Characterization of the receptor for keratinocyte growth factor. Evidence for multiple fibroblast growth factor receptors

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. KGF exhibits potent mitogenic activity for a variety of epithelial cell types but is distinct from other known FGFs in that it is not mitogenic for fibroblasts or endothelial cells. We report saturable specific binding of 125I-KGF to surface receptors on intact Balb/MK mouse epidermal keratinocytes. 125I-KGF binding was completed efficiently by acidic FGF (aFGF) but with 20-fold lower efficiency by basic FGF (bFGF). The pattern of 125I-acidic FGF binding and competition on Balb/MK keratinocytes and NIH/3T3 fibroblasts suggests that these cell types possess related but distinct FGF receptors. Scatchard analysis of 125I-KGF binding suggested major and minor high affinity receptor components (KD = 400 and 25 pM, respectively) as well as a third high capacity/low affinity heparin-like component. Covalent affinity cross-linking of 125I-KGF to its receptor on Balb/MK cells revealed two species of 115 and 140 kDa. KGF also stimulated the rapid tyrosine phosphorylation of a 90-kDa protein in Balb/MK cells but not in NIH/3T3 fibroblasts. Together these results indicate that Balb/MK keratinocytes possess high affinity KGF receptors to which the FGFs may also bind. However, these receptors are distinct from the receptor(s) for aFGF and bFGF on NIH/3T3 fibroblasts, which fail to interact with KGF.     

Heparan sulfate proteoglycan modulates keratinocyte growth factor signaling through interaction with both ligand and receptor

Keratinocyte growth factor (KGF) is an unusual fibroblast growth factor (FGF) family member in that its activity is largely restricted to epithelial cells, and added heparin/heparan sulfate inhibits its activity in most cell types. The effects of heparan sulfate proteoglycan (HSPG) on binding and signaling by acidic FGF (aFGF) and KGF via the KGFR were studied using surface-bound and soluble receptor isoforms expressed in wild type and mutant Chinese hamster ovary (CHO) cells lacking HSPG. Low concentrations of added heparin (1 microgram/mL) enhanced the affinity of ligand binding to surface-bound KGFR in CHO mutants, as well as ligand-stimulated MAP kinase activation and c-fos induction, but had little effect on binding or signaling in wild type CHO cells. Higher heparin concentrations inhibited KGF, but not aFGF, binding and signaling. In addition to the known interaction between HSPG and KGF, we found that the KGFR also bound heparin. The biphasic effect of heparin on KGF, but not aFGF, binding and signaling suggests that occupancy of the HSPG binding site on the KGFR may specifically inhibit KGF signaling. In contrast to events on the cell surface, added heparin was not required for high-affinity soluble KGF-KGFR interaction. These results suggest that high-affinity ligand binding is an intrinsic property of the receptor, and that the difference between the HSPG-dependent ligand binding to receptor on cell surfaces and the HSPG-independent binding to soluble receptor may be due to other molecule(s) present on cell surfaces.     

Basic and acidic fibroblast growth factors modulate the epidermal growth factor receptor by a protein kinase C-independent pathway

Human acidic and basic fibroblast growth factors (aFGF and bFGF) inhibit epidermal growth factor (EGF) receptor binding in mouse Swiss 3T3 cells. Scatchard analysis indicates that aFGF and bFGF cause a decrease in the high affinity EGF receptor population, similar to that observed for activators of protein kinase C such as phorbol esters, platelet-derived growth factor (PDGF) and bombesin. However, unlike phorbol esters, aFGF and bFGF inhibit EGF binding in protein kinase C-deficient cells. The time course and dose response of inhibition of EGF binding by both aFGF and bFGF are very similar, with an ID50 of approximately 0.10 ng/ml. In contrast to bombesin but like PDGF, neither aFGF nor bFGF act on the EGF receptor through a pertussis toxin-sensitive G protein. These results indicate that both acidic and basic FGF depress high affinity EGF binding in Swiss 3T3 cells with similar potency through a protein kinase C/Gi-independent pathway.     

