Cyclic AMP distinguishes between two functions of acidic FGF in a rat bladder carcinoma cell line

The rat bladder carcinoma cell line NBT-II exhibits two completely different responses to acidic FGF (aFGF): at high cell density, aFGF is a potent mitogen whereas at low cell density, aFGF acts as a scattering agent that can convert the epithelial NBT-II cells into fibroblastic- like, motile cells. The basis of the dual action of aFGF has been approached by using substances interfering with the transducing pathways known to be activated by growth factors. Genistein and tyrphostin, two inhibitors of tyrosine kinases, inhibit both cell scattering and mitogenesis induced by aFGF. Conversely, sodium orthovanadate, a potent inhibitor of tyrosine phosphatases can reproduce the two effects of aFGF, indicating that protein tyrosine phosphorylations are determinant in the two pathways. In contrast, transforming growth factor (TGF)-beta 1 is a strong inhibitor of DNA synthesis induced by aFGF but has no effect on cell scattering, providing evidence that the two pathways are divergent. In an attempt to determine the specificity of the pathways of aFGF we found that the level of cAMP, which can be externally elevated, is of pivotal importance in distinguishing between the two transducing pathways leading to either DNA replication or cell dispersion. Forskolin, 8- bromo cAMP, dibutyryl-cAMP, and cholera toxin are all capable of potentiating the mitogenic effect of aFGF while strongly inhibiting its scattering action. Moreover, addition of any of these substances to NBT- II cells converted into fibroblasts immediately induces their reversion towards an epithelial phenotype. These findings support a role for cAMP as a modulator of the effects of aFGF. Moreover, basal cAMP synthesis, which is not affected by aFGF, is higher in sparse than in dense cultures indicating that the level of cAMP depends on the status of the cell. Altogether, these results suggest that establishment and maintenance of the epithelial state require a precise regulation of cAMP level.     

Alternative splicing in fibroblast growth factor receptor 2 is associated with induced epithelial-mesenchymal transition in rat bladder carcinoma cells.

We described previously that acidic fibroblast growth factor (aFGF), but not basic fibroblast growth factor (bFGF), can induce the rat carcinoma cell line NBT-II to undergo a rapid and reversible transition from epithelial to mesenchymal phenotype (EMT). We now find that NBT-II EMT is stimulated by keratinocyte growth factor (KGF) in cells grown at low density. Accordingly, a high-affinity receptor showing 98% homology to mouse FGF receptor 2b/KGF receptor was cloned and sequenced from NBT-II cells. Northern analysis indicated that mRNA for FGF receptor 2b/KGF receptor was drastically down-regulated within 1 wk in aFGF-induced mesenchymal NBT-II cells. This decrease coincided with an up-regulation of FGF receptor 2c/Bek, a KGF-insensitive, alternatively spliced form of FGF receptor 2b/KGF receptor. Functional studies confirmed that KGF could not maintain EMT induction on mesenchymal NBT-II cells. FGF receptor 1 and FGF receptor 2c/Bek could also support EMT induction when transfected into NBT-II cells in response to aFGF or bFGF. Such transfected cells could bind bFGF as well as aFGF. Therefore, EMT can be induced through different FGF receptors, but EMT may also regulate FGF receptor expression itself.     

E-cadherin expression during the acidic FGF-induced dispersion of a rat bladder carcinoma cell line

Cell dissociation and acquisition of cell motility are major events in morphogenesis, wound repair, and cancer invasion and metastasis. We have used the NBT-II bladder carcinoma cell line as a model system to study the mechanisms of these events. Upon exposure to acidic fibroblast growth factor (aFGF), NBT-II cells undergo morphological changes that resemble those described in epithelial-mesenchymal transitions, i.e., dissociation of some or all polygonal epithelial cells and their transformation into motile, fibroblastic-like cells. The disruption of intercellular contacts, which accompanies cell dissociation and acquisition of motility, is correlated with a redistribution of E-cadherin, a Ca(2+)-dependent cell adhesion molecule, over the entire cell surface and within the cytoplasm. However, these modifications are not accompanied by a reduction of the intercellular adhesiveness or a loss of E-cadherin expression. Moreover, the formation of intercellular contacts between fibroblastic-like NBT-II cells results in the relocation of epithelial cadherin (E-cadherin) immunoreactivity on lateral membranes, but is not sufficient to abrogate cell motility. Finally, the overexpression of E-cadherin by NBT-II cells stably transfected with a plasmid containing the mouse E-cadherin cDNA does not impair the scattering effect of aFGF, indicating that high levels of E-cadherin expression do not prevent cells from disrupting their intercellular connections. Altogether, these results suggest that the scattering activity of aFGF is not mediated by direct modulations of E-cadherin expression.     

