FRAG1, a gene that potently activates fibroblast growth factor receptor by C-terminal fusion through chromosomal rearrangement.

Abstract

A constitutively active form of fibroblast growth factor 2 (FGFR2) was identified in rat osteosarcoma (ROS) cells by an expression cloning strategy. Unlike other tyrosine kinase receptors activated by N-terminal truncation in tumors, this receptor, FGFR2-ROS, contains an altered C terminus generated from chromosomal rearrangement with a novel gene, designated FGFR activating gene 1 (FRAGJ). While the removal of the C terminus slightly activates FGFR2, the presence of the FRAGI sequence drastically stimulates the transforming activity and autophosphorylation of the receptor. FGFR2-ROS is expressed as a unusually large protein and is highly phosphorylated in NIH 3T3 transfectants. FRAGJ is ubiquitously expressed and encodes a predicted protein of 28 kDa lacking significant structural similarity to known proteins. Epitope-tagged FRAG1 protein showed a perinuclear localization by immunofluorescence staining. The highly activated state of FGFR2-ROS appears to be attributed to constitutive dimer formation and higher phosphorylation level as well as possibly altered subcellular localization. These results indicate a unique mechanism of receptor activation by a C terminus alteration through a chromosomal fusion with FRA4G).The fibroblast growth factors (FGFs) are a family of related proteins with roles in mitogenesis, differentiation, wound healing, and organogenesis (reviewed in ref. 1). Nine FGFs, acidic FGF (aFGF)/FGF-1, basic FGF/FGF-2, INT-2/ FGF-3, HST/FGF-4, FGF-5, HST-2/FGF-6, keratinocyte growth factor (KGF)/FGF-7, androgen-induced growth factor/FGF-8, and glia-activating factor/FGF-9, have been identified and cloned. While most of the members can stimulate the growth of fibroblasts, KGF/FGF-7 and glia-activating factor/ FGF-9 are involved in epithelial and glial cell proliferation, respectively (2, 3). The biological responses of the FGFs are mediated through specific high affinity receptor tyrosine kinases (reviewed in ref. 4). These receptors, FGFR1/Flg, FGFR2/Bek, FGFR3, and FGFR4, encode structurally related proteins (5-8). The diversity of FGFRs are increased by alternative RNA splicing, which generates additional isoforms of FGFRs (9-11). In particular, the FGFR2/BEK gene encodes two receptor species, FGFR2 and KGF receptor (KGFR), which differ with respect to ligand-binding specificity; FGFR2 binds aFGF and basic FGF, whereas KGFR binds aFGF and KGF (9). These receptors differ only in 49 amino acids in their third Ig-like domains.We have developed an efficient expression cloning system which we have used to isolate several unique transforming cDNAs (12, 13). Here we report the isolation of a constitutively active FGFR2 with an altered C terminus by expression cloning with a rat osteosarcoma (ROS) cDNA library. This FGFR2 isoform (FGFR2-ROS) resulted from a chromosomal rearrangement of the FGFR2 gene with a novel gene, designated FGFR activating gene 1 (FRAG1), in ROS cells. Activation of FGFR2-ROS through C-terminal fusion with FRAG1 suggests a third activation mechanism of receptor tyrosine kin...

Identification of a novel activated form of the keratinocyte growth factor receptor by expression cloning from parathyroid adenoma tissue

“…Together with other known variants, K-SAM-IIC3 (Itoh et al, 1994) and KGFR-ET (Lorenzi et al, 1996), our ®ndings suggest that four KGFR isoforms with di erent C-termini are generated by alternative splicing (Figure 6a). The C-terminal domain of K-SAM-IIC3 is encoded by the C3 exon, which contains a translation termination codon and polyadenylation sequence (Itoh et al, 1994).…”

“…We have previously found that alterations of the Cterminus of KGFR/FGFR2 can stimulate the receptor's transforming activity (Lorenzi et al, 1996). Therefore, we examined a possibility that PA-1 parathyroid adenoma might express KGFR with a similar C-terminal variation.…”

“…These results indicate that the altered C-terminus of KGFR-PA results in activation of the receptor without ligand binding. Another C-terminal variant of KGFR, KGFR-ET (early termination) (Lorenzi et al, 1996), also showed relatively high transforming activity, when its FGFR2-form, FGFR2-ET, was used to compare the activity with FGFR2-WT (Figure 2). …”

“…We have previously reported a transforming isoform of FGFR2 generated by chromosomal rearrangement with a novel gene, FRAG1, at the C-terminus of the receptor (Lorenzi et al, 1996). The FGFR2-FRAG1 recombinant protein showed increased transforming activity due, in part, to the constitutive dimerization of the receptor.…”

Determinants for transformation induced by the Axl receptor tyrosine kinase

“…Whether this increased transforming ability resulted from higher steady state levels of the Axl protein or a functional di erence of the chimeric receptor is unclear. Possible mechanisms for this augmented transforming ability include enhanced protein stability, altered substrate selection, perturbation of receptor turnover, and altered intramolecular interactions (Kornilova et al, 1996;Lorenzi et al, 1996;O'Connor et al, 1997). Nevertheless, it remains consistent that transformation by any form of Axl is associated with receptor overexpression.…”