成纤维细胞生长因子的双受体系统

成纤维细胞生长因子(FGFs)对于细胞代谢的刺激作用是通过双受体系统（dual-receptor system）介导的.该系统包括一个酪氨酸激酶受体家族(FGFRs)及肝素硫酸蛋白多糖(HSPG).目前已知有4种FGFRs基因,其转录过程表现出剪切多样性.FGFs与FGFRs的结合表现出交叉特异性.HSPG可促进FGFs与FGFRs的结合和受体二聚体的形成,并增强FGFs对细胞调控的精度.FGFRs通过激活不同下游信号分子影响细胞有丝分裂、神经细胞轴突生长、胚胎发育等.     

Cellular signaling by fibroblast growth factor receptors

The 22 members of the fibroblast growth factor (FGF) family of growth factors mediate their cellular responses by binding to and activating the different isoforms encoded by the four receptor tyrosine kinases (RTKs) designated FGFR1, FGFR2, FGFR3 and FGFR4. Unlike other growth factors, FGFs act in concert with heparin or heparan sulfate proteoglycan (HSPG) to activate FGFRs and to induce the pleiotropic responses that lead to the variety of cellular responses induced by this large family of growth factors. A variety of human skeletal dysplasias have been linked to specific point mutations in FGFR1, FGFR2 and FGFR3 leading to severe impairment in cranial, digital and skeletal development. Gain of function mutations in FGFRs were also identified in a variety of human cancers such as myeloproliferative syndromes, lymphomas, prostate and breast cancers as well as other malignant diseases. The binding of FGF and HSPG to the extracellular ligand domain of FGFR induces receptor dimerization, activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the receptor molecule. A variety of signaling proteins are phosphorylated in response to FGF stimulation including Shc, phospholipase-Cgamma, STAT1, Gab1 and FRS2alpha leading to stimulation of intracellular signaling pathways that control cell proliferation, cell differentiation, cell migration, cell survival and cell shape. The docking proteins FRS2alpha and FRS2beta are major mediators of the Ras/MAPK and PI-3 kinase/Akt signaling pathways as well as negative feedback mechanisms that fine-tune the signal that is initiated at the cell surface following FGFR stimulation.     

Binding of heparin/heparan sulfate to fibroblast growth factor receptor 4

Fibroblast growth factors (FGFs) are heparin-binding polypeptides that affect the growth, differentiation, and migration of many cell types. FGFs signal by binding and activating cell surface FGF receptors (FGFRs) with intracellular tyrosine kinase domains. The signaling involves ligand-induced receptor dimerization and autophosphorylation, followed by downstream transfer of the signal. The sulfated glycosaminoglycans heparin and heparan sulfate bind both FGFs and FGFRs and enhance FGF signaling by mediating complex formation between the growth factor and receptor components. Whereas the heparin/heparan sulfate structures involved in FGF binding have been studied in some detail, little information has been available on saccharide structures mediating binding to FGFRs. We have performed structural characterization of heparin/heparan sulfate oligosaccharides with affinity toward FGFR4. The binding of heparin oligosaccharides to FGFR4 increased with increasing fragment length, the minimal binding domains being contained within eight monosaccharide units. The FGFR4-binding saccharide domains contained both 2-O-sulfated iduronic acid and 6-O-sulfated N-sulfoglucosamine residues, as shown by experiments with selectively desulfated heparin, compositional disaccharide analysis, and a novel exoenzyme-based sequence analysis of heparan sulfate oligosaccharides. Structurally distinct heparan sulfate octasaccharides differed in binding to FGFR4. Sequence analysis suggested that the affinity of the interaction depended on the number of 6-O-sulfate groups but not on their precise location.     

Specificity for fibroblast growth factors determined by heparan sulfate in a binary complex with the receptor kinase.

