Craig A. Dionne

Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic fibroblast growth factors.

The fibroblast growth factor (FGF) family consists of at least seven closely related polypeptide mitogens which exert their activities by binding and activation of specific cell surface receptors. Unanswered questions have been whether there are multiple FGF receptors and what factors determine binding specificity and biological response. We report the complete cDNA cloning of two human genes previously designated flg and bek. These genes encode two similar but distinct cell surface receptors comprised of an extracellular domain with three immunoglobulin‐like regions, a single transmembrane domain, and a cytoplasmic portion containing a tyrosine kinase domain with a typical kinase insert. The expression of these two cDNAs in transfected NIH 3T3 cells led to the biosynthesis of proteins of 150 kd and 135 kd for flg and bek, respectively. Direct binding experiments with radiolabeled acidic FGF (aFGF) or basic FGF (bFGF), inhibition of binding with native growth factors, and Scatchard analysis of the binding data indicated that bek and flg bind either aFGF or bFGF with dissociation constants of (2‐15) x 10(‐11) M. The high affinity binding of two distinct growth factors to each of two different receptors represents a unique double redundancy without precedence among polypeptide growth factor‐receptor interactions.

Ligand-induced transphosphorylation between different FGF receptors.

Recent evidence shows that different fibroblast growth factors (FGF) bind with similar high affinities to two FGF receptors (FGFR) called flg and bek. In order to explore the mechanism of FGFR tyrosine autophosphorylation, we have generated cell lines which co‐express a kinase‐negative mutant of FGFR and an active form of FGFR. The following transfected NIH 3T3 cells were generated: (i) cells which express a shorter truncated form of bek (two Ig domains) together with a kinase‐negative mutant of full length bek (bek K517A), (ii) cells which express wild‐type bek together with kinase‐negative flg (flg K514A) and (iii) cells co‐expressing wild‐type flg together with bek K517A. Immunoprecipitations with either bek‐or flg‐specific antisera followed by immunoblotting indicated that the double transfectants express the desired receptor species. The addition of acidic FGF (aFGF) to the various cell lines followed by immunoprecipitation with anti‐FGFR antibodies and immunoblotting with anti‐phosphotyrosine specific antibodies indicated that aFGF induces tyrosine phosphorylation of the kinase‐negative FGFR mutants. These results show that tyrosine autophosphorylation of the kinase‐negative FGFR is mediated by a transphosphorylation mechanism and that both homologous (bek‐‐‐‐bek) and heterologous (bek‐‐‐‐flg and flg‐‐‐‐bek) transphosphorylation occurs in living cells. Recent evidence shows that tyrosine autophosphorylation of receptors with tyrosine kinase activities is essential for mediating interactions with signaling molecules. Therefore, heterologous transphosphorylation could amplify the response of cells to various forms of FGFs and their cognate receptors.

Temporal, differential and regional expression of mRNA for basic fibroblast growth factor in the developing and adult rat brain

The expression of basic fibroblast growth factor (bFGF) mRNA and bFGF receptor mRNA was investigated in developing rat brain. In embryonic rat brain days 13-21 (E13-E21), an abundant 1.8 kb bFGF mRNA was detected. Expression of 1.8 kb bFGF mRNA was the highest at E17 to E19 and was relatively undetectable 20 days after birth. However, very little mitogenic activity was associated with prenatal brain. On the other hand, multiple bFGF mRNA species of 6.0, 3.7, 2.5, 1.8, 1.6, 1.4 and 1.0 kb were detected in total adult rat brain and a significant amount of mitogenic activity was present. Differential and spatial bFGF mRNA expression was found in different parts of developing rat brain. Embryonic hypothalamus was found to contain the 1.8 kb bFGF mRNA while the 6.0 kb bFGF mRNA transcript was predominant in adult hypothalamus. Adult pituitary and cortex transcribed the lower molecular weight mRNAs but not the 6.0 kb mRNA. Expression of high-affinity bFGF receptor (flg) mRNA was found to be temporally regulated. flg 4.3 kb mRNA expression was high in embryonic rat brain (E13-E19). There appears to be coordinate expression between the 1.8 kb bFGF mRNA and flg. These results suggest that the expression of basic FGF mRNA is complex since it is both temporally and differentially regulated with different species being expressed at different times in development.

