Two alternative exons, BEK and K-SAM, code for part of the ligand binding site of fibroblast growth factor receptor 2. Splicing of these exons is mutually exclusive, and the choice between them is made in a tissuespecific manner. We identify here pre-mRNA sequences involved in controlling splicing of the K-SAM exon. The short K-SAM exon sequence 5-TAGGGCAGGC-3 inhibits splicing of the exon. This inhibition can be overcome by mutating either the exon's 5 or 3 splice site to make it correspond more closely to the relevant consensus sequence. Two separate sequence elements in the intron immediately downstream of the K-SAM exon, one of which is a sequence rich in pyrimidines, are both needed for efficient K-SAM exon splicing. This is no longer the case if either the exon's 5 or 3 splice site is reinforced. Furthermore, if the exon inhibitory sequence is removed, the intron sequences are not required for splicing of the K-SAM exon in a cell line which normally splices this exon. At least three elements are thus involved in controlling splicing of the K-SAM exon: suboptimal 5 and 3 splice sites, an exon inhibitory sequence, and intron activating sequences.Alternative splicing is a mechanism frequently used by cells for the production of several proteins from one gene (29,33). When subject to control, this mechanism allows the production of similar but distinct versions of a protein in a cell-typespecific manner. Alternative splicing of the fibroblast growth factor receptor 2 (FGFR-2) gene pre-mRNA is a particularly attractive example of this phenomenon (9,21,30,50). The FGFR-2 extracellular domain is composed of three immunoglobulin-like domains, the third of which is particularly important for ligand binding (30, 50). The carboxy-terminal moiety of the third immunoglobulin-like domain is encoded by two alternative exons (9, 30, 50), K-SAM (or IIIb) and BEK (or IIIc). Splicing of these exons is mutually exclusive and is controlled both in cell lines and during development (9,30,31). Epithelial cells use mainly the K-SAM exon and synthesize a receptor with high affinity for keratinocyte growth factor. Other cell types use mainly the BEK exon and synthesize a high-affinity receptor for basic fibroblast growth factor. That the splicing choice is linked to the cell type is illustrated by the observation that oncoprotein fos activation in epithelial cells induces an epithelio-mesenchymal conversion and changes the exon spliced from K-SAM to BEK (36). Controlling the splicing of the FGFR-2 pre-mRNA may be very important for the cell, as a change in the splicing pattern has been correlated with malignant progression of rat prostate cells (48).This control is presumably exerted at the level of construction of the spliceosome, which is composed of four small nuclear ribonucleoproteins (snRNPs) as well as many associated splicing factors (19,37). Early steps in spliceosome formation are the recognition of the 5Ј splice site sequence by U1 snRNP and the recognition of the branch point sequence, associated with the 3Ј splice site...

The fibroblast growth factor receptor 2 gene contains a pair of mutually exclusive alternative exons, one of which (K-SAM) is spliced specifically in epithelial cells. We have described previously (F. Del Gatto and R. Breathnach, Mol. Cell. Biol. 15:4825-4834, 1995) some elements controlling K-SAM exon splicing, namely weak exon splice sites, an exon-repressing sequence, and an intron-activating sequence. We identify here two additional sequences in the intron downstream from the K-SAM exon which activate splicing of the exon. The first sequence (intron-activating sequence 2 [IAS2]) lies 168 to 186 nucleotides downstream from the exon's 5 splice site. The second sequence (intron-activating sequence 3 [IAS3]) lies 933 to 1,052 nucleotides downstream from the exon's 5 splice site. IAS3 is a complex region composed of several parts, one of which (nucleotides 963 to 983) can potentially form an RNA secondary structure with IAS2. This structure is composed of two stems separated by an asymmetric bulge. Mutations which disrupt either stem decrease activation, while compensatory mutations which reestablish the stem restore activation, either completely or partially, depending on the mutation. We present a model for K-SAM exon splicing involving the intervention of multiple, interdependent pre-mRNA sequence elements.Cells very often make several related yet distinct proteins from a single gene by alternative splicing of the corresponding pre-mRNA (36). This strategy can be exploited to express one form of a protein in some cell types and a different form in other cell types. One example of this behavior is provided by the fibroblast growth factor receptor 2 (FGFR-2) gene (28). The extracellular part of the receptor encoded by this gene is made up of three immunoglobulin-like domains. Two alternative exons, K-SAM and BEK, code for the carboxy-terminal half of the third, membrane-proximal, immunoglobulin-like domain (11). Splicing of the K-SAM exon generates a highaffinity receptor for FGF-1 and FGF-7, while splicing of the BEK exon generates a high-affinity receptor for FGF-1 and FGF-2 (38, 56). The splicing choice is strictly controlled, as certain cell types splice essentially only the K-SAM exon, while others splice essentially only the BEK exon (11,38). As the splicing choice conditions the response of a cell to certain growth factors in its environment, the importance of strict control of splicing is evident during both development and adult life (40,43).Many other cases of alternative exon splicing have been described. For example, mouse c-src pre-mRNA contains two exons spliced only in neurons (7,12,39). The avian troponin T gene contains an optional exon spliced in the embryonic but not the adult heart (41, 54). Alpha-and beta-tropomyosin genes contain pairs of mutually exclusive internal exons spliced with strict tissue specificity (5,14,24,25,32). The pre-mRNA encoding calcitonin and calcitonin gene-related peptide contains six exons, one of which (exon 4) is skipped in a few cell types, including neuronal cell...