A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the FGF receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation and supports the binding of activating FGF to self-associated FGFR. Here we show that in contrast to heparin, cellular heparan sulfate forms a binary complex with FGFR that discriminates between FGF-1 and FGF-2. FGFR type 4 (FGFR4) in liver parenchymal cells binds only FGF-1, whereas FGFR1 binds FGF-1 and FGF-2 equally. Cell-free complexes of heparin and recombinant FGFR4 bound FGF-1 and FGF-2 equally. However, in contrast to FGFR1, when recombinant FGFR4 was expressed back in epithelial cells by transfection, it failed to bind FGF-2 unless heparan sulfate was depressed by chlorate or heparinase treatment. Isolated heparan sulfate proteoglycan (HSPG) from liver cells in cell-free complexes with FGFR4 restored the specificity for FGF-1 and supported the binding of both FGF-1 and FGF-2 when complexed with FGFR1. In contrast, FGF-2 bound equally well to complexes of both FGFR1 and FGFR4 formed with endothelial cell-derived HSPG, but the endothelial HSPG was deficient for the binding of FGF-1 to both FGFR complexes. These data suggest that a heparan sulfate subunit is a cell type- and FGFR-specific determinant of the selectivity of the FGFR signaling complex for FGF. In a physiological context, the heparan sulfate subunit may limit the redundancy among the current 18 FGF polypeptides for the 4 known FGFR.     

Fibroblast growth factor receptor signalling is dictated by specific heparan sulphate saccharides

Signalling by fibroblast growth factors (FGFs) through FGF receptors (FGFRs) depends on the cell-surface polysaccharide heparan sulphate (HS) [1] [2]. HS has an ordered domain structure of highly diverse saccharide motifs that present unique displays of sulphate, carboxyl and hydroxyl groups [3]. These motifs interact with many proteins, particularly growth factors. HS binds both to FGFs [4] [5] [6] and FGFRs [7], and probably activates signalling by facilitating ligand-induced receptor dimerisation [8] [9]. Nevertheless, the extent to which specific HS saccharide sequences play a regulatory role has not been established. By screening a library of structurally diverse HS decasaccharides in bioassays of FGF signalling mediated by three different FGFR isoforms, we found that saccharides showed specificity for both ligands and receptors; some saccharides selectively activated FGF signalling through different FGFR isoforms, others acted as negative regulators. We conclude that HS saccharides play critical roles in dictating the specificity of ligand-receptor interactions in FGFR signalling. Controlled alterations in HS structures [10] would provide a mechanism for regulation of cellular responsiveness to growth factors that bind HS.     

Structural basis for fibroblast growth factor receptor activation

FGF signaling plays a ubiquitous role in human biology as a regulator of embryonic development, homeostasis and regenerative processes. In addition, aberrant FGF signaling leads to diverse human pathologies including skeletal, olfactory, and metabolic disorders as well as cancer. FGFs execute their pleiotropic biological actions by binding, dimerizing and activating cell surface FGF receptors (FGFRs). Proper regulation of FGF-FGFR binding specificity is essential for the regulation of FGF signaling and is achieved through primary sequence variations among the 18 FGFs and seven FGFRs. The severity of human skeletal syndromes arising from mutations that violate FGF-FGFR specificity is a testament to the importance of maintaining precision in FGF-FGFR specificity. The discovery that heparin/heparan sulfate (HS) proteoglycans are required for FGF signaling led to numerous models for FGFR dimerization and heralded one of the most controversial issues in FGF signaling. Recent crystallographic analyses have led to two fundamentally different models for FGFR dimerization. These models differ in both the stoichiometry and minimal length of heparin required for dimerization, the quaternary arrangement of FGF, FGFR and heparin in the dimer, and in the mechanism of 1:1 FGF-FGFR recognition and specificity. In this review, we provide an overview of recent structural and biochemical studies used to differentiate between the two crystallographic models. Interestingly, the structural and biophysical analyses of naturally occurring pathogenic FGFR mutations have provided the most compelling and unbiased evidences for the correct mechanisms for FGF-FGFR dimerization and binding specificity. The structural analyses of different FGF-FGFR complexes have also shed light on the intricate mechanisms determining FGF-FGFR binding specificity and promiscuity and also provide a plausible explanation for the molecular basis of a large number craniosynostosis mutations.     

