity and down-regulates beta-receptors only in cells where alpha-receptors are present in addition to beta-receptors. Thus, the different functional capacities of PDGF isoforms on glioma cells fit with their known receptor-binding specificities and are compatible with the hypothesis that the isoforms act by inducing dimeric receptor complexes. When data on PDGF A- and B-chains, as well as alpha- and beta-receptor expression are compiled and the pattern of receptor binding specificity is taken into account, the majority of glioma cell lines are found to have a phenotype that makes autocrine stimulation possible.     

Down-regulation of cellular platelet-derived growth factor receptors induced by an activated neu receptor tyrosine kinase.

The functional integration of growth factor signaling occurs at several levels in target cells. One of the most proximal mechanisms is receptor transmodulation, by which one activated receptor can regulate the expression of other receptors in the same cells. Well-established transregulatory loops involve platelet-derived growth factor (PDGF) down-regulation of epidermal growth factor (EGF) receptors and beta-type transforming growth factors modulation of PDGF receptors. We have studied the relationship between neu tyrosine kinase activation and the expression of the PDGF receptors in transfected NIH/3T3 cells. Expression of the neu oncogene, but not of the neu proto-oncogene, was associated with a decrease of PDGF alpha- and beta-receptors on the cell surface, as measured by [125-I]PDGF-AA and -BB binding. These results were corroborated by metabolic labeling and immunoprecipitation of the PDGF beta-receptors. PDGF alpha- and beta-receptor mRNAs were strongly decreased in the neu oncogene-transformed cells in comparison with control cells expressing the neu proto-oncogene. Down-regulation of the PDGF receptors and their mRNAs was also observed after EGF treatment of cells expressing a chimeric EGF receptor/neu receptor, where the neu tyrosine kinase is activated by EGF binding. These results show that the neu tyrosine kinase can down-modulate PDGF receptor expression, and the effect is mediated via decreased PDGF receptor mRNA levels.     

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.     

Identification of three amino acids in the platelet-derived growth factor (PDGF) B-chain that are important for binding to the PDGF beta-receptor.

Platelet-derived growth factor (PDGF) is a disulfide-linked dimeric protein composed of two homologous polypeptide chains denoted A and B. Two types of PDGF receptors, alpha and beta, have been characterized. Whereas PDGF-AA binds only to PDGF alpha-receptors, PDGF-BB binds to both receptor types with high affinity. To map the regions of the PDGF B-chain that confer its ability to bind with high affinity to the PDGF beta-receptor, we expressed PDGF A/B-chain chimeras in COS cells and analyzed them with regard to PDGF alpha- and beta-receptor binding. A systematic analysis revealed that replacement of Asn-115, Arg-154, and Ile-158 of the PDGF B-chain with the corresponding A-chain amino acids led to a dramatic decrease in the affinity for the beta-receptor. Conversely, introduction of B-chain amino acids into the A-chain in the region spanning from Asn-115 to Ile-158 yielded a product with high affinity for the beta-receptor. These data thus indicate that Asn-115, Arg-154, and Ile-158 are likely to be part of the active site of the PDGF B-chain.     

Expression of platelet-derived growth factor-beta receptors on human fibroblasts. Regulation by recombinant platelet-derived growth factor-BB, IL-1, and tumor necrosis factor-alpha.

