The complete amino acid sequence of the shorter form of human basic fibroblast growth factor receptor deduced from its cDNA

We have isolated a full-length cDNA for human basic fibroblast growth factor (bFGF) receptor-like protein from a human placenta cDNA library. Determination of the nucleotide sequence of the cDNA allows elucidation of the complete amino acid sequence of the receptor (731 amino acids) which has two extracellular immunoglobulin-like domains, a transmembrane domain and an intracellular tyrosine kinase domain. The receptor has remarkable amino acid similarity (98% identity) to the shorter form of murine bFGF receptor reported recently (H.H.Reid et al. (1990) Proc.Natl.Acad.Sci. USA 87, 1596-1600). The receptor described here is expected to be the shorter form of human bFGF receptor.     

Expression and transforming activity of a variant of the heparin-binding fibroblast growth factor receptor (flg) gene resulting from splicing of the alpha exon at an alternate 3'-acceptor site.

Splicing at an alternate 3'-acceptor site results in deletion of a CCCAG in the 5'-sequence of the exon coding for the NH2-terminal immunoglobulin-like disulfide loop of the heparin-binding fibroblast growth factor receptor (flg) alpha isoform. The result is an in-frame stop codon 138 base pairs after the first flg consensus translational initiation site. The next more favorable site predicts the same two loop intracellular receptor isoform, gamma, which was predicted from two different human cDNAs that arise by alternate use of two exons at the same site. Although expressed in normal tissue, the gamma mRNA is increased in rat prostate tumors and confers ability of anchorage-dependent cells expressing non-secreted heparin-binding fibroblast growth factors to grow in soft agar.     

Cloning, expression and tissue distribution of the gene encoding rat fibroblast growth factor receptor subtype 4

The rat homologue of the gene encoding the fibroblast growth factor receptor subtype 4 (FGFR4) was cloned from rat lung mRNA, and the cDNA sequence was found to be 95% similar and 92% identical to the human homologue. Northern blot analysis of adult rat tissues demonstrated that a 3.1-kb mRNA encoding FGFR4 is detectable only in the lung and kidney. The receptor variant described here encodes two potential immunoglobulin-like domains, 21 hydrophobic amino acids encoding a potential transmembrane domain, and a split tyrosine kinase motif. However, the acidic box and hydrophobic signal peptide domains are not present in this cDNA isolate.     

Expression and immunochemical analysis of rat and human fibroblast growth factor receptor (flg) isoforms.

Potentially 96 splice variants among four genes that code for the human heparin-binding fibroblast growth factor receptor family complicate study of structure, metabolism, and function of single isoforms in mammalian cells. As an alternative, we expressed structural subdomains and isoforms of the flg receptor gene in bacteria and baculoviral-infected insect cells. We developed and characterized a panel of 16 isoform and domain-specific polyclonal and monoclonal antibodies. The panel of antibodies was used to distinguish mature glycosylated ligand-binding and kinase-active and -inactive recombinant isoforms in baculoviral insect cells and transfected mammalian cells and natural isoforms in rat prostate and human liver cells. The results revealed a cell type-specific expression of the flg gene and isoforms that result from combinations of splice variations. Reactive epitopes of monoclonal antibodies against both the three (alpha) and two (beta) immunoglobulin-like disulfide loop extracellular domain isoforms were mapped by cross-reactivity with synthetic polypeptide sequences and deletion mutants expressed in bacteria. The native alpha and beta receptor isoforms differed in display of shared epitopes and suggested that the NH2-terminal Loop I and COOH-terminal Loops II and III of the alpha isoform are interactive. Although the common Loops II and III appear qualitatively sufficient for ligand binding, the results suggest that tertiary relationships among loops in the three and two loop isoforms are distinct and, therefore, the two isoforms may have distinct activities. Spatial models for arrangement of immunoglobulin-like loops in the extracellular domain of the two isoforms are presented.     

A novel fibroblast growth factor receptor-5 preferentially expressed in the pancreas(1)

Using the polymerase chain reaction on human embryonic cDNAs, we isolated a cDNA encoding a novel 504 amino acid protein, termed fibroblast growth factor receptor (FGFR)-5, which is highly homologous to known FGFRs. The NH(2)-terminal portion of FGFR5 contains a putative secretory signal sequence, three typical immunoglobulin-like domains, six cysteines, and an acidic box, but no HAV motif. The COOH-terminal portion of FGFR5 contains one transmembrane domain but no intracellular kinase domain. Recombinant FGFR5 expressed in COS-7 cells is not secreted, but recombinant truncated FGFR5 lacking the predicted transmembrane domain is secreted. Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) do not bind to FGFR5. Among 23 adult human tissues, FGFR5 mRNA is preferentially expressed in the pancreas. These results suggest that FGFR5 may provide a binding site for some other fibroblast growth factors and may regulate some pancreatic function.     

