Murine neural precursor cells and cell lines derived from them are stimulated by members of the heparin-binding fibroblast growth factor (FGF) family. The activity of FGF is regulated by heparan sulfate proteoglycans (HSPGs), and this interaction is an essential prerequisite for the binding of growth factor to the signal transducing receptors. Messenger RNA for FGF-2 was detectable in the neuroepithelium at embryonic day 9, and the HSPGs produced by these cells at this time preferentially bound FGF-2. However, at embryonic day 11, when messenger RNA for FGF-1 was first detectable, there was a switch in the binding specificity of the HSPG to FGF-1. Thus, a single species of HSPG undergoes a rapid, tightly controlled change in growth factor-binding specificity concomitant with the temporal expression of the FGFs.

Heparan sulfate (HS) glycosaminoglycans are essential modulators of fibroblast growth factor (FGF) activity and appear to act by coupling particular forms of FGF to appropriate FGF receptors. During neural development, one particular HS proteoglycan is able to rapidly switch its potentiating activity from FGF-2, as neural precursor cell proliferation occurs, to FGF-1, as neuronal differentiation occurs. Using various analytical techniques, including chemical and enzymatic cleavage, low pressure chromatography, and strong anionexchange high performance liquid chromatography, we have analyzed the different HSs expressed during these crucial developmental stages. There are distinct alterations in patterns of 6-O-sulfation, total chain length, and the number of sulfated domains of the HS from the more mature embryonic brain. These changes correlate with a switch in the ability of the HS to potentiate the actions of FGF-1 in triggering cell differentiation. It thus appears that each HS pool is designed to function in the modulation of an intricate interaction with a specific growth factor and its cognate receptor, and suggests tightly regulated expression of specific, bioactive disaccharide sequences. The data can be used to construct a simple model of controlled variations in HS chain structure which have functional consequences at a crucial stage of neuronal maturation.Heparin-binding fibroblast growth factors (FGFs) 1 are essential regulators of mitogenesis and differentiation for the precursor cells of the mammalian central nervous system (1). As in many tissues, the bioactivity of the FGFs is partially regulated by the glycosaminoglycan heparan sulfate (HS). These carbohydrate chains are normally found attached to core proteins and cells lacking these heparan sulfate proteoglycans (HSPGs) are unable to transduce an FGF signal (2, 3). One current hypothesis is that HSs serve to couple FGFs to specific HSbinding regions on competent FGF receptors (FGFRs) to form activating ternary complexes (4, 5). We have previously purified two different species of HSPG from embryonic murine neuroepithelia: from embryonic day 10 (E10) cells, when an HSPG with an affinity for FGF-2 was found and whose sugar chains are herein designated as HS2, and from E12 neuroepithelial cells, when an HSPG with an affinity for FGF-1 is expressed, and whose sugar chains are called HS1 (6, 7). This switch in HSPG activating activity was coincident with a similar switch in FGF expression. As the HSPG core protein carrying the two HS species appeared to be the same (6) then presumably differences in HS structure between E10 and E12 must account for the change in FGF specificity.As specific HS species are essential to FGF activation, knowledge of the structures is clearly crucial to understanding their potentiating functions. Variation in HS species arises from the synthesis of non-random, highly sulfated sequences of sugar residues which are separated by unsulfated regions of disaccharides containing N-acetylated glucosamine. The initial conversion...

Inverted duplications have been observed to be a common feature of gene amplification in mammalian cells and appear to be generated as a primary event in the amplification process (Ford etal., 1985; Ford and Fried, 1986). The structural features of the amplified inverted duplication, containing the polyoma virus oncogene middle T-antigen, were analysed in transformed 3B rat cells. No unusual sequences such as transposition elements were detected at the site of the inversion. The inversion was generated by a simple illegitimate recombination event in which only a single nucleotide directly at the point of the inversion cannot be accounted for from the sequence of the two parental strands. Possible structural (hairpin formation) and sequence (rich AT) features may have been involved in the illegitimate recombination event at the inversion join. In the cellular DNA near one of its joins with polyoma virus DNA an unusual sequence of 198 bp composed of 99 consecutive purine-pyrimidine pairs has been detected. A model for the generation of amplified DNA containing inverted duplications is proposed.

No abstract

Heparan sulfate proteoglycans are thought to be obligatory for receptor binding and subsequent mitogenic activity of basic fibroblast growth factor (FGF-2). In a previous study (Nurcombe V., Ford, M. D., Wildschut, J., Bartlett, P. F. (1993) Science 260, 103-106) we have shown that primary cultures of mouse neuroepithelial cells and a cell line derived from them, 2.3D, secrete a heparan sulfate proteoglycan with a high affinity for FGF-2. In this study, a combination of affinity chromatography and gel chromatography was used to further isolate heparan sulfate side chains with high affinity for FGF-2. These active chains had an average molecular weight of 18,000 -20,000. In order to determine whether heparan sulfate chains with specificity for FGF-2 also displayed selectivity for the different FGF receptors, peptides designed to the heparin-binding region of the receptors were used in competitive inhibition studies. The structure of the predicted heparinbinding domain of the FGF receptor 1 was modeled on the basis of its presumed secondary and tertiary structure homology with immunoglobulin loops. These results suggested that many of the basic residues within the second immunoglobulin loop of the FGF receptor 1 form a basic domain in the molecule and therefore form part of a heparin-binding site. Peptides homologous to this region of FGF receptor 1 were shown to inhibit mitogenesis in 2.3D cells, while those to FGF receptor types 2, 3, and 4 did not. A reverse transcriptase-polymerase chain reaction assay designed to detect expression of the four FGF receptors types demonstrated that FGF receptors 1 and 3 were present on the 2.3D cell line but that receptors 2 and 4 were not. These findings indicate that unique heparan sulfate domains interact with specific cell-surface receptors to direct cellular responses.The fibroblast growth factor (FGF) 1 family consists of at least nine members including acidic FGF (FGF-1) and basic FGF (FGF-2), which are known to control the proliferation, migration, and differentiation of a broad variety of cell types, including vertebrate neuroepithelial cells (1). The biological effects of the FGFs are derived from their interactions with specific, high affinity cell-surface receptors (2, 3). The FGF receptor family consists of at least four types: FGFR1 (flg) (4, 5), FGFR2 (bek) (6), FGFR3 (7), and FGFR4 (8, 9). These integral transmembrane proteins have been identified within the mammalian central nervous system (10). Three of these receptors, FGFR1, FGFR2, and FGFR3, can transduce the FGF-2 signal in vitro (6, 11, 12) and can be alternatively spliced to produce translation products with either two or three immunoglobulin domains with a variably spliced C-terminal region within the third immunoglobulin domain (isoforms) that binds FGF-2 with different affinities (13-15). The temporal and spatial regulation of these isoforms suggest that they are likely to play a major role in the specificity of bioactivation of the FGFs in different tissues throughout the body (16 -18). FGFs are also...