Keratinocyte growth factor receptor ligands target the receptor to different intracellular pathways

The keratinocyte growth factor receptor (KGFR)/fibroblast growth factor receptor 2b is activated by high-affinity-specific interaction with two different ligands, keratinocyte growth factor (KGF)/fibroblast growth factor (FGF)7 and FGF10/KGF2, which are characterized by an opposite requirement of heparan sulfate proteoglycans and heparin for binding to the receptor. We investigated here the possible different endocytic trafficking of KGFR, induced by the two ligands. Immunofluorescence and immunoelectron microscopy analysis showed that KGFR internalization triggered by either KGF or FGF10 occurs through clathrin-coated pits. Immunofluorescence confocal microscopy using endocytic markers as well as tumor susceptibility gene 101 (TSG101) silencing demonstrated that KGF drives KGFR to the degradative pathway, while FGF10 targets the receptor to the recycling endosomes. Biochemical analysis showed that KGFR is ubiquitinated and degraded after KGF treatment but not after FGF10 treatment, and that the alternative fate of KGFR might depend on the different ability of the receptor to phosphorylate the fibroblast growth factor receptor substrate 2 (FRS2) substrate and to recruit the ubiquitin ligase c-Cbl. The recycling endocytic pathway followed by KGFR upon FGF10 stimulation correlates with the higher mitogenic activity exerted by this ligand on epithelial cells compared with KGF, suggesting that the two ligands may play different functional roles through the regulation of the receptor endocytic transport.     

Alternative patterns of mitogenesis and cell scattering induced by acidic FGF as a function of cell density in a rat bladder carcinoma cell line.

The dual function exerted by acidic fibroblast growth factor (aFGF) in a rat bladder carcinoma cell line has been explored under two different conditions of culture density. At low cell density, aFGF promotes the epithelium-to-mesenchyme transition of NBT-II cells characterized by cell dissociation, morphological changes toward a fibroblastic-like phenotype, and acquisition of cell motility. Under these conditions, NBT-II cells are unresponsive to the growth-promoting effect of aFGF. At high cell density, aFGF is a potent mitogenic factor, but its scattering activity is essentially abrogated. Slight modifications in the binding of aFGF to its specific receptors were observed at high cell density; these changes correlated with a downregulation of receptors with no apparent change in their molecular form. NBT-II cells located at the edge of artificial wounds mimicked the behavior of subconfluent cells, because they did not proliferate upon aFGF treatment. Furthermore, in large-sized NBT-II colonies, peripheral cells were the first to dissociate in response to aFGF. Altogether, our results suggest that the cellular response to multifunctional growth factors might depend on the localization within the responding cell population.     

Paracrine effects of bFGF and KGF on the process of mouse blastocyst implantation

Implantation is a complex process that requires the interaction of the blastocyst, and subsequently, that of the developing embryos with the endometrium. Several growth factors and cytokines are involved in implantation, but the details of their actions as related to the regulation of blastocyst implantation remain unclear. In the present study, the RT-PCR method was used to determine the gene expression of basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), FGF receptor 1 (FGFR1), FGF receptor 2 (FGFR2), and KGF receptor (KGFR) in mouse embryos and in the stromal and epithelial cells of the uterine endometrium. Basic FGF and KGF mRNA were expressed in the endometrial cells, but were not expressed in the embryos. The mRNAs of receptors for bFGF and KGF were expressed in the blastocysts and in the in vitro implanting embryos, suggesting that bFGF and KGF may exert paracrine effects on blastocyst implantation. In this mouse model of blastocyst implantation, it was found that transforming growth factor α (TGF-α) at the concentrations of 1 ng/ml and 10 ng/ml significantly enhanced the blastocyst attachment and trophoblast spreading and increased trophoblast surface area. Relatively high concentrations of bFGF (100–500 ng/ml) significantly enhanced the rates of blastocyst attachment and of trophoblast spreading and promoted the expansion of the surface area of the implanting embryos. Unlike the rates of blastocyst attachment and trophoblast spreading, the surface area of the spreading embryos was significantly increased by addition of KGF (1–100 ng/ml). These results suggest that the bFGF and KGF derived from the endometrial cells exert paracrine effects on the process of implantation by stimulating trophoblast outgrowth through their cognate receptors. Mol. Reprod. Dev. 50:54–62, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Ligand Binding by Fibroblast Growth Factor Receptors Investigated Using Chimeric Receptor Molecules

Abstract

In order to map in detail the ligand binding sites of fibroblast growth factor receptor 2 (FGFR2) and keratinocyte growth factor receptor (KGFR), we have generated receptor molecules that are chimeric within the membrane proximal sequence that varies between them. The chimeric molecules are found to bind aFGF with a greater than 5-fold difference in affinity, indicating that there is coupling between the chimeric regions with respect to aFGF binding. Further, binding of bFGF and KGF is abolished in the chimeras, showing that the binding site for these ligands requires the whole of the 48- or 50- amino acid variable sequence to be intact. Direct interactions between the different regions exchanged in the chimeras are most probably involved in forming KGF or bFGF binding sites.     