The fibroblast growth factor receptor acid box is essential for interactions with N-cadherin and all of the major isoforms of neural cell adhesion molecule

Interactions between the neural cell adhesion molecules NCAM and N-cadherin with the fibroblast growth factor receptor (FGFR) are important for a number of developmental events and have also been implicated in tumor progression. The factors regulating these interactions are not known. We have used co-immunoprecipitation and co-clustering paradigms to show that both adhesion molecules can interact with the 3Ig IIIC isoform of the FGFR1 in a number of cell types. Interestingly, whereas the interaction can be seen over most of the cell surface, it is not seen at points of cell-cell contact where the adhesion molecules accumulate at stable junctions. We also demonstrate for the first time that all of the major isoforms of NCAM can interact with the FGFR. Using deletion mutagenesis we have found that the adhesion molecule/FGFR interaction can withstand the removal of most of any one of the FGFR immunoglobulin-like domains (D1-D3). In contrast, the FGFR interaction with N-cadherin and NCAM (but not FGF) is absolutely dependant on the presence of the acid box motif that can be found in the linker region between D1 and D2. As this motif can be spliced out of all four FGFRs, it suggests that this is one mechanism that can regulate the interaction of the receptor with different ligand classes.     

The Acidic Domain and First Immunoglobulin-Like Loop of Fibroblast Growth Factor Receptor 2 Modulate Downstream Signaling through Glycosaminoglycan Modification

Fibroblast growth factor receptors (FGFRs) are membrane-spanning tyrosine kinases that have been implicated in a variety of biological processes including mitogenesis, cell migration, development, and differentiation. We identified a unique isoform of FGFR2 expressed as a diffuse band with an unusually large molecular mass. This receptor is modified by glycosaminoglycan at a Ser residue located immediately N terminal to the acidic box, a stretch of acidic amino acids. The acidic box and the glycosaminoglycan modification site are encoded by an alternative exon of the FGFR2 gene. The acidic box appears to play an important role in glycosaminoglycan modification, and the presence of this domain is required for modification by heparan sulfate glycosaminoglycan. Moreover, the presence of the first immunoglobulin-like domain encoded by another alternative exon abrogated the modification. The high-affinity receptor with heparan sulfate modification enhanced receptor autophosphorylation, substrate phosphorylation, and ternary complex factor-independent gene expression. It also sustained mitogen-activated protein kinase activity and increased eventual DNA synthesis, a long-term response to fibroblast growth factor stimulation, at physiological ligand concentrations. We propose a novel regulation mechanism of FGFR2 signal transduction through glycosaminoglycan modification.     

The fifth immunoglobulin-like domain of the Kit receptor is required for proteolytic cleavage from the cell surface

Stem cell factor (SCF) initiates its biological effects by binding to its receptor Kit. Cell surface Kit is proteolytically cleaved to generate soluble Kit. Structure-function analysis of the extracellular region of Kit has implicated the first three immunoglobulin-like domains in SCF binding, and the fourth immunoglobulin-like domain in receptor dimerization. However, the role of the fifth immunoglobulin-like domain is unknown. To test the hypothesis that the fifth immunoglobulin-like domain is important for proteolytic cleavage of Kit from the cell surface, we constructed a mutant form of Kit in which the first four immunoglobulin-like domains are linked to the transmembrane and cytoplasmic domains (designated Kit-Del5). Kit-wild type (Kit-WT) and Kit-Del5 were expressed in the murine mast cell line IC2. Flow cytometry demonstrated that both Kit-WT and Kit-Del5 are displayed on the IC2 cell surface, and immunoblotting confirmed the presence of Kit proteins of the expected molecular weights, 154 kDa and 134 kDa, respectively. Although IC2-Kit-WT cells proteolytically cleave cell surface Kit, generating a 98 kDa soluble form of Kit, IC2-Kit-Del5 cells do not. These findings demonstrate that the fifth immunoglobulin-like domain of Kit is required for proteolytic cleavage of Kit from the cell surface.     