Acidic fibroblast growth factor autocrine system as a mediator of calcium-regulated parathyroid cell growth.

Both parathyroid hormone secretion and cell growth are negatively regulated by extracellular calcium in parathyroid cells. The mechanism of growth regulation by calcium has been unknown. Previously, we reported that clonal parathyroid cells (PT-r cells) bear two high affinity receptors for acidic fibroblast growth factor (aFGF) and that at least a subpopulation of the receptors with a higher molecular mass carries heparan sulfate (HS) glycosaminoglycan chains which give the receptor higher affinity (Sakaguchi, K., Yanagishita, M., Takeuchi, Y., and Aurbach, G. D. (1991) J. Biol. Chem. 266, 7270-7278). Here, I have found that the parathyroid cells expressed aFGF and that aFGF receptors with lower affinity apparently translocated in response to changing extracellular calcium concentrations. Expression of both aFGF mRNA and peptide was suppressed by calcium. Cells had more ligand-accessible receptors on the cell surface at lower calcium concentrations. This apparent translocation was temperature-dependent but independent of de novo protein synthesis. Heparin or HS glycosaminoglycans are a prerequisite for the FGF receptor encoded by flg gene to bind basic FGF (Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P., and Ornitz, D. M. (1991) Cell 64, 841-848). In PT-r cells, major cellular HS proteoglycans redistribute between intracellular and extracellular compartments with more HS proteoglycans expressed on the cell surface at lower calcium concentrations (Takeuchi, Y., Sakaguchi, K., Yanagishita, M., Aurbach, G. D., and Hascall, V. C. (1990) J. Biol. Chem. 265, 13661-13668). However, this redistribution of HS proteoglycans cannot explain the difference in bindability of radiolabeled aFGF to its receptors in different calcium concentrations, since addition of heparin did not change the binding of radiolabeled aFGF to the receptors either at high or low calcium conditions. In concordance with the apparent translocation of aFGF receptors, thymidine incorporation was stimulated by decreasing extracellular calcium concentrations with further stimulation by added aFGF. Anti-aFGF antibody inhibited thymidine incorporation by more than 32% in the cells exposed to 0.05 mM Ca2+ shortly before adding [3H]thymidine, whereas the incorporation was not significantly affected by the antibody at 0.7 mM Ca2+. Cell growth was also stimulated by low calcium. Anti-aFGF antibody inhibited cell growth significantly only at low calcium concentrations. From these observations, an aFGF autocrine system including the apparent translocation of aFGF receptors may explain, if not entirely, the mechanism by which calcium regulates parathyroid cell growth.     

Activation of a transfected FGFR-1 receptor in Madin-Darby epithelial cells results in a reversible loss of epithelial properties

Basic fibroblast growth factor (bFGF) is a potent mitogen for a wide variety of cell types derived from mesoderm and neuroectoderm. The activity of bFGF is mediated by several types of closely related receptors belonging to the tyrosine-kinase family of receptors. We have found that Madin-Darby epithelial cells (MDCK) do not seem to produce bFGF or bFGF receptors. High level expression of human bFGF cDNA in these cells did not produce any mitogenic or morphological effects. Expression of the mouse-derived cDNA encoding FGF receptor-1 (FGFR-1) in MDCK cells resulted in the acquisition of a fibroblast-like morphology when the transfected cells were cultured at low density in the presence of 0.6% fetal calf serum and 20 ng/ml bFGF. Acidic fibroblast growth factor (aFGF) also induced these morphological changes but not keratinocyte growth factor. The morphological effect was not accompanied by increased bFGF-induced cell proliferation and did not result in the loss of epithelial cell markers such as cytokeratins. However, the morphological transition was accompanied by changes in the intracellular distribution of actin. In spite of these changes the transfected cells formed monolayers even in the presence of bFGF. Coexpression of bFGF and FGFR-1 in the MDCK cells resulted in similar morphological effects that were not dependent upon exogenous bFGF. These morphological effects were mimicked by exposure of MDCK cells to either orthovanadate or phorbol ester. Parental and FGFR-1 -expressing MDCK cells formed monolayers tht displayed high electrical resistance. Incubation of monolayers of FGFR-1-transfected cells with bFGF resulted in the loss of trans-epithelial resitance. Monolayers of parental MDCK cells did not lose their trans-epithelial resistance in response to bFGF, although exposure to phorbol ester did result in the loss of their trans-epithelial resistance, indicating that the effects on the trans-epithelial resistance are mediated by protein kinase C activation. Interestingly, orthovanadate did not cause a loss of transepithelial resistance, suggesting that the loss of trans-epithelial resistance is separable from the morphological transition. © 1995 Wiley-Liss, Inc.     

