Identification of Residues Important Both for Primary Receptor Binding and Specificity in Fibroblast Growth Factor7 (original) (raw)
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Journal of Biological Chemistry, 1999
The fibroblast growth factor (FGF) family plays a key role in a multitude of physiological and pathological processes. The activities of FGFs are mediated by a family of tyrosine kinase receptors, designated FGFRs. The mechanism by which FGFs induce receptor activation is controversial. Despite their structural similarity, FGFs display distinct receptor binding characteristics and cell type specificity. Previous studies with FGF-2 identified a low affinity receptor binding site that is located within a loop connecting its 9th and 10th -strands. The corresponding residues in the other family members are highly variable, and it was proposed that the variability might confer on FGFs unique receptor binding characteristics. We studied the role of this loop in FGF-7 by both site-directed mutagenesis and loop replacement. Unlike the other members of the FGF family, FGF-7 recognizes only one FGFR isoform and is, therefore, ideal for studies of how the specificity in the FGF-FGFR interaction is conferred at the structural level. Point mutations in the loop of FGF-7 did not change receptor binding affinity but resulted in reduced mitogenic potency and reduced ability to induce receptor-mediated phosphorylation events. These results suggest that the loop of FGF-7 fulfills the role of low affinity binding site required for receptor activation. The observation that it is possible to uncouple FGF-7 receptor binding and biological activity favors a bivalent model for FGFR dimerization, and it may be clinically relevant to the design of FGF-7 antagonists. Reciprocal loop replacement between FGF-7 and FGF-2 had no effect on their known receptor binding affinities nor did it alter their known specificity in eliciting a mitogenic response. In conclusion, these results suggest that, despite the diversity in the loop structure of FGF-2 and FGF-7, the loop has a similar function in both growth factors.
Journal of Biological Chemistry, 1995
Basic fibroblast growth factor (FGF) and keratinocyte growth factor (KGF) are structurally related fibroblast growth factors, yet they exhibit distinct receptor binding specificity. Basic FGF binds with high affinity to FGFR1, FGFR2, and FGFR4, whereas KGF does not interact with these receptors and can only bind an isoform of FGFR2 known as the KGFR. Basic FGF binds KGFR but with lower affinity than KGF. In order to identify domains that confer this specificity, four reciprocal chimeras were generated between the two growth factors and were analyzed for receptor recognition and biological activity. The chimeras are designated BK1 (bFGF 1-54 :KGF 91-194 ), BK2 (bFGF 1-74 :KGF 111-194 ), KB1 (KGF 31-90 :bFGF 55-155 ), and KB2 (KGF 31-110 :bFGF 75-155 ). The two BK chimera similarly interacted with FGFR1 and FGFR4 but differed from each other with respect to KGFR recognition. BK1 displayed a slightly better affinity for KGFR than BK2 and induced a higher level of DNA synthesis in keratinocytes compared with bFGF and BK2. A neutralizing monoclonal antibody directed against bFGF specifically neutralized the biological activity of the BK chimeras. The reciprocal chimeras, KB1 and KB2, exhibited KGF-like receptor binding and activation properties. However, KB2 displayed higher affinity for KGFR and was significantly more potent mitogen than KB1. Altogether, our results suggest that the amino-terminal part of KGF and bFGF plays an important role in determining their receptor binding specificity. In addition, the results point to the contribution of a segment from the middle part of KGF (residues 91-110) for recognition and activation of the KGFR, as the two chimeras containing these residues (BK1 and KB2) displayed an enhanced interaction with the KGFR.
FEBS Letters, 2003
Receptor binding speci¢city is an essential element in regulating the diverse activities of ¢broblast growth factors (FGFs). FGF7 is ideal to study how this speci¢city is conferred at the structural level, as it interacts exclusively with one isoform of the FGF-receptor (FGFR) family, known as FGFR2IIIb. Previous mutational analysis suggested the importance of the L L4/L L5 loop of FGF7 in speci¢c receptor recognition. Here a theoretical model of FGFR2IIIb/FGF7 complex showed that this loop interacts with the FGFR2IIIb unique exon. In addition, the model revealed new residues that either directly interact with the FGFR2IIIb unique exon (Asp63, Leu142) or facilitate this interaction (Arg65). Mutations in these residues reduced both receptor binding a⁄nity and biological activity of FGF7. Altogether, these results provide the basis for understanding how receptor-binding speci¢city of FGF7 is conferred at the structural level. ß
The Journal of biological chemistry, 1990
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 11...
The Journal of biological chemistry, 1993
A cDNA predicted to encode a transmembrane tyrosine kinase receptor with sequence features characteristic of known fibroblast growth factor (FGF) receptors was isolated from an expression library constructed from the human mammary epithelial cell line B5/589. This cDNA, designated cl44, encodes a product of 803 amino acid residues and was readily distinguishable from known FGF receptors. During the course of our studies, Partanen et al. (Partanen, J., Makela, T. P., Eerola, E., Korhonen, J., Hirvonen, H., Claesson, W. L., and Alitalo, K. (1991) EMBO J. 10, 1347-1354) isolated a new FGF receptor, designated FGFR4, from the human leukemia cell line, K562. Its amino acid sequence is identical to that of cl44 with the exception of 1 residue. The 5'-untranslated sequences of the two cDNAs diverged far upstream of the initiation codon. A myoblast line, L6E9, which lacks FGF receptors, was utilized to express high levels of FGFR4. We found, in contrast to Partenen et al., who reported ...