Stimulation of human fibroblasts by platelet-derived growth factor (PDGF)-BB leads to a down-regulation of PDGF beta-receptors and a concomitant appearance of intracellular granular accumulations of receptors, as determined by stainings with the mAb PDGFR-B2. The granules contained both the ligand and PDGF beta-receptors, as revealed by double-immunofluorescence staining, and were formed in response to PDGF-BB but not in response to other cytokines tested. The formation of intracellular PDGF beta-receptor granules was dependent on PDGF-BB concentration and time of stimulation. The granular PDGF beta-receptor staining on cells treated with PDGF-BB for 1 h at 37 degrees C was used to investigate the effects of macrophage-derived cytokines on PDGF beta-receptor expression. The number of PDGF beta-receptor granules was found to be reduced in fibroblasts grown for 48 h in the presence of PDGF-BB, TNF-alpha, or IL-1; PDGF-AA under the same conditions had no effect. The reduction observed was paralleled by a decrease in cell surface expression of PDGF beta-receptors, measured as binding of 125I-PDGF-BB and of the PDGFR-B2 antibody. Furthermore, both TNF-alpha and IL-1 decreased the detergent-extractable pool of PDGF-beta receptors in the fibroblasts, as revealed by immunoblotting of detergent cell extracts. Finally, the decrease in PDGF beta-receptors after culturing of the cells in the presence of TNF-alpha and IL-1 was accompanied by a decreased incorporation of [3H]thymidine in response to PDGF-BB stimulation. In conclusion, our data suggest that certain macrophage-derived cytokines can modulate the expression of PDGF beta-receptors by cultured fibroblasts, which may contribute in part to their reduced responsiveness to PDGF.     

Tyr-716 in the platelet-derived growth factor beta-receptor kinase insert is involved in GRB2 binding and Ras activation.

Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.     

Characterization of the receptor for platelet-derived growth factor on human fibroblasts. Demonstration of an intimate relationship with a 185,000-Dalton substrate for the platelet-derived growth factor-stimulated kinase

The receptor for platelet-derived growth factor (PDGF) on human foreskin fibroblasts has been characterized. The molecular weight of the PDGF-receptor complex was estimated by affinity labeling techniques to about 200,000, as determined by sodium dodecyl sulfate-gel electrophoresis performed under reducing conditions. Subtraction of the Mr of reduced PDGF (18,000 to 15,000) gives a Mr for the receptor proper of 185,000 (+/- 10,000). The mobility in sodium dodecyl sulfate-gel electrophoresis was similar whether or not reducing agents were present, suggesting that the receptor may be a single chain protein. The hydrodynamic size of the 125I-PDGF-receptor complex after solubilization with Triton X-100, corresponded to a Mr of approximately 320,000, as determined by gel chromatography. Subtraction of the Mr contributions from Triton X-100 and PDGF, respectively, gives a Mr of approximately 200,000 for the receptor itself, an estimate in good agreement with the value obtained from the affinity-labeling experiments. Several lectins were analyzed for their ability to inhibit binding of 125I-PDGF to its receptor. It was found that wheat germ agglutinin and a lectin from Crotalaria juncea were effective inhibitors and that their inhibitory effects could be neutralized by N-acetylglucosamine and galactose, respectively, suggesting that the receptor contains these sugars. The properties of the receptor were compared with those of a 185,000-Da component, being the major substrate for the membrane-bound PDGF-stimulated kinase. It was found that the 185,000-Da component behaved similar to the PDGF receptor in sodium dodecyl sulfate-gel electrophoresis, performed with or without reducing agents present. Further, the 185,000-Da component co-eluted with the PDGF receptor on a Sepharose 6B column, and had affinity for the same lectins that inhibited the binding of 125I-PDGF to its receptor. Finally, the 185,000-Da component had affinity for PDGF immobilized on Sepharose beads, suggesting that it has PDGF-binding activity. We conclude that the PDGF receptor and the 185,000-Da substrate for the PDGF-dependent kinase are intimately related and probably identical molecules.     

Cellular response to platelet-derived growth factor (PDGF)-AB after down-regulation of PDGF alpha-receptors. Evidence that functional binding does not require alpha-receptors.