The cell surface proteoglycan syndecan-1 mediates fibroblast growth factor-2 binding and activity

Binding of fibroblast growth factors (FGFs) to receptor tyrosine kinases (FGFRs) and signaling is facilitated by binding of FGF to heparan sulfate proteoglycans (HSPGs). There are multiple families of HSPGs, including extracellular and cell surface forms. An important and potentially controversial question is whether cell surface forms of HSPGs act as positive or negative regulators of FGF signaling. This study examines the ability of the cell surface HSPG syndecan-1 to regulate FGF binding and signaling. HSPG-deficient Raji lymphoma cells, expressing a transfected syndecan-1 cDNA (Raji S1 cells), were used as HSPG “donor” cells. BaF3 cells, expressing an FGFR1 cDNA (FR1C-11 cells), were used as FGFR “reporter” cells. Using Raji S1 cells preincubated with FGF, it was found that they formed heterotypic aggregates with FR1C-11 cells in the presence of FGF-2, but not FGF-1. In addition, the FR1C-11 cells demonstrated FGF-2, but not FGF-1, dependent survival when cultured on fixed Raji S1 cells. Thus, Raji syndecan-1 (1) differentially regulates the binding and signaling of FGFs 1 and 2 and (2) acts as a positive regulator of FGF-2 signaling. J. Cell. Physiol. 174:310–321, 1998. © 1998 Wiley-Liss, Inc.     

Ligand dependent and independent internalization and nuclear translocation of fibroblast growth factor (FGF) receptor 1

Basic fibroblast growth factor (FGF-2) is one of the prototype members of a rapidly expanding family of polypeptides. FGF-2 acts on cells via a dual-receptor system consisting of high-affinity tyrosine kinase receptors (FGFR) and low-affinity receptors comprised of heparan sulfate proteoglycans. Following ligand binding and subsequent internalization, both FGF-2 and FGFR1 are translocated to the nucleus where they have activities distinct from those expressed at the cell surface. Despite the growing number of growth factors and receptors shown to translocate to the nucleus, little is known about the mechanisms of internalization and translocation and how these processes are regulated. In the studies reported in this paper, we examined the roles of clathrin-dependent and -independent endocytosis in the uptake of FGFR1 and one of its ligands, FGF-2. While the uptake of FGF-2 occurred at least partly by a caveolar-dependent mechanism, that of FGFR1 was independent of both caveolae and coated pits. Surprisingly, neither the uptake of FGF-2 nor FGFR1 required the activity of the receptor tyrosine kinase. In addition, we identified a cell cycle-dependent pathway of FGFR1 nuclear translocation that appears to be independent of ligand binding.     

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.     

Heparan sulfate is required for interaction and activation of the epithelial cell fibroblast growth factor receptor-2IIIb with stromal-derived fibroblast growth factor-7

Summary Fibroblast growth factor-7 (FGF-7) and a specific splice variant of the FGF tyrosine kinase receptor family (FGFR2IIIb) constitute a paracrine signaling system from stroma to epithelium. Different effects of the manipulation of cellular heparan sulfates and heparin on activities of FGF-7 relative to FGF-1 in epithelial cells suggest that pericellular heparan sulfates may regulate the activity of FGF-7 by a different mechanism than other FGFs. In this report, we employ the heparan sulfate-binding protein, protamine sulfate, to reversibly block cellular heparan sulfates. Protamine sulfate, which does not bind significantly to FGF-7 or FGFR2IIIb, inhibited FGF-7 activities, but not those of epidermal growth factor. The inhibition was overcome by increasing the concentrations of FGF-7 or heparin. Heparin was essential for binding of FGF-7 to recombinant FGFR2IIIb expressed in insect cells or FGFR2IIIb purified away from cell products. These results suggest that, similar to other FGF polypeptides, heparan sulfate within the pericellular matrix is required for activity of FGF-7. Differences in response to heparin and alterations in the BULK heparan sulfate content of cells likely reflect FGF-specific differences in the cellular repertoire of multivalent heparan sulfate chains required for assembly and activation of the FGF signal transduction complex.     

Potential mechanisms for the regulation of growth factor binding by heparin

Heparin and heparan sulfate proteoglycans (HSPG) bind many soluble growth factors and this binding is now recognized as an important mechanism for modulation of cell activity. Fibroblast growth factor-2 (FGF-2) is one of the best characterized of the heparin-binding growth factors and it has been shown experimentally that heparin regulation of FGF-2 activity is dependent on the level of cell HSPG and the concentration of heparin. In this paper, we explore, using mathematical modeling, proposed mechanisms for heparin regulation and determine how they impact FGF receptor binding. We demonstrate that the experimentally observed receptor binding phenomena can be reproduced if cells (1) express heparin-binding cell surface molecules and if either (2) these heparin binding sites are FGFR and bind heparin and FGF-2-heparin complexes or (3) are surface molecules able to bind FGF-2 and couple with FGF-2 receptors to form high-affinity FGF-2-bound surface complexes. The ability of heparin to directly interact with the FGFR and bind FGF-2 in the absence of this coupling function was not sufficient to explain heparin activity. These findings have implications with regard to regulation of heparin-binding growth factors and could help guide the development of highly specific growth regulatory molecules through specific regulation by heparin and HSPG.     

Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family

In mammals, fibroblast growth factors (FGFs) are encoded by 22 genes. FGFs bind and activate alternatively spliced forms of four tyrosine kinase FGF receptors (FGFRs 1-4). The spatial and temporal expression patterns of FGFs and FGFRs and the ability of specific ligand-receptor pairs to actively signal are important factors regulating FGF activity in a variety of biological processes. FGF signaling activity is regulated by the binding specificity of ligands and receptors and is modulated by extrinsic cofactors such as heparan sulfate proteoglycans. In previous studies, we have engineered BaF3 cell lines to express the seven principal FGFRs and used these cell lines to determine the receptor binding specificity of FGFs 1-9 by using relative mitogenic activity as the readout. Here we have extended these semiquantitative studies to assess the receptor binding specificity of the remaining FGFs 10-23. This study completes the mitogenesis-based comparison of receptor specificity of the entire FGF family under standard conditions and should help in interpreting and predicting in vivo biological activity.     

Similarities and differences between the effects of heparin and glypican-1 on the bioactivity of acidic fibroblast growth factor and the keratinocyte growth factor

The keratinocyte growth factor (KGF or FGF-7) is unique among its family members both in its target cell specificity and its inhibition by the addition of heparin and the native heparan-sulfate proteoglycan (HSPG), glypican-1 in cells expressing endogenous HSPGs. FGF-1, which binds the FGF-7 receptor with a similar affinity as FGF-7, is stimulated by both molecules. In the present study, we investigated the modulation of FGF-7 activities by heparin and glypican-1 in HS-free background utilizing either HS-deficient cells expressing the FGF-7 receptor (designated BaF/KGFR cells) or soluble extracellular domain of the receptor. At physiological concentrations of FGF-7, heparin was required for high affinity receptor binding and for signaling in BaF/KGFR cells. In contrast, binding of FGF-7 to the soluble form of the receptor did not require heparin. However, high concentrations of heparin inhibited the binding of FGF-7 to both the cell surface and the soluble receptor, similar to the reported effect of heparin in cells expressing endogenous HSPGs. The difference in heparin dependence for high affinity interaction between the cell surface and soluble receptor may be due to other molecule(s) present on cell surfaces. Glypican-1 differed from heparin in that it stimulated FGF-1 but not FGF-7 activities in BaF/KGFR cells. Glypican-1 abrogated the stimulatory effect of heparin, and heparin reversed the inhibitory effect of glypican-1, indicating that this HSPG inhibits FGF-7 activities by acting, most likely, as a competitive inhibitor of stimulatory HSPG species for FGF-7. The regulatory effect of glypican-1 is mediated at the level of interaction with the growth factor as glypican-1 did not bind the KGFR. The effect of heparin and glypican-1 on FGF-1 and FGF-7 oligomerization was studied employing high and physiological concentrations of growth factors. We did not find a correlation between the effects of these glycosaminoglycans on FGFs biological activity and oligomerization. Altogether, our findings argue against the heparin-linked dimer presentation model as key in FGFR activation, and support the notion that HSPGs primarily affect high affinity interaction of FGFs with their receptors.     

Identification of residues important both for primary receptor binding and specificity in fibroblast growth factor-7

Fibroblast growth factors (FGFs) mediate a multitude of physiological and pathological processes by activating a family of tyrosine kinase receptors (FGFRs). Each FGFR binds to a unique subset of FGFs and ligand binding specificity is essential in regulating FGF activity. FGF-7 recognizes one FGFR isoform known as the FGFR2 IIIb isoform or keratinocyte growth factor receptor (KGFR), whereas FGF-2 binds well to FGFR1, FGFR2, and FGFR4 but interacts poorly with KGFR. Previously, mutations in FGF-2 identified a set of residues that are important for high affinity receptor binding, known as the primary receptor-binding site. FGF-7 contains this primary site as well as a region that restricts interaction with FGFR1. The sequences that confer on FGF-7 its specific binding to KGFR have not been identified. By utilizing domain swapping and site-directed mutagenesis we have found that the loop connecting the beta4-beta5 strands of FGF-7 contributes to high affinity receptor binding and is critical for KGFR recognition. Replacement of this loop with the homologous loop from FGF-2 dramatically reduced both the affinity of FGF-7 for KGFR and its biological potency but did not result in the ability to bind FGFR1. Point mutations in residues comprising this loop of FGF-7 reduced both binding affinity and biological potency. The reciprocal loop replacement mutant (FGF2-L4/7) retained FGF-2 like affinity for FGFR1 and for KGFR. Our results show that topologically similar regions in these two FGFs have different roles in regulating receptor binding specificity and suggest that specificity may require the concerted action of distinct regions of an FGF.     

