Faculty Bibliography

The role of heparin or heparan sulfates in the interaction of basic fibroblast growth factor (bFGF) with its high affinity receptor were investigated using purified extracellular ligand-binding region of FGF receptor-1 (FGFR-1) and intact receptors expressed in a myeloid cell line (32D) that does not express detectable levels of heparan sulfate proteoglycans or in Chinese hamster ovary (CHO) cell mutants defective in heparan sulfate synthesis. The purified extracellular domain of FGFR-1 formed complexes with 125I-bFGF both in the presence or absence of heparin. Intact FGFR-1 expressed in 32D cells also bound the same amount of 125I-bFGF in the presence or absence of heparin when saturating concentrations of bFGF were used. Varying the concentration of 125I-bFGF showed that heparin increased the amount of 125I-bFGF bound at low bFGF concentrations and increased the affinity of bFGF for its receptor by about 3-fold. To eliminate the possibility of alteration of bFGF properties through the chemical modification reactions, bFGF was labeled biosynthetically. The binding of biosynthetically labeled bFGF to FGFR-1 also did not require heparin. When FGFR-1 or FGFR-2 were expressed in mutant CHO cells deficient in heparan sulfate synthesis, the cells also bound 125I-bFGF in the absence of heparin, and the addition of heparin increased the affinity of bFGF for its receptors 2-3-fold. Thus, heparin or heparan sulfate is not required for the binding of bFGF to its receptors but increases the binding affinity to a moderate degree. Finally, the requirement for heparin in signal transduction through the receptor was investigated. Expression of c-fos mRNA was induced by bFGF in 32D cells expressing FGFR-1 to the same extent in the presence or absence of heparin

The fibroblast growth factor receptor 1 (flg) contains eight acidic amino acids between the first and second immunoglobulin domain. This report examines the role of the acidic domain in the interaction of the flg receptor with its ligands. We observed a marked inhibition of binding of bFGF to the receptor when the acidic domain was completely deleted, but mutants with two and four amino acids deleted (flg delta A2 and flg delta A4, respectively) still bound the ligand. After addition of a bifunctional cross-linking reagent, cross-linked complexes (between bFGF and receptor) with the expected size were observed in cells expressing mutants lacking two or four acidic residues, but not in cells expressing mutants lacking six or eight acidic residues. Immunoprecipitation with anti-flg antibody followed by electrophoresis produced a band of 90 Kd in tunicamycin-treated cells expressing the mutant as well as the wild-type receptors, indicating that the inhibition of binding was not due to defective expression of the protein. The ability of flg delta A8 to mediate a mitogenic response to FGFs was also greatly reduced when this mutated receptor was expressed in receptor-negative cells. The effect of replacing the acidic amino acids with lysine residues was also studied. Binding of bFGF to cells transfected with a plasmid encoding a mutated protein with four amino acid substitutions was totally inhibited, but an eight amino acid substitution did not alter ligand binding to the receptor. In this case the mutation with four amino acids substitution caused a drastic impairment of protein expression. Thus the acidic domain of the FGFR-1 plays an essential role in receptor function, either because it is important for a stable protein configuration or for ligand-receptor interaction

The temperature-sensitive BN51 cell cycle mutant of BHK cells arrests in G1 at the nonpermissive temperature (39.5 degrees C). We have previously reported cloning the gene which complements this mutation. The complementing gene encodes a highly charged protein with a basic amino-terminal domain and an acidic carboxyl-terminal domain. We have recently found that the predicted BN51 protein shows significant homology to the 53 kilodalton subunit of RNA polymerase C (III) from Saccharomyces cerevisiae. Consistent with this, antibodies raised to fusion proteins containing BN51 coding sequences and antipeptide antibodies reveal that the BN51 gene encodes a 48 kilodalton protein which appears to be located primarily in the nucleus following subcellular fractionation and by immunohistochemistry. Analysis of RNA polymerase III activity in temperature-sensitive BN51 cells by nuclear runoff transcription assay reveals a marked drop in RNA polymerase III transcription after 48 h at the nonpermissive temperature (39.5 degrees C). This is correlated with a significant decrease in low molecular weight RNAs after 48 h at 39.5 degrees C. In addition, RNA polymerase III activity in S100 extracts of BN51 cells is more sensitive to heat inactivation at 39 degrees C than control extracts from BHK cells. When the yeast gene is introduced into the BN51 cells in a mammalian expression vector, it weakly complements the BN51 mutation in that it prevents cell death at 39.5 degrees C. The mechanism by which inhibition of RNA polymerase III activity leads to arrest in G1 is unclear but is not due to a marked decrease in total protein synthesis