Signal transduction defect appears to be the cause of rat prostate cancer cell fibroblast growth factor insensitivity.

Using monolayer cultures of clonally isolated C3 and T5 rat prostate cancer cells, we determined that acidic (aFGF) and basic (bFGF) fibroblast growth factors profoundly enhanced T5 cell thymidine incorporation with half-maximum stimulation at 0.53 and 0.35 ng/ml, respectively. In contrast, aFGF or bFGF enhancement of C3 cell thymidine incorporation was about 5% of that of T5 cells, and effects were principally mitogen concentration independent. Saturation analyses and cross-linking studies established that both C3 and T5 cells contained high-affinity FGF receptors of 120 and 145 kilodaltons and that receptor content and Kd of C3 and T5 cells were comparable. aFGF or bFGF stimulation of T5 cell thymidine incorporation profoundly decreased as cell plating density was reduced from 1.5 x 10(5) to 1.0 x 10(4) cells/well. The modest response of C3 cells to either aFGF or bFGF also decreased as cell plating density was reduced. Because heparin preserves FGF biological activity and enhances bFGF binding to high-affinity FGF receptors, we examined the effect of heparin on FGF stimulation of C3 cell thymidine incorporation. We found that changes in cell plating density and/or medium heparin concentration had variable, inconsistent effects. These were C3 cell plating density associated and included inhibition or modest enhancement of FGF effects. Binding analyses established that high-affinity bFGF binding of C3 and T5 cells immediately prior to assessing FGF-stimulated thymidine incorporation was comparable and independent of cell plating density, implying that C3 cell FGF insensitivity was not attributable to differences in C3 and T5 cell FGF receptor content at the time of mitogen stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of heparan sulfate proteoglycan as a high affinity receptor for acidic fibroblast growth factor (aFGF) in a parathyroid cell line.

We have characterized two high affinity acidic fibroblast growth factor (aFGF) receptors in a rat parathyroid cell line (PT-r). Affinity labeling with 125I-aFGF showed that these two receptors, apparent molecular masses, 150 and 130 kDa, respectively, display higher affinity for aFGF than for bFGF. The 150-kDa receptor bears a heparan sulfate chain(s), demonstrated by a decrease in size of 15-20 kDa with heparitinase digestion after affinity labeling. Heparitinase digestion before affinity labeling markedly reduced the intensity of the 150 kDa species. Scatchard analysis showed two different high affinity binding sites (Kd of 3.9 pM with 180 sites/cell and Kd of 110 pM with 5800 sites/cell). The higher affinity site was completely eliminated by digestion with heparitinase before adding labeled aFGF; the lower affinity site was unaffected. In ion exchange chromatography after metabolic labeling of the cells with [3H]glucosamine and affinity labeling with 125I-aFGF, the larger receptor-ligand complex, 165 kDa, eluted with approximately 0.5 M NaCl, typical eluting conditions for heparan sulfate proteoglycans. Both of the receptor-ligand complexes were smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than two major heparan sulfate proteoglycans, HSPG I and II, which we characterized in this cell line previously (Yanagishita, M., Brandi, M. L., and Sakaguchi, K. (1989) J. Biol. Chem. 264, 15714-15720). Both receptors have similar N-linked oligosaccharide and sialic acid contents, shown by analysis of affinity-labeled receptors upon digestion with glycopeptidase F and with neuraminidase. All together, these results suggest that PT-r cells bear two distinct high affinity receptors for aFGF, a 150-kDa receptor which is a heparan sulfate proteoglycan and another that is a glycoprotein. The heparan sulfate glycosaminoglycan moiety of the 150- kDa receptor is critical for high affinity binding of aFGF. These findings contrast with current concepts derived from other systems, suggesting that heparan sulfate glycosaminoglycans/proteoglycans function as a reservoir source for FGF or as a group of low affinity binding sites.     

pp60c-src is a positive regulator of growth factor-induced cell scattering in a rat bladder carcinoma cell line