Platelet-derived growth factor (PDGF) and its receptor exist in multiple forms. PDGF exists in three dimeric combinations of A and B subunit chains, which are the products of separate genes. The PDGF receptor is similarly encoded by genes for two distinct receptor proteins, alpha and beta. A recent model proposed PDGF binding involves the association of the two receptor proteins into three possible dimeric forms. An essential prediction of that model is that PDGF alpha-receptors are required for cells to bind and respond to the heterodimeric AB isoform of PDGF. In contrast, we found both binding and functional response to PDGF-AB was retained in Balb/c-3T3 cells after PDGF alpha-receptors had been down-regulated by PDGF-AA pretreatment. The observation that PDGF-AB could still elicit these responses suggests that at 37 degrees C, PDGF-AB may bind directly to beta-receptors in either monomeric or dimeric forms and that initial receptor activation may occur independently of the formation of alpha beta-receptor heterodimers.     

Characterization of baculovirus-expressed human alpha and beta platelet-derived growth factor receptors.

In an effort to biochemically characterize PDGF receptors and their mechanism of activation, recombinant baculovirus vectors containing the cDNAs of the human alpha PDGF receptor or beta PDGF receptor were engineered. Characterization of recombinant PDGF receptor expression in infected Sf9 insect cells by immunoblot analysis with specific PDGF receptor peptide antisera revealed that the alpha and beta PDGF receptor gene products were translated as 160- and 165-kDa transmembrane proteins, respectively. Ligand binding analysis demonstrated saturable, high-affinity binding of either 125I-labeled PDGF AA or 125I-labeled PDGF BB to Sf9 cells expressing the recombinant alpha PDGF receptor. In contrast, recombinant beta PDGF receptor expressing Sf9 cells showed high-affinity binding only for PDGF BB. Analysis of the kinetics of PDGF receptor expression demonstrated that receptor number increased dramatically from 24- to 48-h postinfection. Early in infection, the PDGF receptors were present in low numbers, lacked tyrosine phosphorylation, and exhibited ligand-dependent tyrosine phosphorylation. However, with increasing time postinfection and increasing receptor number, the PDGF receptors became constitutively tyrosine-phosphorylated in serum-free culture medium. Cross-linking studies revealed that receptor activation involved ligand-independent receptor dimer formation at high receptor number. Thus, these results strongly suggest that PDGF stabilizes and increases the frequency of PDGF receptor interaction, which ultimately results in PDGF receptor activation and intracellular signaling.     

Effects of platelet-derived growth factor on phosphorylation of the epidermal growth factor receptor in human skin fibroblasts

Heterologous regulation of the epidermal growth factor (EGF) receptor by platelet-derived growth factor (PDGF) was studied in FS4 human skin fibroblasts. The addition of PDGF to FS4 cells inhibited high affinity binding of 125I-EGF and stimulated phosphorylation of the EGF receptor. Phosphopeptide analysis by high performance liquid chromatography revealed that PDGF treatment of cells increased phosphorylation at several distinct sites of the EGF receptor. However, PDGF did not stimulate phosphorylation of threonine 654, a residue previously shown to be phosphorylated when protein kinase C is activated. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also stimulated phosphorylation of the same peptides from the EGF receptor as PDGF, and, in addition, induced phosphorylation of threonine 654. TPA inhibited both high and low affinity 125I-EGF binding by these cells. PDGF treatment of cells had no effect on EGF-dependent, tyrosine-specific autophosphorylation of the receptor, whereas TPA treatment was inhibitory. TPA, but not PDGF, stimulated phosphorylation of a Mr = 80,000 protein, known to be a substrate for protein kinase C, even though PDGF appeared to mediate breakdown of phosphoinositides. These data suggest that regulation of EGF receptor function by PDGF and TPA are distinct in these cells, even though some elements of regulation are shared. The results differ from those previously reported for a human lung fibroblast isolate, indicating that cell type-specific differences may exist in metabolism of the EGF receptor.     

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.     