Quantitative assessment of FGF regulation by cell surface heparan sulfates

Heparin/heparan sulfate-like glycosaminoglycans (HSGAGs) modulate the activity of the fibroblast growth factor (FGF) family of proteins. Through interactions with both FGFs and FGF receptors (FGFRs), HSGAGs mediate FGF-FGFR binding and oligomerization leading to FGFR phosphorylation and initiation of intracellular signaling cascades. We describe a methodology to examine the impact of heparan sulfate fine structure and source on FGF-mediated signaling. Mitogenic assays using BaF3 cells transfected with specific FGFR isoforms allow for the quantification of FGF1 and FGF2 induced responses independent of conflicting influences. As such, this system enables a systematic investigation into the role of cell surface HSGAGs on FGF signaling. We demonstrate this approach using cell surface-derived HSGAGs and find that distinct HSGAGs elicit differential FGF response patterns through FGFR1c and FGFR3c. We conclude that this assay system can be used to probe the ability of distinct HSGAG species to regulate the activity of specific FGF-FGFR pairs.     

Not all perlecans are created equal: interactions with fibroblast growth factor (FGF) 2 and FGF receptors

Human basement membrane heparan sulfate proteoglycan (HSPG) perlecan binds and activates fibroblast growth factor (FGF)-2 through its heparan sulfate (HS) chains. Here we show that perlecans immunopurified from three cellular sources possess different HS structures and subsequently different FGF-2 binding and activating capabilities. Perlecan isolated from human umbilical arterial endothelial cells (HUAEC) and a continuous endothelial cell line (C11 STH) bound similar amounts of FGF-2 either alone or complexed with FGFRalpha1-IIIc or FGFR3alpha-IIIc. Both perlecans stimulated the growth of BaF3 cell lines expressing FGFR1b/c; however, only HUAEC perlecan stimulated those cells expressing FGFR3c, suggesting that the source of perlecan confers FGF and FGFR binding specificity. Despite these differences in FGF-2 activation, the level of 2-O- and 6-O-sulfation was similar for both perlecans. Interestingly, perlecan isolated from a colon carcinoma cell line that was capable of binding FGF-2 was incapable of activating any BaF3 cell line unless the HS was removed from the protein core. The HS chains also exhibited greater bioactivity after digestion with heparinase III. Collectively, these data clearly demonstrate that the bioactivity of HS decorating a single PG is dependent on its cell source and that subtle changes in structure including secondary interactions have a profound effect on biological activity.     

Heparin differentially regulates the interaction of fibroblast growth factor-4 with FGF receptors 1 and 2.

Fibroblast growth factor-4 (FGF4), like other FGFs, shares a high affinity for the anionic glycosaminoglycans heparin and heparan sulfate (HS), which in turn enhance FGF-receptor (FGFR) binding and activation. Here we demonstrate using a cell free system that, at low concentrations of heparin, FGF4 binds only to FGFR-2, while much higher heparin levels are required for binding to FGFR-1. Chemical crosslinking of radiolabeled FGF4 to the soluble FGF receptors confirms the preferential formation of FGF4-FGFR-2 complexes under restricted heparin availability, with maximal ligand-receptor interactions at almost 20-fold lower heparin concentrations then those required for the affinity labeling of FGFR-1. In accordance, HS-deficient cells expressing FGFR-2 proliferate in response to FGF4 at extremely low exogenous heparin concentrations, while FGFR-1 expressing cells are completely unresponsive under the same conditions. We suggest that FGFR-2 is the preferred receptor for FGF4 under restricted HS conditions and that the bioavailability of structurally distinct HS motifs may differentially control receptor specificity of FGF4 in vivo.     

