Review Article ISOFORMS of RECEPTORS of FIBROBLAST GROWTH FACTORS†

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Gong S-G. Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, Ontario, Canada, M5G 1G6. E-mail: [email protected]. Tel: 416-979-4900 x 4588; Fax: (416) 979-4936



This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jcp.24649] Additional Supporting Information may be found in the online version of this article. Received 13 August 2013; Revised 02 April 2014; Accepted 10 April 2014 Journal of Cellular Physiology © 2014 Wiley Periodicals, Inc. DOI: 10.1002/jcp.24649

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Abstract The breadth and scope of Fibroblast Growth Factor signaling is immense, with documentation of its role in almost every organism and system studied so far. FGF ligands signal through a family of four distinct tyrosine kinase receptors, the FGF receptors (FGFRs). One contribution to the diversity of function and signaling of FGFs and their receptors arises from the numerous alternative splicing variants that have been documented in the FGFR literature. The present review discusses the types and roles of alternatively spliced variants of the FGFR family members and the significant impact of alternative splicing on the physiological functions of five broad classes of FGFR isoforms. Some characterized known regulatory mechanisms of alternative splicing and future directions in studies of FGFR alternative splicing are also discussed. Presence, absence, and/or the combination of specific exons within each FGFR protein impart upon each individual isoform its unique function and expression pattern during normal function and in diseased states (e.g., in cancers and birth defects). A better understanding of the diversity of FGF signaling in different developmental contexts and diseased states can be achieved through increased knowledge of the presence of specific FGFR isoforms and their impact on downstream signaling and functions. Modern high-throughput techniques afford an opportunity to explore the distribution and function of isoforms of FGFR during development and in diseases.

TABLE OF CONTENTS 1. Introduction 2. Key structural and functional domains of FGFRs 3. Genomic organization of FGFRs 4. Types of isoforms 4.1. Use of alternate exons 4.2. Deletions/changes in specific exons in the extracellular domains (inclusions or exclusions of exons) 4.2.1. Absence of IgI and/or Acid box domains 4.2.2. Lacking exons 8/9 and 10 4.3. Soluble receptors 4.4. Deletions in C-terminal ends 4.5. Absence or presence of specific amino acids

5. FGFR isoforms and cancers 5.1. Switch in expression between alternatively spliced isoforms 2

5.2. Expression of distinct isoforms 6. FGFR isoforms and human birth defects

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7. Concluding remarks and perspectives

Keywords: • Alternative splicing • Exon inclusion and exclusion • FGF signaling • Tissue-specific expression •

Cancers

1. Introduction Fibroblast Growth Factor (FGF) ligands signal through a family of four distinct tyrosine kinase receptors, the FGF receptors (e.g. of recent reviews, Dorey and Amaya, 2010; Tomlinson and Knowles, 2010; Wesche et al., 2011). Four FGF receptors (FGFR1-4) have been identified and serve at least twenty-two FGF ligands (Johnson and Williams, 1993; McKeehan et al., 1998). The breadth and scope of Fgf signaling is immense, with documentation of its role in almost every organism and system studied so far. One contribution to the diversity of function and signaling of FGFs and their receptors arises from the unusual structure and complexity of the receptors of FGFs, further amplified by the numerous alternative splicing variants documented in the FGFR literature. The goal of the present review is to discuss the types and roles of alternatively spliced variants of the FGFR family members. A summary of the key domains of FGFRs, genomic organization and the ascribed functions of each domain will first be described. The description will set the stage for a better understanding of the groupings of the different classes of FGFR isoforms and their functions. The involvement of some of these isoforms during tumorigenesis is also discussed. The reader is encouraged to read several excellent general and specific reviews on FGFs and Fgf signaling (e.g., Guillemot and Zimmer, 2011; Marie et al., 2012; Wesche et al., 2011). 2. Key structural and functional domains of FGFRs All four FGF receptors share the same general protein structure characteristic of most receptor tyrosine kinases (Johnson and Williams, 1993). There is a 5’ non-translated sequence, an aminoterminal signal sequence, three extracellular immunoglobulin (Ig) domains (IgI, IgII, or IgIII), a single membrane-spanning region, and an intracellular split tyrosine kinase domain (Fig. 1) (Johnson et al., 1990; Plotnikov et al., 2000). The intracellular part of the receptor includes the juxtamembrane domain, the split tyrosine kinase domain, and a short carboxy-terminal tail. The extracellular region of the protein has important roles in regulating the affinity and specificity of ligand binding. The N-terminal domain of IgII, a region highly conserved across all isoforms (Table I), exhibits a 18-residue domain binding site for heparin/heparan sulfate (HS), factors essential for high affinity FGF/FGFR binding and formation of an active receptor signaling complex (Kan et al., 1993; McKeehan and Kan, 1994; Nugent and Edelman, 1992; 3