We have previously shown that asparagine synthetase (AS) mRNA expression can be dramatically up-regulated by asparagine deprivation in ts11 cells, mutants of BHK hamster cells which encode a temperature-sensitive AS. The expression of AS mRNA was also induced upon starvation for one of several essential amino acids in HeLa cells. We also showed that regulation of AS mRNA expression by amino acid concentration has both transcriptional and posttranscriptional components. Here we report the analysis of the elements in the human AS promoter region important for its basal activity and activation by amino acid starvation. Our results indicate that a DNA fragment spanning from nucleotides -164 to +44 of the AS promoter is sufficient for uninduced and induced gene expression. Mutations in a region located 15 to 30 bp downstream from the major transcription start site that shows good homology to a sequence in the first exon of c-fos implicated as a negative regulatory element resulted in a significant increase in basal gene expression but did not affect regulation. Interestingly, this region binds single-stranded-DNA-binding proteins that are specific for the AS coding strand. Mutations in either one of two putative binding sites for transcription factor Sp1, in a region of approximately 60 bp where many minor RNA start sites are located, or at the major transcription start site decreased promoter activity, but significant induction by amino acid starvation was still observed. Strikingly, mutations centered around nucleotide -68 not only decreased the basal promoter activity but also abolished amino acid regulation. This DNA region contains the sequence 5'-CATGATG-3', which we call the amino acid response element (AARE), that can bind a factor(s) present in HeLa cells nuclear extracts that is not capable of binding to an AS promoter with mutations or deletions of the AARE. This finding is in line with the hypothesis that transcriptional activation of AS gene expression is mediated through the binding of a positive regulatory element. We did not detect changes in the level of binding of this factor to the AARE by using nuclear extracts from HeLa cells grown under starved conditions, suggesting that activation of this factor(s) results from posttranslational modification or complexing with other proteins that do not affect its DNA-binding properties

The K-FGF/HST (FGF-4) growth factor is a member of the FGF family which is efficiently secreted and contains a single N-linked glycosylation signal. To study the role of glycosylation in the secretion of K-FGF, we mutated the human K-fgf cDNA to eliminate the glycosylation signal and the mutated cDNA was cloned into a mammalian expression vector. Studies of immunoprecipitation from the conditioned medium of cells expressing this plasmid revealed that the lack of glycosylation did not impair secretion, however the unglycosylated protein was immediately cleaved into two NH2-terminally truncated peptides of 13 and 15 kD, which appeared to be more biologically active than the wild-type protein. These two proteins also showed higher heparin binding affinity than that of wt K-FGF. We have expressed in bacteria the larger of these two proteins (K140), in which the NH2-terminal 36 amino acids present in the mature form of K-FGF have been deleted. Mitogenicity assays on several cell lines showed that purified recombinant K140 had approximately five times higher biological activity than wild-type recombinant K-FGF. Studies of receptor binding showed that K140 had higher affinity than wt K-FGF for two of the four members of FGF receptor's family, specifically for FGFR-1 (flg) and FGFR-2 (bek). K140 also had increased heparin binding ability, but this property does not appear to be responsible for the increased affinity for FGF receptors. Thus removal of the NH2-terminal 36 amino acids from the mature K-FGF produces growth factor molecules with an altered conformation, resulting in higher heparin affinity, and more efficient binding to FGF receptors. Although it is not clear whether cleavage of K-FGF to generate K140 occurs in vivo, this could represent a novel mechanism of modulation of growth factor activity

The K-fgf/hst oncogene encodes a growth factor of the fibroblast growth factor (FGF) family and transforms cells through an autocrine mechanism which requires extracellular activation of its receptor(s). To identify the cell and tissue targets of K-fgf oncogenic potential in vivo, we constructed a recombinant retrovirus carrying the human K-fgf cDNA and injected it, together with helper Moloney murine leukemia virus, into immunocompetent as well as nude mice. The original construct was highly transforming in tissue culture but produced no detectable pathologies in vivo with the exception of a single fibrosarcoma which arose after a long latency. The virus produced by this tumor appears to have undergone a complex series of recombination events involving the helper Moloney murine leukemia virus. It encodes an Env/K-FGF fusion protein whose expression is under the control of a hybrid long terminal repeat. This virus (designated MFS, for meningeal fibrosarcoma) induces tumors in mice with high frequency and short latency. These neoplasms consist of aggressive fibrosarcomas of soft tissue as well as diffuse meningeal tumors originating from the dura mater that surround the whole central nervous system and cause severe hydrocephalus. The Env/K-FGF fusion protein expressed by the MFS virus has retained all of the biological properties of native K-FGF, including secretion, mitogenic activity, heparin binding, and neutralization by anti-K-FGF antibodies. These and other results indicate that the tumors induced by the MFS virus result from the oncogenic potential of K-FGF

Fibroblast growth factors (FGFs), such as basic FGF, have been implicated in the growth of Kaposi's sarcoma (KS) cells in vitro. In the evaluation of the expression of the various genes of the different members of the FGF family and their receptors in fresh KS tissue specimens, int-2 was found to be expressed in more than half of the KS tumors examined. Using reverse transcription PCR, the expression of int-2 was detected in 21 of 38 (55.2%) fresh KS biopsy specimens. In contrast, int-2 mRNA transcripts were not found in normal appearing skin from the same patients except in one sample which was obtained from an AIDS patient with disseminated KS lesions. Sequence data confirmed that the amplified sequences were derived from int-2 mRNA with proper splicing. In addition, 12 nucleic acid alterations were identified in eight out of nine KS tumor samples sequenced. Using immunohistochemical methods, int-2 protein was detected in some of the spindle-shaped tumor cells surrounding the abnormal endothelial-lined vascular slits histologically characteristic of KS. Int-2 specific immunostaining was shown to be present in both the nuclei and cytoplasm of these spindle cells but was more pronounced in the nuclei. Neither amplification nor gross rearrangement of the int-2 gene was detected in KS lesions by Southern blot analysis. These results suggest that the expression of int-2 may play a role in the pathogenesis KS by stimulating local angiogenesis and cell proliferation