Heparan sulfate upregulates platelet-derived growth factor receptors on human lung fibroblasts

Heparan sulfate is a molecule that possesses a large structural variability and which has been shown to inhibit the proliferation of fibroblasts in vitro. The aim of this study was to determine whether the anti-proliferative effects of heparan sulfate were exerted by regulation of the activity of the platelet-derived growth factor and/or of the platelet-derived growth factor receptors. Both l-iduronate-rich, anti-proliferative and the l-iduronate-poor, non-anti-proliferative heparan sulfate species, were incubated with confluent human embryonic lung fibroblasts for 24 h. The mRNA levels for PDGF-AA, PDGF-BB, and their receptors were measured. Binding studies were performed with [125I]-PDGF-BB and [125I]-EGF for 2 h at 4 degreesC in cultures preincubated with both types of heparan sulfate for 24 h. In separate experiments, cultures were incubated together with heparan sulfate and [125I]-PDGF-BB for 2 h at 4 degreesC. Increases of two- to threefold in the mRNA levels for both the alpha- and the beta-receptors of PDGF was obtained after treatment with both types of heparan sulfate, whereas the mRNA levels of both the PDGF-AA and the PDGF-BB were essentially unaffected. A sixfold increase in binding was only noted for [125I]- PDGF-BB in cultures pre-treated with the anti-proliferative heparan sulfate for 24 h, whereas no effect was noted with use of the non-anti- proliferative heparan sulfate. Incubating the [125I]-PDGF-BB and the anti-proliferative heparan sulfate together for 2 h resulted in a smaller, threefold increase in binding. This indicates that the anti- proliferative heparan sulfate both stabilizes and increases expression of the PDGF receptors. To investigate whether the increased number of PDGF receptors could affect cell activity, cells were preincubated with anti-proliferative heparan sulfate and then treated with PDGF-BB. This resulted in an increase in mitogenicity compared to cells treated only with PDGF-BB. Neither an increase in binding for [125I-EGF] nor an increase in the mitogenic response of EGF could be observed in cultures pre-treated with the anti-proliferative heparan sulfate. The results indicate that the extracellular matrix itself may regulate important biological phenomena such as cell proliferation and matrix production through affecting the expression of receptors of PDGF, which initiate both stimulatory and inhibitory signals.      

Differential expression of platelet-derived growth factor alpha- and beta- receptors on fat-storing cells and endothelial cells of rat liver.

Fat-storing cells and endothelial cells of the liver sinusoids play important roles in the biosynthesis and degradation of hyaluronan (HYA). These cells were isolated from rat liver by a simple and rapid procedure involving: (1) cell separation by centrifugation on a Nycodenz gradient, after dispersion of the liver cells by collagenase perfusion; (2) further purification of the cells by centrifugation on a discontinuous Percoll gradient; and (3) culturing of the cells, taking advantage of the different time that seeded cells need for attachment to plastic surfaces. We have determined the effects of two isoforms of platelet-derived growth factor (PDGF), PDGF-BB and PDGF-AA, on HYA production by the original fat-storing cells, as well as by fat-storing cells which had changed in vitro to myofibroblast-like cells. PDGF-BB was found to stimulate HYA synthesis in both types of cells with a maximal response equal to that obtained with 10% fetal calf serum. PDGF-AA had no stimulatory effect on HYA production. Fat-storing cells and their modified myofibroblast-like phenotype bound specifically to 125I-PDGF-BB, but not to 125I-PDGF-AA, indicating that they had PDGF beta-receptors, but not alpha-receptors. In contrast, liver endothelial cells were found to have PDGF alpha-receptors, but not beta-receptors.     

Design and synthesis of a novel biotinylated photoreactive insulin for receptor analysis.