The fibroblast growth factor receptor-2 gene contains a pair of alternative exons, K-SAM and BEK, which are spliced in a cell type specific manner. We have shown previously that a 10 nucleotide sequence within the K-SAM exon exerts a negative effect on K-SAM exon splicing independent of cell type. We demonstrate here that this sequence works autonomously, as it can repress splicing of a heterologous exon, the EIIIb alternative exon of the rat fibronectin gene. By introducing point mutations into the 10 nucleotide sequence, we have shown that the functional portion is limited to 4 nucleotides, TAGG, the dinucleotide AG of which is particularly important. This short sequence may participate in the control of splicing of exons carrying it, provided that they carry weak splice sites.

The fibroblast growth factor receptor 2 gene pre-mRNA can be spliced by using either the K-SAM exon or the BEK exon. The exon chosen has a profound influence on the ligand-binding specificity of the receptor obtained. Cells make a choice between the two alternative exons by controlling use of both exons. Using fibroblast growth factor receptor 2 minigenes, we have shown that in cells normally using the K-SAM exon, the BEK exon is not used efficiently even in the absence of the K-SAM exon. This is because these cells apparently express a titratable repressor of BEK exon use. In cells normally using the BEK exon, the K-SAM exon is not used efficiently even in the absence of a functional BEK exon. Three purines in the K-SAM polypyrimidine tract are at least in part responsible for this, as their mutation to pyrimidines leads to efficient use of the K-SAM exon, while mutating the BEK polypyrimidine tract to include these purines stops BEK exon use.Multiple alternative splicing events lead to synthesis from the fibroblast growth factor (FGF) receptor 2 (FGFR-2) gene of a family of receptors differing in defined parts of their extra-and intracellular domains (reviewed in references 19 and 21). In its first described version, FGFR-2 contains an extracellular domain made up of three immunoglobulin-like domains (Ig domains), with a stretch of consecutive acidic residues, the acid box, separating the first two Ig domains. A particularly interesting alternative splice concerns sequences of the mRNA coding for the carboxy-terminal half of the third Ig domain, as this region of the receptor appears to be part of the ligand-binding site. Two alternative exons (K-SAM and BEK) code for this part of FGFR-2 (4,20,29,40). Use of the K-SAM exon results in synthesis of a high-affinity receptor for acidic FGF and keratinocyte growth factor (KGF), while use of the BEK exon yields a high-affinity receptor for acidic FGF and basic FGF (16,29,40). Correct control of the BEK-K-SAM splicing choice appears important, since this choice can influence a cell's response to growth factors that it produces itself as well as to those present in its environment. Consistent with this view, we and others have shown that a given cell line uses predominantly one of the two alternative exons, use of the other exon being sufficiently rare that it cannot be detected in a reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis (4, 29). Thus, epithelial cells express the K-SAM form of FGFR-2 but do not produce KGF, an epithelial cell-specific growth factor, while fibroblasts, which secrete KGF, express the BEK form. The consequences of a "wrong" choice can be disastrous: forced expression of the K-SAM receptor form in fibroblasts producing KGF leads to transformation (30).We are interested in determining the mechanisms involved in discrimination between the BEK and K-SAM exons. Pre-mRNA sequence elements representing potential targets for control of splicing include the 5' and 3' splice sites, the branch point sequence, and the associated polypy...