The NBT-II rat carcinoma cell line exhibits two mutually exclusive responses to FGF-1 and EGF, entering mitosis at cell confluency while undergoing an epithelium-to-mesenchyme transition (EMT) when cultured at subconfluency. EMT is characterized by acquisition of cell motility, modifications of cell morphology, and cell dissociation correlating with the loss of desmosomes from cellular cortex. The pleiotropic effects of EGF and FGF-1 on NBT-II cells suggest that multiple signaling pathways may be activated. We demonstrate here that growth factor activation is linked to at least two intracellular signaling pathways. One pathway leading to EMT involves an early and sustained stimulation of pp60c-src kinase activity, which is not observed during the growth factor-induced entry into the cell cycle. Overexpression of normal c-src causes a subpopulation of cells to undergo spontaneous EMT and sensitizes the rest of the population to the scattering activity of EGF and FGF-1 without affecting their mitogenic responsiveness. Addition of cholera toxin, a cAMP-elevating agent, severely perturbs growth factor induction of EMT without altering pp60c-src activation, therefore demonstrating that cAMP blockade takes place downstream or independently of pp60c-src. On the other hand, overexpression of a mutated, constitutively activated form of pp60c-src does not block cell dispersion while strongly inhibiting growth factor-induced entry into cell division. Moreover, stable transfection of a dominant negative mutant of c-src inhibits the scattering response without affecting mitogenesis induced by the growth factors. Altogether, these results suggest a role for pp60c-src in epithelial cell scattering and indicate that pp60c-src might contribute unequally to the two separate biological activities engendered by a single signal.     

Influence of cell density and receptor number on the binding and distribution of cell surface epidermal growth factor receptors

Summary Previous studies have shown that cell density influences the expression of receptors for at least four growth factors. The data presented in this report demonstrate that epidermal growth factor receptors are regulated differently on cells expressing over a million receptors as opposed to cells expressing approximately fivefold fewer receptors. Specifically, we show that BT-20, MDA-468, and A-431-R1 cells, which exhibit a large number of epidermal growth factor receptors, preferentially down-regulate the high affinity class of these receptors as cell density increases. In addition, we show that these cells express cell surface epidermal growth factor receptors that are localized predominately to the periphery of the cells. In contrast, A-549 and BSC-1 cells, which exhibit fewer cell surface epidermal growth factor receptors and which reduce all affinity classes of epidermal growth factor receptors as cell density increases, exhibit a diffuse cell surface distribution of these receptors at both low and high densities.     

Ligand specificity and heparin dependence of fibroblast growth factor receptors 1 and 3.

The heparin-binding growth factors include a family of seven structurally related proteins that can potentially interact with four known high affinity receptors. We have cloned the murine homologues of fibroblast growth factor receptors 1 and 3 (mFR1 and mFR3). To define the ligand specificity of these receptors, we have characterized their binding properties with respect to acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) and their biologic activity with respect to aFGF, bFGF, FGF-4/K-FGF, and FGF-5. Unlike mFR1, which binds both aFGF and bFGF, mFR3 preferentially binds aFGF. mFR3-mediated mitogenicity also favors aFGF and FGF-4 with a 10-12-fold lower response to bFGF and no response to FGF-5. Both receptor binding and growth factor-mediated mitogenicity are dependent on heparin. Heparin-binding growth factor activity can thus be regulated by proteoglycans and by the type of FGF receptor expressed on the target cell.     

The proliferative and morphologic responses of a colon carcinoma cell line (LIM 1215) require the production of two autocrine factors.

The role of autocrine growth factors in tumor cell growth has been difficult to prove. Our results indicate that more than one autocrine factor is required for the autonomous growth of the LIM 1215 colonic carcinoma cell line. Furthermore, the morphologic changes induced by epidermal growth factor (EGF) are also density dependent and appear to require a synergistic autocrine factor. The serum-free proliferation of the colonic carcinoma cell line LIM 1215 depends on cell density and the presence of EGF (A. Sizeland, S. Bol, and A.W. Burgess, Growth Factors 4:129-143, 1991). At cell densities below 10(4)/cm2, conditioned medium (from cells at a density of 10(5)/cm2) was required for the cells to elicit a mitogenic response to exogenous EGF. At higher cell densities (10(5)/cm2), the cells were independent of both exogenous EGF and conditioned medium. In addition, the EGF receptor was found to be phosphorylated on tyrosine in LIM 1215 cells proliferating at high density, suggesting that the autocrine production of transforming growth factor alpha (TGF alpha) and subsequent ligation to the EGF receptor was occurring. The proliferation of cells at high density was partly inhibited by TGF alpha antibodies but was almost completely inhibited by an antisense oligonucleotide to TGF alpha. The antisense inhibition could be overcome by the addition of EGF, indicating that the effect of the antisense TGF alpha oligonucleotide was on the production of autocrine TGF alpha. LIM 1215 cells were also observed to undergo morphologic changes (spreading and actin cable organization) in response to EGF. These changes were density dependent, but they occurred with a cell density dependence different from that of the proliferative response. These results suggest two possibilities: that the morphologic changes and proliferative responses have different sensitivities to the autocrine factors or that the actions of the autocrine factors are mediated through different signal transduction pathways.     