A novel form of fibroblast growth factor receptor 2. Alternative splicing of the third immunoglobulin-like domain confers ligand binding specificity.

The fibroblast growth factor receptor 2 (FGFR2) gene is expressed as alternatively spliced mRNAs that encode bacterially expressed kinase, the keratinocyte growth factor receptor, or K-sam. We have now isolated a novel FGFR2 cDNA that is identical with the previously cloned human bacterially expressed kinase, except in the third immunoglobulin-like domain. The ligand binding properties of FGFR2 were studied by expressing the protein in rat L6 muscle myoblasts. Unlike human bacterially expressed kinase which binds acidic and basic FGF with similar affinities, FGFR2 bound acidic FGF with approximately 1000-fold higher affinity than basic FGF. These results indicate that alternative splicing of the FGFR2 gene in the region encoding the carboxyl-terminal half of the third immunoglobulin domain determines the ligand specificity of this group of receptors.     

Identification of the ligand-binding regions in the macrophage colony-stimulating factor receptor extracellular domain.

The c-fms gene encodes the receptor for the macrophage colony-stimulating factor (M-CSF), and its extracellular domain consists of five immunoglobulin-like subdomains. To identify which of the five immunoglobulin-like regions are involved in ligand binding, we polymerase chain reaction-cloned five segments of the extracellular domain of the murine c-fms gene, each starting with the normal initiation codon and containing successive additions of the immunoglobulin-like subdomains. These protein segments are designated A, B, C, D, and E and contain, from the N-terminal end, either one, two, three, four, or all five immunoglobulin-like subdomains, respectively. Each segment was expressed as a secreted soluble protein from a baculovirus expression vector in Sf9 insect cells. In addition, segments A, B, C, and E were produced as soluble alkaline phosphatase fusion proteins, as was a segment containing only the fourth and fifth immunoglobulin domains. These segments of the Fms extracellular domain were used to assess M-CSF binding by competition radioimmunoassays, plate binding immunoassays, and immunoprecipitation analyses. The results indicated that the first two N-terminal immunoglobulin-like domains did not interact with M-CSF but, in combination with the third immunoglobulin-like domain, provided high-affinity M-CSF binding. The fourth and fifth immunoglobulin-like domains near the cell membrane did not exhibit M-CSF binding and may inhibit interaction of M-CSF with the first three immunoglobulin domains. These results suggest that the three N-terminal immunoglobulin-like domains constitute the high-affinity M-CSF binding region and that the fourth and fifth immunoglobulin-like domains may perform functions other than ligand binding.     

Diverse forms of a receptor for acidic and basic fibroblast growth factors.

We recently reported the isolation of a chicken cDNA clone encoding a basic fibroblast growth factor (FGF) receptor that has three immunoglobulinlike domains in the extracellular region. We have now identified four unique human cDNA clones encoding previously unknown FGF receptor variants which contain only two immunoglobulinlike domains. Two of the human clones encode membrane-spanning receptors, and two encode putative secreted forms. Both the three- and two-immunoglobulinlike-domain forms mediate biological responsiveness to acidic and basic FGF. Thus, the first immunoglobulinlike domain of the three-domain form may have a function other than binding of acidic and basic FGF.     

Expression and purification of recombinant human fibroblast growth factor receptor in Escherichia coli

Human fibroblast growth factor receptor (FGFR) is responsible for multifunctional signaling that regulates developmental processes. The three immunoglobulin-like extracellular domains of FGFR (D1, D2, and D3) include the determinants of ligand binding and specificity for fibroblast growth factor and heparan sulfate. D1 and the D1-D2 linker with a contiguous stretch of acidic amino acids are known to be involved in auto-inhibitory regulation. In an effort to gain a better understanding of the role of D1 and the linker in FGFR regulation, we have subcloned, overexpressed, and purified the extracellular fragments, D1-D2 and D1-D3, of FGFR1 in Escherichia coli. The recombinant proteins were produced in an insoluble form and were renatured using a dropwise or on-column refolding method. In addition, D2-D3 was coexpressed with chaperones to test the possibility that the presence of chaperones might enhance refolding efficiencies. A combination of immobilized nickel and heparin affinity chromatography and size-exclusion chromatography resulted in the purification of recombinant ectodomain proteins D1-D2 and D1-D3 of high purity for structural studies.     