B1-(4-Azido-salicyloyl)-[B1-biocytin,B2-lysine]insulin was synthesized by double Edman degradation of A1,B29-Msc2-insulin and stepwise acylation at the N-terminus of the B-chain. This derivative is homogeneous in RP-HPLC and has a biological in vitro activity of 20% and receptor binding of 15%, relative to insulin. Radioiodination and HPLC gave the B1-labelled 125I-derivative (I) as well as the 4 isomers with 125I-labelled tyrosine (A14, A19, B16, B26). UV-induced crosslinking of I with insulin receptors led to specific labelling of the alpha-subunit (Mr 130,000). The peptide bond LysB2-AspB3 is completely cleavable by trypsin (EC 3.4.21.4). I is thus a new tool for the analysis of the hormone-binding region by making possible the isolation of tryptic, biotinylated receptor fragments labelled by the dipeptide 125I-4-azidosalicyloyl-biocytinyl-Lys.     

Regulation of transferrin receptor expression at the cell surface by insulin-like growth factors, epidermal growth factor and platelet-derived growth factor.

Addition of platelet-derived growth factor (PDGF), recombinant insulin-like growth factor I (rIGF-I) or epidermal growth factor (EGF) to BALB/c 3T3 fibroblasts causes a marked increase in the binding of [125I]diferric transferrin to cell surface receptors. This effect is very rapid and is complete within 5 min. The effect of EGF is transient, with [125I]diferric transferrin binding returning to control values within 25 min. In contrast, PDGF and rIGF-I cause a prolonged stimulation of [125I]diferric transferrin binding that could be observed for up to 2 h. The increase in the binding of [125I]diferric transferrin caused by growth factors was investigated by analysis of the binding isotherm. Epidermal growth factor, PDGF and rIGF-I were found to increase the cell surface expression of transferrin receptors rather than to alter the affinity of the transferrin receptors. This result was confirmed in human fibroblasts by the demonstration that EGF, PDGF and rIGF-I could stimulate the binding of a monoclonal antibody directed against the transferrin receptor (OKT9) to the cell surface. Furthermore, PDGF and rIGF-I stimulated the sustained uptake of [59Fe]diferric transferrin by BALB/c 3T3 fibroblasts, while EGF transiently increased uptake. Thus the effect of these growth factors to increase the cell surface expression of the transferrin receptor appears to have an important physiological consequence.     

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.     

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)     

Intracellular (Golgi) receptors for insulinlike peptides in rat liver

An insulinlike peptide (ILAs) has been isolated in our laboratory from human serum. The binding of 123I-labeled ILAs was studied in subcellular fractions from rat liver and found to be much greater in microsomes than purified plasmalemma. The high level of microsomal binding was due to a particular enrichment of binding sites in Golgi elements. Binding to Golgi was time and temperature dependent and was augmented by an increase of either subcellular fraction or 125I-labeled ILAs in accordance with a mass action process. Degradation of 125I-labeled ILAs was greatest in the Golgi vesicle fraction and was reduced by incubation at 4 degrees C. Bound 125I-labeled ILAs could be eluted and was found to retain integrity. Binding was pH dependent with a broad optimum at pH 7.7-8.5. Dissociation of bound 125I-labeled ILAs was time and temperature dependent. It was greater at 37 than 4 degrees C, and was uninfluenced by unlabeled ILAs. The ILAs receptor was stable at 4 degrees C but was markedly decreased by preincubation at 37 degrees C. The binding of 125I-labeled ILAs was inhibited by unlabeled ILAs and related insulinlike peptides (the insulinlike growth factors, IGF-1 and IGF-2) in a dose-dependent manner. Insulin and its analogues had only a partially inhibitory effect, and structurally unrelated peptides were without inhibitory efficacy. In contrast ILAs and IGF-1 and IGF-2 inhibited 125I-labeled insulin binding to its receptors in a dose-dependent fashion. These observations identify a receptor for insulinlike peptides in the Golgi elements of rat liver. It is distinct from the insulin receptor previously observed in these elements. The dual interaction of ILAs and other insulinlike peptides with both the insulin receptor and their own unique receptor constitutes the presumed biochemical basis for the two types of action effected by this family of peptides, namely, an effect on metabolism comparable to insulin and an effect on cellular anabolism and growth.