Repression of myogenic differentiation by aFGF, bFGF, and K-FGF is dependent on cellular heparan sulfate

We have proposed a model in which fibroblast growth factor (FGF) signalling requires the interaction of FGF with at least two FGF receptors, a heparan sulfate proteoglycan (HSPG) and a tyrosine kinase. Since FGF may be a key mediator of skeletal muscle differentiation, we examined the synthesis of glycosaminoglycans in MM14 skeletal muscle myoblasts and their participation in FGF signalling. Proliferating and differentiated MM14 cells exhibit similar levels of HSPG, while differentiated cells exhibit reduced levels of chondroitin sulfate proteoglycans and heparan sulfate chains. HSPGs, including syndecan, present in proliferating cells bind bFGF, while the majority of chondroitin sulfate and heparan sulfate chains do not. Treatment of skeletal muscle cells with chlorate, a reversible inhibitor of glycosaminoglycan sulfation, was used to examine the requirement of sulfated proteoglycans for FGF signalling. Chlorate treatment reduced glycosaminoglycan sulfation by 90% and binding of FGF to high affinity sites by 80%. Chlorate treatment of MM14 myoblasts abrogated the biological activity of acidic, basic, and Kaposi's sarcoma FGFs resulting in terminal differentiation. Chlorate inhibition of FGF signalling was reversed by the simultaneous addition of sodium sulfate or heparin. Further support for a direct role of heparan sulfate proteoglycans in fibroblast growth factor signal transduction was demonstrated by the ability of heparitinase to inhibit basic FGF binding and biological activity. These results suggest that activation of FGF receptors by acidic, basic or Kaposi's sarcoma FGF requires simultaneous binding to a HSPG and the tyrosine kinase receptor. Skeletal muscle differentiation in vivo may be dependent on FGFs, FGF tyrosine kinase receptors, and HSPGs. The regulation of these molecules may then be expected to have important implications for skeletal muscle development and regeneration.     

Mapping ligand binding domains in chimeric fibroblast growth factor receptor molecules. Multiple regions determine ligand binding specificity

Fibroblast growth factors (FGFs) mediate essential cellular functions by activating one of four alternatively spliced FGF receptors (FGFRs). To determine the mechanism regulating ligand binding affinity and specificity, soluble FGFR1 and FGFR3 binding domains were compared for activity. FGFR1 bound well to FGF2 but poorly to FGF8 and FGF9. In contrast, FGFR3 bound well to FGF8 and FGF9 but poorly to FGF2. The differential ligand binding specificity of these two receptors was exploited to map specific ligand binding regions in mutant and chimeric receptor molecules. Deletion of immunoglobulin-like (Ig) domain I did not effect ligand binding, thus localizing the binding region(s) to the distal two Ig domains. Mapping studies identified two regions that contribute to FGF binding. Additionally, FGF2 binding showed positive cooperativity, suggesting the presence of two binding sites on a single FGFR or two interacting sites on an FGFR dimer. Analysis of FGF8 and FGF9 binding to chimeric receptors showed that a broad region spanning Ig domain II and sequences further N-terminal determines binding specificity for these ligands. These data demonstrate that multiple regions of the FGFR regulate ligand binding specificity and that these regions are distinct with respect to different members of the FGF family.     

Fibroblast growth factor receptor-4 shows novel features in genomic structure, ligand binding and signal transduction.

Fibroblast growth factor (FGF) receptor (FGFR) gene family consists of at least four receptor tyrosine kinases that transduce signals important in a variety of developmental and physiological processes related to cell growth and differentiation. Here we have characterized the binding of different FGFs to FGFR-4. Our results establish an FGF binding profile for FGFR-4 with aFGF having the highest affinity, followed by K-FGF/hst-1 and bFGF. In addition, FGF-6 was found to bind to FGFR-4 in ligand competition experiments. Interestingly, the FGFR-4 gene was found to encode only the prototype receptor in a region where both FGFR-1 and FGFR-2 show alternative splicing leading to differences in their ligand binding specificities and to secreted forms of these receptors. Ligands binding to FGFR-4 induced receptor autophosphorylation and phosphorylation of a set of cellular polypeptides, which differed from those phosphorylated in FGFR-1-expressing cells. Specifically, the FGFR-1-expressing cells showed a considerably more extensive tyrosine phosphorylation of PLC-gamma than the FGFR-4-expressing cells. Structural and functional specificity within the FGFR family exemplified by FGFR-4 may help to explain how FGFs perform their diverse functions.