The mutations in FGFR2-associated craniosynostoses are clustered in five structural elements of immunoglobulin-like domain III of the receptor

Exons 5 and 7 of the fibroblast growth factor receptor 2 (FGFR2) gene code for immunoglobulin-like domain III (IgIII) and for the region connecting the second and the third Ig domain of the receptor. Numerous mutations in these two exons have been shown to cause various craniosynostotic syndromes. Here, we describe three previously unrecognized mutations at amino acid positions 276, 301, and 314, in one nonspecific craniosynostosis and in two Crouzon patients. We also present a polypeptide model of IgIII of FGFR2. The known mutations involve five distinct structural elements of the receptor. The changes within these elements affect receptor function by various mechanisms, including altered dimerization, truncation, increased mobility between Ig domains, disintegration of IgIII, and alteration of the ligand-binding site. Received: 30 June 1997 / Accepted: 31 October 1997     

A novel type I fibroblast growth factor receptor activates mitogenic signaling in the absence of detectable tyrosine phosphorylation of FRS2

A novel variant of the fibroblast growth factor receptor type 1 (FGFR-1) was identified in human placental RNA. In this receptor (FGFR-1L) portions of the second and third immunoglobulin-like (Ig-like) domains are deleted. To determine whether FGFR-1L was functional, full-length variant (pSV/FGFR-1L) and wild-type (pSV/FGFR-1) receptors were stably transfected into rat L6 myoblasts cells. Transfected L6 clones expressed respective proteins and bound (125)I-labeled FGF-2 with K(d) values of 99 pm (FGFR-1) and 26 pm (FGFR-1L). FGF-1 and FGF-2 competed efficiently with (125)I-FGF-2 for binding to FGFR-1 and FGFR-1L, whereas FGF-4 was less efficient. FGF-1, FGF-2, and FGF-4 enhanced mitogen-activated protein kinase (MAPK) activity, increased steady-state c-fos mRNA levels, and stimulated proliferation through either receptor, whereas KGF was without effect. FGFR-1 expressing clones exhibited ligand-induced tyrosine phosphorylation of fibroblast growth factor receptor substrate 2 (FRS2), a 90-kDa adaptor protein that links FGFR-1 activation to the MAPK cascade. In contrast, tyrosine phosphorylation of FRS2 was not evident with FGFR-1L. In addition, phospholipase C-gamma was not tyrosine phosphorylated via activated FGFR-1L. These findings indicate that FGFR-1L binds FGF-1 and FGF-2 with high affinity and is capable of mitogenic signaling, but may activate MAPK to occur via non-classical signaling intermediates.     

FGFR-4, a novel acidic fibroblast growth factor receptor with a distinct expression pattern.

We have previously identified two novel members of the fibroblast growth factor receptor (FGFR) gene family expressed in K562 erythroleukemia cells. Here we report cDNA cloning and analysis of one of these genes, named FGFR-4. The deduced amino acid sequence of FGFR-4 is 55% identical with both previously characterized FGFRs, flg and bek, and has the structural characteristics of a FGFR family member including three immunoglobulin-like domains in its extracellular part. Antibodies raised against the carboxy terminus of FGFR-4 detected 95 and 110 kd glycoproteins with a protein backbone of 88 kd in COS cells transfected with a FGFR-4 cDNA expression vector. The FGFR-4 protein expressed in COS cells could also be affinity-labeled with radioiodinated acidic FGF. Furthermore, ligand binding experiments demonstrated that FGFR-4 binds acidic FGF with high affinity but does not bind basic FGF. FGFR-4 is expressed as a 3.0 kb mRNA in the adrenal, lung, kidney, liver, pancreas, intestine, striated muscle and spleen tissues of human fetuses. The expression pattern of FGFR-4 is distinct from that of flg and bek and the yet additional member of the same gene family, FGFR-3, which we have also cloned from the K562 leukemia cells. Our results suggest that FGFR-4 along with other fibroblast growth factor receptors performs cell lineage and tissue-specific functions.     

