The expression of two isoforms of the human fibroblast growth factor receptor (flg) is directed by alternative splicing.

Structural analysis of the cDNA for the human fibroblast growth factor receptor (flg) revealed the existence of a larger and a shorter isoform of the receptor. The larger form has three extracellular immunoglobulin-like domains. On the other hand, the shorter form deletes the first (the most external) immunoglobulin-like domain region. Two consecutive amino acids (Arg Met) between the first and second immunoglobulin-like domains are sometimes deleted from the shorter form. In this paper, we isolated and analyzed the gene for the human fibroblast growth factor receptor. Organization of the gene revealed that the isoforms are produced by two different types of alternative splicing (the cassette and internal donor types) from the common gene. In human placenta, the shorter form is expressed as the major isoform.     

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.     

Isolation from mouse fibroblasts of a cDNA encoding a new form of the fibroblast growth factor receptor (flg).

Structural definition of the receptors for neurotropic and angiogenic modulators such as fibroblast growth factors and related polypeptides will yield insight into the mechanisms that control early development, embryogenesis, organogenesis, wound repair and neovessel formation. We isolated 3 murine cDNAs encoding different binding domains of these receptors (flg). Comparison of these ectoplasmic portions showed that two of the forms corresponded to previously described murine molecules whereas the third one had a different ectoplasmic portion generated by specific changes in two regions. Interestingly, expression of this third form seems to be restricted in its tissue distribution. Such modifications could influence the ligand specificity of the different receptors and/or their binding affinity.     

Expression of basic fibroblast growth factor in human gastric carcinomas.

The expression of mRNA for the basic fibroblast growth factor (FGF) gene was examined in seven human gastric carcinoma cell lines and in tissue from 29 gastric carcinomas together with the adjacent normal mucosa. Among the seven gastric carcinoma cell lines, the MKN45 cell line expressed mRNA for the basic FGF gene. Basic FGF protein production was confirmed by flow cytometric analysis and immunohistochemistry. Among the surgical specimens, 16 (55%) of 29 gastric carcinomas showed higher levels of basic FGF mRNA than the normal mucosa. Interestingly, in scirrhous gastric carcinomas characterized by their fibrous stroma and rapid growth, 9 (69%) of 13, samples examined revealed higher levels of basic FGF mRNA than normal mucosa, whereas only 3 (33%) of the 9 well differentiated adenocarcinomas studied produced similar results. Immunohistochemically, basic FGF protein was localized in tumor cells. These results suggest that basic FGF produced by tumor cells may play an important role in producing fibrosis and angiogenesis in gastric carcinomas.     

Expression of fibroblast growth factor receptor 3 by fibroblast growth factor 2 in cultured chick embryo chondrocytes

Although fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 3 (FGFR3) both inhibit longitudinal bone growth, little is known about the relationship between FGF2 and FGFR3. Accordingly, the current study examined the expression of FGFR3 mRNA after the administration of FGF2 using cultured chondrocytes from day 17 chick embryos to evaluate the relationship between FGF2 and FGFR3. The chondrocytes were isolated from the caudal one-third portion (LS) of sterna, peripheral regions (USP) and central core regions (USC) of the cephalic portion of the sterna, and lower portion of the proximal tibial growth plate (Ti) of day 17 chick embryo. The expression of FGFR1, FGFR3, and type II and X collagen mRNA in the chondrocytes from the LS, USP, USC, and Ti was determined. FGFR1 was not expressed in the LS and USP chondrocytes, yet strongly expressed in the USC and Ti chondrocytes. With a treatment of FGF2, the expression of FGFR1 slightly increased in the USC chondrocytes and was not related with the concentration of FGF2 in the Ti chondrocytes. FGFR3 was expressed in all the chondrocyte types, yet strongly increased in the LS, USC, USP, and Ti in that order according to the concentration of FGF2. For the LS and USP chondrocytes, the expression of FGFR3 with FGF2 increased in a 4-day culture, yet decreased in a 6-day culture, whereas for the USC chondrocytes, the expression of FGFR3 mRNA with FGF2 increased in a 2-day culture, yet decreased in a 4-day culture, suggesting that the hypertrophic chondrocytes were more numerous and sensitive compared to the proliferative chondrocytes. For all the chondrocyte types, FGF2 appeared to be up-regulated to FGFR3, as the expression of FGFR3 mRNA increased with a higher concentration of FGF2 until a peak level. In conclusion, FGF2 was found to up-regulate to FGFR3 until the peak level of FGFR3 mRNA expression, while in hypertrophic chondrocytes, FGFR3 appeared to cause the differentiaton of chondrocytes, resulting in the inhibition of longitudinal bone growth after the peak level of FGFR3 mRNA expression.     

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.     

Expression of basic fibroblast growth factor in human gastric carcinomas

The expression of mRNA for the basic fibroblast growth factor (FGF) gene was examined in seven human gastric carcinoma cell lines and in tissue from 29 gastric carcinomas together with the adjacent normal mucosa. Among the seven gastric carcinoma cell lines, the MKN45 cell line expressed mRNA for the basic FGF gene. Basic FGF protein production was confirmed by flow cytometric analysis and immunohistochemistry. Among the surgical specimens, 16 (55%) of 29 gastric carcinomas showed higher levels of basic FGF mRNA than the normal mucosa. Interestingly, in scirr-hous gastric carcinomas characterized by their fibrous stroma and rapid growth, 9 (69%) of 13, samples examined revealed higher levels of basic FGF mRNA than normal mucosa, whereas only 3 (33%) of the 9 well differentiated adenocarcinomas studied produced similar results. Immunohistochemically, basic FGF protein was localized in tumor cells. These results suggest that basic FGF produced by tumor cells may play an important role in producing fibrosis and angiogenesis in gastric carcinomas.     