BEK, a receptor for multiple members of the fibroblast growth factor (FGF) family, maps to human chromosome 10q25.3→q26

The gene for the fibroblast growth factor receptor BEK was assigned to human chromosome 10 by applying polymerase chain reaction techniques to DNAs from a panel of human x rodent somatic cell hybrids. The gene was further localized to 10q25.3----q26 by in situ hybridization.

Effect of heparin on the binding affinity of acidic FGF for the cloned human FGF receptors, flg and bek.

Heparin potentiates the mitogenic activity of acidic fibroblast growth factor (aFGF) by 20-100 fold but mechanisms detailing this potentiation have not yet been fully elucidated. We report that heparin increases the binding affinity of aFGF for the two cloned and overexpressed human FGF receptors, flg and bek, by 2-3 fold. This increase in binding affinity, together with previous data demonstrating a 3-5 fold increase in the stability of aFGF, are likely to account for a significant portion of heparins potentiation of aFGF activity observed in biological assay systems.

Acidic Fibroblast Growth Factor Accelerates the Healing of Acetic-Acid-lnduced Gastric Ulcers in Rats

Acidic fibroblast growth factor (aFGF) was evaluated for the healing of acetic-acid-induced gastric ulcers in rats. The effect of aFGF on angiogenesis in the gastric ulcer bed was determined by the carmine dye infusion method, while its effect on gastric acid secretion was assessed in chronic gastric fistula rats. Oral treatment with aFGF, in the presence of heparin, reduced (ED50 value = 30.2 micrograms/kg/day) the acetic-acid-induced gastric ulcer area, when assessed 1 week later. aFGF was about 1,333-fold more potent than famotidine for healing such ulcers. At a dose of 200 micrograms/kg/day, aFGF increased the carmine density 3-fold and correspondingly reduced (80%) the gastric ulcer area. Thus, the ulcer healing effect of this agent involves angiogenesis in the gastric ulcer bed. This effect of aFGF appears to be unrelated to an inhibition of gastric acid secretion, as it was ineffective in chronic gastric fistula rats. In summary, oral aFGF significantly accelerates the healing of experimental gastric ulcers in rats. It may be a potent and effective agent for the treatment of peptic ulcers in humans.

The Use of Polymerase Chain Reaction for Chromosome Assignment

The polymerase chain reaction (PCR) approach to chromosome assignment consists of analyzing the profile of specific amplification products of the gene of interest using genomic DNAs from a panel of somatic cell hybrids as templates (see Fig 1). Each hybrid somatic cell line contains the normal complement of chromosomes of one species, along with one or more additional chromosomes from a second species (1,2). The PCR approach has obvious advantages over the conventional Southern blotting method of chromosome assignment, including speed, sensitivity, and conservation of the genomic DNAs, which are difficult and labor intensive to generate.

Chromosome assignment by polymerase chain reaction techniques.

Publisher Summary This chapter describes the application of the technique for the chromosome assignment of the human BEK gene, which codes for a tyrosine-kinase-linked receptor for fibroblast growth factors. Instead of Southern analysis of somatic cell hybrid DNAs, the chapter chooses to polymerase chain reaction (PCR0 amplify the human gene of interest in each of the DNA samples. Like most analytical PCR techniques, the objective of this application is the detection of minute amounts of specific nucleic acid sequences in different samples and therefore requires high specificity and sensitivity. It is always useful, if possible, to verify the results of PCR-generated assignment by comparison to the results obtained for another gene mapped to the same chromosome. Theoretically, two genes on the same chromosome should cosegregate. Similarly, the product should be available from the appropriate chromosome specific genomic DNA library. Even with the extra controls and verifications, chromosome assignment by PCR technique is much faster than by conventional methods and is much more conservative of the valuable somatic cell hybrid DNA.