Internalization of growth factor-receptor complexes under the influence of antibodies initiates cell apoptosis in vitro

Antibodies against growth factors like IGF1, IGF2, aFGF, bFGF and, to a certain extent, TGF alpha and EGF were shown to cause apoptosis of normal and tumorigenic cells while apoptotic cell death could be prevented neither by single growth factors nor by serum. Antibodies to growth factors caused apoptosis by interacting with growth factors bound to their receptors on the cell surface. The phenomenon is likely to be associated with active internalization of growth factor receptors loaded with ligands. Apparently these activated receptors are essential for cell survival and their disappearance from the cell surface initiates apoptosis.     

Nature of the interaction of growth factors with suramin.

Suramin inhibits the binding of a variety of growth factors to their cell surface receptors. The direct interaction of suramin with acidic fibroblast growth factor has been detected by the enhancement of the drug's fluorescence in the presence of the protein with the maximum effect occurring at a molar ratio of suramin to aFGF of 2:1. This interaction stabilizes aFGF to thermal denaturation and partially protects a free thiol in its polyanion binding site from oxidation. The binding of suramin to aFGF also induces aggregation of the growth factor to at least a hexameric state as detected by static and dynamic light scattering as well as by gel filtration studies. Both CD and amide I' FTIR spectra of aFGF in the presence and absence of suramin suggest that the drug may also be causing a small conformational change in the growth factor. Suramin produces an even greater aggregation of bFGF and PDGF but not of EGF or IGF-1. Evidence for a suramin-induced conformational change in IGF-1 but not EGF is found by CD, however. It is concluded that suramin binds to many growth factors and that this induces microaggregation and, in some cases, conformational changes. In the case of aFGF, suramin interacts at or near its heparin binding site. The relationship between these phenomena and the anti-growth factor activity of suramin remains to be clearly elucidated.     

The cell adhesion receptor carcinoembryonic antigen-related cell adhesion molecule 1 regulates nucleocytoplasmic trafficking of DNA polymerase delta-interacting protein 38

The homophilic cell-cell adhesion receptor CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1, CD66a) acts as a regulator of contact-dependent cell survival, differentiation, and growth. It is involved in the control of proliferation in hematopoietic and epithelial cells and can act as a tumor suppressor. In this study, we identify DNA polymerase delta-interacting protein 38 (PDIP38) as a novel binding partner for CEACAM1-L and CEACAM1-S. We show that PDIP38 can occur in the nucleus, in the cytoplasm and at the plasma membrane in NBT-II, IEC18, RBE, and HeLa cells and that the distribution in NBT-II cells is influenced by the confluency of the cells. We also demonstrate that the interaction of CEACAM1 and PDIP38 is of functional importance in NBT-II cells, which co-express the long and the short CEACAM1 isoform. In subconfluent, proliferating NBT-II cells, perturbation of CEACAM1 by antibody clustering induces increased binding to PDIP38 and results in rapid recruitment of PDIP38 to the plasma membrane. The same treatment of confluent, quiescent NBT-II cells leads to a different response, i.e. translocation of PDIP38 to the nucleus. Together, our data show that PDIP38 can shuttle between the cytoplasmic and the nuclear compartments and that its subcellular localization is regulated by CEACAM1, implicating that PDIP38 may constitute a novel downstream target of CEACAM1 signaling.     