Expression of cDNA encoding human basic fibroblast growth factor in E. coli

The cDNA encoding human basic fibroblast growth factor was expressed in E. coli under the control of trp promoter. Bacterially synthesized hbFGF was highly purified using a heparin affinity HPLC column. By this chromatography, hbFGF was eluted as four distinct forms, which were indistinguishable by SDS polyacrylamide gel electrophoresis, amino acid composition, and partial terminal sequence analysis. These molecules stimulated the growth of fibroblasts and endothelial cells although their specific activities varied. The angiogenesis activity of these molecules was also confirmed.     

Genomic organization of the human fibroblast growth factor receptor 2 (FGFR2) gene and comparative analysis of the human FGFR gene family

The human fibroblast growth factor receptor (FGFR) genes play important roles in normal vertebrate development. Mutations in the human FGFR2 gene have been associated with many craniosynostotic syndromes and malformations, including Crouzon, Pfeiffer, Apert, Jackson-Weiss, Beare-Stevenson cutis gyrata, and Antley-Bixler syndromes, and Kleeblaatschadel (cloverleaf skull) deformity. The mutations identified to date are concentrated in the previously characterized region of FGFR2 that codes for the extracellular IgIII domain of the receptor protein. The search for mutations in other regions of the gene, however, has been hindered by lack of knowledge of the genomic structure. Using a combination of genomic library screening, long-range PCR, and genomic walking, we have characterized the genomic structure of nearly the entire human FGFR2 gene, including a delineation of the organization and size of all introns and exons and determination of the DNA sequences at the intron/exon boundaries. Comparative analysis of the human FGFR gene family reveals that the genomic organization of the FGFRs is relatively conserved. Moreover, alignment of the amino acid sequences shows that the four corresponding proteins share 46% identity overall, with up to 70% identity between individual pairs of FGFR proteins. However, the FGFR2 gene contains an additional exon not found in other members of the family, and it also has much larger intronic sequences throughout the gene. Remarkable similarities in genomic organization, intron/exon boundaries, and intron sizes are found between the human and mouse FGFR2 genes. Knowledge gained from this study of the human FGFR2 gene structure may prove useful in future screening studies designed to find additional mutations associated with craniosynostotic syndromes, and in understanding the molecular and cell biology of this receptor family.     

Cysteine-rich fibroblast growth factor receptor alters secretion and intracellular routing of fibroblast growth factor 3

Expression of the cysteine-rich fibroblast growth factor (FGF) receptor (CFR) in COS-1 cells strongly inhibits the secretion of co-expressed FGF3. By using a column retention assay and affinity chromatography, we demonstrate that at physiological salt concentrations FGF3 binds with strong affinity to CFR in vivo and in vitro. Furthermore, to show that FGF3 binds to CFR in vivo, truncation mutants of CFR with changed subcellular distributions were shown to cause a similar redistribution of FGF3. Although CFR is a 150-kDa integral membrane glycoprotein that is primarily located in the Golgi apparatus, we show here that in COS-1 cells a substantial proportion of CFR is secreted. This is due to a carboxyl-terminal proteolytic cleavage that releases the intraluminal portion of the protein for secretion. However, the apparent size of the integral membrane and secreted CFR appears similar, since the loss of protein mass is balanced by a gain of complex carbohydrates. The released CFR is associated with the extracellular matrix through its affinity for glycosaminoglycans. These findings show that CFR can modulate the secretion of FGF3 and may control its biological activity by regulating its secretion.     

Expression of acidic fibroblast growth factor mRNA in the developing and adult rat brain.

We have localized acidic fibroblast growth factor (aFGF) mRNA in the developing and adult rat brain using in situ hybridization histochemistry. Prenatally, hybridization to aFGF mRNA was observed throughout the brain, with the strongest signal associated with cells of the developing cortical plate. Postnatally, labeling was localized to specific neuronal populations. In the hippocampus, labeling of the pyramidal cell layer and dentate granule cells was observed and became progressively more intense with maturation. Labeling was also observed in both the external and internal granule cell layers of the developing cerebellum. Pyramidal cells of the neocortex as well as neurons of the substantia nigra and locus ceruleus also express aFGF. This pattern persists into adulthood, although the intensity of the labeling is significantly reduced in the adult brain. These patterns of hybridization correlate with specific developmental events and suggest that aFGF plays a significant role in both central nervous system development and neuronal viability in the adult brain.     

A truncated form of fibroblast growth factor receptor 1 inhibits signal transduction by multiple types of fibroblast growth factor receptor.

A truncated form of the type 1 fibroblast growth factor receptor (FGFR1) lacking most of its cytoplasmic domain was tested for its ability to inhibit signal transduction by each of three different wild-type FGFRs (FGFR1, 2, and 3). When the truncated FGFR1 was expressed in Xenopus oocytes in excess of each wild-type FGFR, mobilization of intracellular calcium mediated by the wild-type FGFRs was completely blocked. The truncated FGFR did not inhibit signal transduction by the co-expressed platelet-derived growth factor beta-receptor. A form of truncated FGFR1 which lacked the first immunoglobulin-like domain also inhibited signal transduction by wild-type FGFRs. Truncated FGFR formed complexes with wild-type FGFR in the presence of basic FGF in intact cells. These observations were consistent with the hypothesis that the truncated FGFR interacted with wild-type FGFRs to form nonfunctional heterodimers, thus eliminating the signaling by the wild-type FGFRs. The observation that signaling by multiple types of FGFR can be blocked by a single type of truncated FGFR suggests that the different types of FGFR can interact with each other in ligand-mediated complexes. These findings provide a molecular basis for inhibiting the actions of FGFs in vivo.