The Zinc-Finger Protein Slug Causes Desmosome Dissociation, an Initial and Necessary Step for Growth Factor–induced Epithelial–Mesenchymal Transition

Epithelial–mesenchymal transition (EMT) is an essential morphogenetic process during embryonic development. It can be induced in vitro by hepatocyte growth factor/scatter factor (HGF/SF), or by FGF-1 in our NBT-II cell model for EMT. We tested for a central role in EMT of a zinc-finger protein called Slug. Slug mRNA and protein levels were increased transiently in FGF-1–treated NBT-II cells. Transient or stable transfection of Slug cDNA in NBT-II cells resulted in a striking disappearance of the desmosomal markers desmoplakin and desmoglein from cell–cell contact areas, mimicking the initial steps of FGF-1 or HGF/SF- induced EMT. Stable transfectant cells expressed Slug protein and were less epithelial, with increased cell spreading and cell–cell separation in subconfluent cultures. Interestingly, NBT-II cells transfected with antisense Slug cDNA were able to resist EMT induction by FGF-1 or even HGF/SF. This antisense effect was suppressed by retransfection with Slug sense cDNA. Our results indicate that Slug induces the first phase of growth factor–induced EMT, including desmosome dissociation, cell spreading, and initiation of cell separation. Moreover, the antisense inhibition experiments suggest that Slug is also necessary for EMT.     

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.     

角质细胞生长因子的研究进展

角质细胞生长因子(KGF)是成纤维细胞生长因子(FGFs)家族的成员,即FGF-7,最初是从人胚胎肺成纤维细胞的培养上清中分离纯化获得的。成熟KGF为一163个氨基酸残基的单链多肽,分子量为26—28KD。KGF由各种来源的间质细胞分泌,受体分布于上皮细胞,其生物学活性是特异性地促进上皮细胞的增殖、迁移和分化。KGF的表达受激素和一些细胞因子的调控。有关研究表明,KGF对肺泡Ⅱ型细胞的增殖以及皮肤、胃肠道粘膜和角膜损伤的修复具有十分重要的作用。     

A confined variable region confers ligand specificity on fibroblast growth factor receptors: implications for the origin of the immunoglobulin fold.

Binding of cellular growth factors to their receptors constitutes a highly specific interaction and the basis for cell and tissue-type specific growth and differentiation. A unique feature of fibroblast growth factor (FGF) receptors is the multitude of structural variants and an unprecedented degree of cross-reactivity between receptors and their various ligands. To examine receptor-ligand specificity within these families of growth factors and receptors, we used genetic engineering to substitute discrete regions between Bek/FGFR2 and the closely related keratinocyte growth factor receptor (KGFR). We demonstrate that a confined, 50 amino acid, variable region within the third immunoglobulin-like domain of Bek and KGFR exclusively determines their ligand binding specificities. Replacing the variable region of Bek/FGFR2 with the corresponding sequence of KGFR resulted in a chimeric receptor which bound KGF and had lost the capacity to bind basic FGF. We present evidence that the two variable sequences are encoded by two distinct exons that map close together in the mouse genome and follow a constant exon, suggesting that the two receptors were derived from a common gene by mutually exclusive alternative mRNA splicing. These results identify the C-terminal half of the third immunoglobulin-like domain of FGF receptors as a major determinant for ligand binding and present a novel genetic mechanism for altering receptor-ligand specificity and generating receptor diversity.     