Structure of a VEGF-VEGF receptor complex determined by electron microscopy

Receptor tyrosine kinases are activated upon ligand-induced dimerization. Here we show that the monomeric extracellular domain of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) has a flexible structure. Binding of VEGF to membrane-distal immunoglobulin-like domains causes receptor dimerization and promotes further interaction between receptor monomers through the membrane-proximal immunoglobulin-like domain 7. By this mechanism, ligand-induced dimerization of VEGFR-2 can be communicated across the membrane, activating the intracellular tyrosine kinase domains.     

miR-301a-3p induced by endoplasmic reticulum stress mediates the occurrence and transmission of trastuzumab resistance in HER2-positive gastric cancer

Trastuzumab resistance negatively influences the clinical efficacy of the therapy for human epidermal growth factor receptor 2 (HER2) positive gastric cancer (GC), and the underlying mechanisms remain elusive. Exploring the mechanisms and finding effective approaches to address trastuzumab resistance are of great necessity. Here, we confirmed that endoplasmic reticulum (ER) stress-induced trastuzumab resistance by up-regulating miR-301a-3p in HER2-positive GC cells. Moreover, we elucidated that miR-301a-3p mediated trastuzumab resistance by down-regulating the expression of leucine-rich repeats and immunoglobulin-like domains containing protein 1 (LRIG1) and subsequently activating the expression of insulin-like growth factor 1 receptor (IGF-1R) and fibroblast growth factor receptor 1 (FGFR1) under ER stress. We also found that intercellular transfer of miR-301a-3p by exosomes disseminated trastuzumab resistance. The present study demonstrated that exosomal miR-301a-3p could serve as a non-invasive biomarker for trastuzumab resistance, which was maybe a novel potential therapeutic target to overcome trastuzumab resistance and improve the curative effect of trastuzumab in HER2-positive GC patients.Subject terms: Cancer therapeutic resistance, Gastric cancer     

Assignment by in situ hybridization of a fibroblast growth factor receptor gene to human chromosome band 10q26

Summary A 2.3-kb cDNA probe for the human bek fibroblast growth factor receptor was used to determine the chromosomal localization of the corresponding gene by in situ hybridization. The results show that this gene, a form of which is amplified in some poorly differentiated stomach cancers, is localized on chromosome region 10q26. The two previously identified fibroblast growth factor receptor genes are thus not on the same chromosome, as the related fig (fms-like gene) fibrovblast growth factor receptor gene has previously been mapped to human chromosome region 8p12.     

1H, 13C and 15N assignments of the four N-terminal domains of human fibrillin-1

Fibrillins are extracellular, disulphide-rich glycoproteins that form 10–12 nm diameter microfibrils in connective tissues. They are found in the majority of higher animals, from jellyfish to humans. Fibrillin microfibrils confer properties of elasticity and strength on connective tissue and regulate growth factor availability in the extracellular matrix (ECM). Mutations in FBN1, the human gene encoding the fibrillin-1 isoform, are linked to several inherited connective tissue disorders. The fibrillin-1 N-terminus forms many functionally-important interactions, both with other fibrillin molecules and various ECM components. In particular, the first four domains, the fibrillin unique N-terminal (FUN) and three epidermal growth factor (EGF)-like domains (FUN-EGF3), are implicated in microfibril assembly and growth factor sequestration. The structure of these domains, which comprise 134 residues, is unknown. We have produced a recombinant fragment corresponding to this region of human fibrillin-1. Here, we report ¹H, ¹³C and ¹⁵N resonance assignments of the FUN-EGF3 fragment. Assignments will facilitate structure determination, analysis of interdomain dynamics and the mapping of interaction surfaces.     

Origin and molecular evolution of receptor tyrosine kinases with immunoglobulin-like domains

Receptor tyrosine kinases (RTKs) are involved in the control of fundamental cellular processes in metazoans. In vertebrates, RTK could be grouped in distinct classes based on the nature of their cognate ligand and modular composition of their extracellular domain. RTK with immunoglobulin-like domains (IG-like RTK) encompass several RTK classes and have been found in early metazoans, including sponges. Evolution of IG-like RTK is characterized by extended molecular and functional diversification, which prompted us to study their evolutionary history. For that purpose, a nonredundant data set including annotated protein sequences of IG-like RTK (n = 85) was built, representing 19 species ranging from sponges to humans. Phylogenetic trees were generated from alignment of conserved regions using maximum likelihood approach. Molecular phylogeny strongly suggests that IG-like RTK diversification occurred according to a complex scenario. In particular, we propose that specific cis duplications of a common ancestor to both platelet-derived growth factor receptor (class III) and vascular endothelial growth factor receptor (class V) families preceded two trans duplications. In contrast, other IG-like RTK genes, like Musk and PTK7, apparently did not evolve by duplications, whereas fibroblast growth factor receptors (class IV) evolved through two rounds of trans duplications. The proposed model of IG-like RTK evolution is supported by high bootstrap values and by the clustering of genes encoding class III and class V RTKs at specific chromosomal locations in mouse and human genomes.