Identification of a novel aFGF-binding peptide with anti-tumor effect on breast cancer from phage display library

It has been reported that acidic fibroblast growth factor (aFGF) is expressed in breast cancer and via interactions with fibroblast growth factor receptors (FGFRs) to promote the stage and grade of the disease. Thus, aFGF/FGFRs have been considered essential targets in breast cancer therapy. We identified a specific aFGF-binding peptide (AGNWTPI, named AP8) from a phage display heptapeptide library with aFGF after four rounds of biopanning. The peptide AP8 contained two (TP) amino acids identical and showed high homology to the peptides of the 182–188 (GTPNPTL) site of high-affinity aFGF receptor FGFR1. Functional analyses indicated that AP8 specifically competed with the corresponding phage clone A8 for binding to aFGF. In addition, AP8 could inhibit aFGF-stimulated cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing PA2G4 and suppressing Cyclin D1 and PCNA, and blocked the aFGF-induced activation of Erk1/2 and Akt kinase in both breast cancer cells and vascular endothelial cells. Therefore, these results indicate that peptide AP8, acting as an aFGF antagonist, is a promising therapeutic agent for the treatment of breast cancer.     

EGF receptor binding studies in endometrial cell culture

Guinea pig endometrial cells were isolated and maintained in cell culture. Our investigation of these cultures for the presence of epidermal growth factor (EGF) binding activity demonstrated that these cells possess EGF receptors. Upon the addition of EGF to growing cell cultures, an increase in the rate of cell growth resulted along with a higher saturation density. The binding of EGF to these cells is saturable at 40-50 ng/ml., and the receptors demonstrate "down-regulation" in response to hormonal challenge at 37 degrees C.     

Identification of a fibroblast growth factor-binding protein in Drosophila melanogaster.

As assessed by competitive binding and protein-crosslinking experiments, Drosophila melanogaster cells possess basic fibroblast growth factor (bFGF)-specific binding proteins that are similar to FGF receptors on vertebrate cells in molecular weight and binding affinity; these D. melanogaster cells, however, have no detectable binding proteins for acidic fibroblast growth factor (aFGF). Consistent with the presence of bFGF-specific binding proteins, D. melanogaster cells degrade bFGF but not aFGF. These results indicate the conservation of heparin-binding growth factors and receptors between vertebrates and D. melanogaster.     

Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic fibroblast growth factors.

The fibroblast growth factor (FGF) family consists of at least seven closely related polypeptide mitogens which exert their activities by binding and activation of specific cell surface receptors. Unanswered questions have been whether there are multiple FGF receptors and what factors determine binding specificity and biological response. We report the complete cDNA cloning of two human genes previously designated flg and bek. These genes encode two similar but distinct cell surface receptors comprised of an extracellular domain with three immunoglobulin-like regions, a single transmembrane domain, and a cytoplasmic portion containing a tyrosine kinase domain with a typical kinase insert. The expression of these two cDNAs in transfected NIH 3T3 cells led to the biosynthesis of proteins of 150 kd and 135 kd for flg and bek, respectively. Direct binding experiments with radiolabeled acidic FGF (aFGF) or basic FGF (bFGF), inhibition of binding with native growth factors, and Scatchard analysis of the binding data indicated that bek and flg bind either aFGF or bFGF with dissociation constants of (2-15) x 10(-11) M. The high affinity binding of two distinct growth factors to each of two different receptors represents a unique double redundancy without precedence among polypeptide growth factor-receptor interactions.     

High and low affinity binding of heparin-binding growth factor to a 130-kDa receptor correlates with stimulation and inhibition of growth of a differentiated human hepatoma cell

The differentiated human hepatoblastoma-derived cell line, HepG2, displayed two classes of specific membrane receptors for heparin-binding growth factor type 1 (HBGF-1). Specific membrane receptors were distinguished from nonreceptor heparin-like binding sites. Receptors with an apparent Kd of 9.2 +/- 0.9 pM and present at 15,000 +/- 900/cell correlated with HBGF-1 stimulation of HepG2 growth. Receptors with an apparent Kd of 2 +/- 0.4 nM and present at 180,000 +/- 18,000/cell correlated with inhibition of growth and changes in secretory products. Other hepatoma cell lines exhibited a simple positive mitogenic response to HBGF-1 and a single class of high affinity binding sites. HBGF-1 covalently cross-linked to hepatoma cell surface polypeptides of apparent mean molecular mass of 130 kilodaltons. At 37 degrees C, receptor-bound HBGF-1 was internalized (t 1/2 = 45 min) but not degraded for up to 6 h. The display of receptors decreased with increased cell density and expression of HBGF-1 mRNA and HBGF-1-like activity in the culture medium. Proliferating normal human hepatocytes also exhibited two classes of binding sites with affinities for HBGF-1 and apparent molecular weight similar to HepG2 cells. These results implicate HBGF-1 or homologues in human hepatoma cell growth and normal liver cell regeneration.