Cell type and tissue distribution of the fibroblast growth factor receptor

A receptor for fibroblast growth factor (aFGF, bFGF) was partially characterized in intact cell cultures, cell plasma membranes, and tissue plasma membrane preparations. Analysis of 24 different cell types from four species identified a 165-kDa FGF receptor present on the cell surface of most mesodermal and neuroectodermal cells. Chemical crosslinking of 125I-aFGF to its cell surface receptor was specifically blocked by a 100-fold molar excess of either aFGF or bFGF. In contrast to the similar molecular weight of FGF receptors, different cell types exhibited significant variations in binding of 125I-aFGF to intact cultures with low values of 8 pM and 700, to high values of 60 pM and 30,000, for the Kd and receptor number per cell, respectively. A binding assay was developed for quantitation of 125I-aFGF binding to cell- and tissue-derived membrane preparations. Membranes prepared from baby hamster kidney cells exhibited a Kd of 55 pM, while a similar Kd of 67 pM was determined for intact baby hamster kidney cells. Although ten different adult bovine tissue membrane preparations and human term placental membranes exhibited no specific binding of 125I-aFGF, FGF receptor was detected in embryonic murine tissues (17 days gestation). These results support the existence, in a variety of cells, of either a common FGF receptor that binds both aFGF and bFGF or closely related FGF receptors that cannot be distinguished by molecular weight. The differential binding of FGF to its receptor in embryonic vs. adult tissues suggests a potentially broad role for FGF in embryonic development and a more restrictive role in the adult.     

Hrs regulates the endocytic sorting of the fibroblast growth factor receptor 2b

The keratinocyte growth factor receptor or fibroblast growth factor receptor 2b (KGFR/FGFR2b) is activated by the specific interaction with the keratinocyte growth factor (KGF/FGF7), which targets the receptor to the degradative pathway, and the fibroblast growth factor 10 (FGF10/KGF2), which drives the receptor to the juxtanuclear recycling route. Hrs plays a key role in the regulation of the endocytic degradative transport of ubiquitinated receptor tyrosine kinases, but the direct involvement of this protein in the regulation of FGFR endocytosis has not been investigated yet. We investigated here the possible role of Hrs in the alternative endocytic pathways of KGFR. Quantitative immunofluorescence microscopy and biochemical analysis showed that both overexpression and siRNA interference of Hrs inhibit the KGF-triggered KGFR degradation, blocking receptor transport to lysosomes and causing its rapid reapparance at the plasma membrane. In contrast, the FGF10-induced KGFR targeting to the recycling compartment is not affected by Hrs overexpression or depletion. Coimmunoprecipitation approaches indicated that Hrs is recruited to KGFR only after KGF treatment, although it is not tyrosine phosphorylated by the ligand. In conclusion, Hrs regulates the KGFR degradative pathway, but not its juxtanuclear recycling transport. In addition, the results suggest that Hrs recruitment to the receptor, but not its ligand-induced phosphorylation, could be required for its function.     

Up-regulation of fibroblast growth factor (FGF) receptor mRNA levels by basic FGF or testosterone in androgen-sensitive mouse mammary tumor cells.

Since we had previously shown that both basic fibroblast growth factor (bFGF) and testosterone stimulate the growth of mouse mammary carcinoma cells (SC-3) in serum-free culture, we tested the effect of bFGF or testosterone on FGF receptor mRNA levels. Northern blot analyses revealed that stimulation with bFGF resulted in a 5-fold increase in FGF receptor mRNA levels at 6-8 h followed by a decline to the unstimulated levels at 24 h. Simultaneous addition of cycloheximide blocked bFGF-induced accumulation of FGF receptor mRNA, although exposure of SC-3 cells to cycloheximide alone caused marginal increase in its basal level. Neither phorbol ester nor forskolin stimulated FGF receptor mRNA expression, but testosterone could raise FGF receptor mRNA levels. To obtain the maximum stimulation, however, testosterone required the longer stimulation period (12 h) than bFGF, suggesting that testosterone-induced FGF receptor mRNA accumulation is mediated through an induction of FGF-like growth factor.     

Acidic and basic fibroblast growth factor bind with differing affinity to the same heparan sulfate proteoglycan on BALB/c 3T3 cells: Implications for potentiation of growth factor action by heparin

Heparan sulfate proteoglycans on the cell surface act as low affinity binding sites for acidic and basic fibroblast growth factor (FGF) [Moscatelli (1887): J Cell Physiol 131:123–130] and play an important role in the interaction of FGF with the FGF receptor (FGFR). In this study, several aspects of the interaction of FGFs with cell surface heparan sulfate proteoglycans were examined. Reciprocal cross blocking studies demonstrated that acidic FGF (aFGF) and basic FGF (bFGF) bind to identical or closely associated heparan sulfate motifs on BALB/c 3T3 cell surface heparan sulfate proteoglycans. However, the binding affinity of the two growth factros for these heparan sulfate proteoglycans differs considerably, competition binding data indicating that aFGF has a 4.7-fold lower affinity than bFGF for 3T3 heparan sulfate proteoglycan. Subsequent studies of dissociation kinetics demonstrated that bFGF dissociates form the FGFR at least 10-fold slower than aFGF, whereas, following removal of cell surface heparan sulfate proteoplycan. Subsequent studies of dissociation kinetic demonstrated that bFGF dissociates from the FGFR at least 10-fold slwer than aFGF, whereas, following removal of cell surface heparan sulfate proteoglycans by heparinase treatment, the dissociation rate of both FGFs is similar and rapid. These results support the concept that cell surface heparan sulfate proteoglycans stabilize the interactio fo FGF with FGFR, possibly by the formatin of a ternary complex. © Wiley-Liss, Inc.     