Biochemical and Biophysical Research Communications, 2005
Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells which belongs to the family of fibroblast growth factor receptors (FGFRs). Following ligand binding, KGFR is rapidly autophosphorylated on specific tyrosine residues in the intracellular domain, recruits substrate proteins, and is rapidly internalized by clathrin-mediated endocytosis. The role of different autophosphorylation sites in FGFRs, and in particular the role of the tyrosine 766 in FGFR1, first identified as PLCc binding site, has been extensively studied. We analyzed here the possible role of the tyrosine 769 in KGFR, corresponding to tyrosine 766 in FGFR1, in the regulation of KGFR signal transduction and MAPK activation as well as in the control of the endocytic process of KGFR. A mutant KGFR in which tyrosine 769 was substituted by phenylalanine was generated and transfected in NIH3T3 and HeLa cells. Our results indicate that tyrosine 769 is required for the binding to KGFR and tyrosine phosphorylation of PLCc as well as for the full activation of MAPKs and for cell proliferation through the regulation of FRS2 tyrosine phosphorylation, suggesting that this residue represents a key regulator of KGFR signal transduction. Our data also show that tyrosine 769 is not involved in the regulation of the endocytic process of KGFR.
Keratinocyte Growth Factor Receptor Ligands Target the Receptor to Different Intracellular Pathways
Traffic, 2007
The keratinocyte growth factor receptor (KGFR)/fibroblast growth factor receptor 2b is activated by high-affinityspecific 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 clathrincoated 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.
Journal of Biological Chemistry, 1994
and the 1Max-Planck-Znstitut fur Biochemie, A m Klopferspitz 18a, 0-82152 Martinsried, Germany Fibroblast growth factors (FGF) regulate the growth and differentiation of cells through complex combinatorial signaling pathways. There are nine ligands that interact with a family of four tyrosine kinase FGF receptors (FGFR). Diversity in FGF signaling is determined in part by the affinity of specific ligand-receptor pairs. Alternative splicing in the FGFR ligand binding domain generates additional receptor isoforms with novel ligand affinities. For example, splicing events in the ligand binding domain of FGFR2 dramatically increases ita affinity for keratinocyte growth factor (KGFIFGF-7). W e have identified an alternatively spliced form of the FGFR3 & A , corresponding to known splice variants of FGFRa 1 and 2. W e demonstrate both by binding studies on genetically engineered soluble receptors and by the mitogenic response of growth factor-dependent cell lines that this splice variant of FGFR3 (FGFRS IIIb), by binding only acidic FGF (aFGF/FGF-l), has the most restricted ligand binding properties of any FGFR thus far described. Furthermore, by constructing a chimeric receptor that contains the homologous exon from FGFR2, we demonstrate that this single domain from FGFR2 is sufficient to confer upon FGFR3 the ability to bind KGF/ FGF-7. The uniquely limited repertoire of ligands that interact with this receptor suggests that a novel ligand for FGFR3 11% exists. Fibroblast growth factors (FGFs)' comprise a family of nine structurally related proteins (1-3). FGFs are expressed in specific spatial and temporal patterns and are involved in several physiologic processes including embryonic development, angiogenesis, wound healing, and tumorigenesis (4-6). Three classes of receptors are known to bind members of the FGF family. These include a family of four transmembrane tyrosine kinase receptors (FGFR) (reviewed in Ref. l), a cysteine-rich transmembrane protein (7), and heparin or heparin sulfate proteoglycans (8). Of these, only the FGFRs have been shown to transmit an intracellular signal upon binding FGF. However,
Journal of Biological Chemistry, 2000
Fibroblast growth factors (FGFs) transmit their signals through four transmembrane receptors that are designated FGFR1-4. Alternative splicing in the extracellular region of FGFR1-3 generates receptor variants with different ligand binding affinities. Thus two types of transmembrane receptors (IIIb and IIIc isoforms) have been identified for FGFR2 and FGFR3, and the existence of analogous variants has been postulated for FGFR1 based on its genomic structure. However, only a single full-length transmembrane FGFR1 variant (FGFR1-IIIc) has been identified so far. Here we describe the cloning of a full-length cDNA encoding FGFR1-IIIb from a mouse skin wound cDNA library. This receptor isoform was expressed at the highest levels in a subset of sebaceous glands of the skin and in neurons of the hippocampus and the cerebellum. FGFR1-IIIb was expressed in L6 rat skeletal muscle myoblasts and used in cross-linking and receptor binding studies. FGF-1 was found to bind the receptor with high affinity, whereas FGF-2,-10, and-7 bound with significantly lower affinities. Despite their apparently similar but low affinities, FGF-10 but not FGF-7 induced the activation of p44/42 mitogen-activated protein kinase in FGFR1-IIIb-expressing L6 myoblasts and stimulated mitogenesis in these cells, demonstrating that this new receptor variant is a functional transmembrane receptor for FGF-10.