Effects of acidic and basic fibroblast growth factors (aFGF, bFGF) on glial precursor cell proliferation: age dependency and brain region specificity.

Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) are present in high levels in most areas of the embryonic rodent brain. To begin to understand the role of these growth factors in brain development, the effects of aFGF and bFGF on dissociated cell cultures prepared from embryonic and neonatal rat brain were studied. Addition of aFGF and heparin or bFGF alone to serum-free cultures of the dissociated Embryonic Day (E) 14.5 mesencephalon stimulates cell proliferation, as judged by [3H]thymidine autoradiography, leading to a maximal 75-fold increase in the total number of cells. This effect is dose-dependent with half-maximal increases at concentrations of about 5-6 ng/ml of aFGF or bFGF and is inhibited by the FGF antagonist HBGF-1U. The effect of aFGF on cell proliferation in cultures prepared from E14.5 mesencephalon is similar to that in cultures prepared from E14.5 cortex. However, in cultures prepared from E14.5 rhombencephalon or diencephalon, the proliferative effect of aFGF is much reduced. In all brain areas studied, the proliferative effect of aFGF declines with increasing age. Immunocytochemical analysis of E14.5 mesencephalic cultures demonstrated that the aFGF-induced increase in cell number is due to the proliferation of A2B5-immunoreactive (IR) glial precursor cells, but not of neuronal precursors, fibroblasts, or microglial cells. Moreover, differentiated glial fibrillary acidic protein-IR astrocytes and 2',3'-cyclic nucleotide 3'-phosphohydrolase-IR oligodendrocytes were not observed in cultures continuously treated with aFGF or bFGF, but were observed in high numbers after removal of the growth factors. These results suggest (1) that aFGF and bFGF are potent mitogens for glial precursor cells in all embryonic brain regions, (2) that the magnitude of the effects of aFGF depends on embryonic age and brain region, and (3) that both growth factors inhibit the differentiation of astrocyte or oligodendrocyte precursors. These observations made in vitro strongly support the hypothesis that FGF plays a critical role in gliogenesis and the timing of glial differentiation in the brain.     

Isolation of a receptor for acidic and basic fibroblast growth factor from embryonic chick

A receptor for acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) was isolated from 7-day embryonic chick. Chromatography of solubilized membrane proteins on wheat germ agglutininagarose and aFGF-Sepharose yielded three major polypeptides migrating at 150, 70, and 45 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides were eluted from aFGF-Sepharose with either 1.0 M NaCl or 100 micrograms/ml heparin, but were not retained on underivatized Sepharose. Cross-linking of 125I-aFGF or 125I-bFGF to either crude membrane preparations or to purified fractions yielded a 165-kDa complex, suggesting the existence of a 150-kDa FGF receptor after subtraction of approximately 15 kDa for 125I-FGF. Addition of excess aFGF or bFGF competed for binding of either 125I-aFGF or 125I-bFGF to FGF receptor preparations. Purified FGF receptor fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to Immobilon membranes, and incubated with 125I-aFGF or 125I-bFGF in order to identify FGF-binding polypeptides. Bound 125I-aFGF and 125I-bFGF were displaced by aFGF and bFGF, but not epidermal growth factor, consistent with the identification of the 150-kDa polypeptide as a receptor for acidic and basic FGF. Treatment of purified FGF receptor fractions with N-glycanase demonstrated that the 150-kDa polypeptide contained approximately 10 kDa of N-linked oligosaccharide. The apparent molecular mass of the 150-kDa polypeptide was unaffected by treatment with heparitinase, indicating that the 150-kDa polypeptide is not a heparan sulfate proteoglycan. Together, these data suggest that the 150-kDa polypeptide is a FGF receptor that may mediate the biological activities of aFGF and bFGF.