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Ornitz, 2000; Rapraeger et al., 1994; Schlessinger et al., 2000; Yayon et al., 1991). The base FGF binding site appears to be mediated by the IgII domain, the IgII/III linker region, and the Nterminus of IgIII. For example, the IgII -IgIII linker region is involved in regulating affinity for both FGF and heparin (Johnson and Williams, 1993; Mohammadi et al., 2005; Plotnikov et al., 1999; Wang et al., 1995). Further specificity of ligand binding is provided by the exclusive alternate splicing of two exons coding for the C-terminal half of IgIII (McKeehan and Kan, 1994; Wang et al., 1999)(see 4.1). One unique characteristic of the extracellular region of FGFRs is the stretch of 4-8 contiguous stretch of glutamate-, aspartate-, and serine-rich sequence situated in the IgI and IgII linker, termed the acid box (AB) (Johnson and Williams, 1993; Figure 1). Although not involved in ligand binding, the IgI and AB/linker region plays an important role in receptor auto-inhibition (Chellaiah et al., 1999; Kalinina et al., 2012). The AB/linker region, being flexible, was originally thought to passively engage ligand- and HS-sites located on the IgII-IgIII region to suppress both ligand- and HS-binding affinity of the receptor (Olsen et al., 2004; Wang et al., 1995). More recent evidence, however, suggests a more active mechanism (Kalinina et al., 2012; Olsen et al., 2004). The use of nuclear magnetic resonance and surface plasmon resonance spectroscopy revealed that the HS binding site (HSS) of IgII, which is positively charged, engaged the negatively charged AB sub-region (Plotnikov et al., 1999). The resultant cis AB:HSS electrostatic interactions in turn encouraged intramolecular interactions of IgI with ligand-binding sites in the IgII-III region to suppress ligand binding (Olsen et al., 2004). The steric interference of AB domain, together with its strong amino acid sequence conservation among FGFR orthologs, highlights the universal role of the AB sub-region in FGFR autoinhibition, acting thus to minimize inadvertent FGF signaling Signaling by FGFR is initiated upon ligand binding, when the FGFRs dimerize and autophosphorylate up to seven tyrosine residues in the intracellular catalytic domain of the receptor to activate downstream signaling cascades which have been well-described in several review papers (e.g., Eswarakumar et al., 2005). Briefly, upon ligand binding, FGFR2 directly activate Phospholipase Cγ and indirectly activate MAPK (mitogen-activated protein kinase). MAPK activation is achieved through recruitment and activation of the docking protein FRS2 (FGFR substrate 2), followed by binding and activation of adaptor proteins GRB2 (growth factor receptor bound 2)/SOS (sons of sevenless1) complex. FRS2-mediated signaling also stimulates phosphatidylinositol 3-kinase/AKT (PI3K/AKT) activity through the adaptor proteins GRB2 and GRB2-associated binding protein 1 (GAB1). Activation of FGFR requires HS proteoglycan coreceptors to regulate signaling potency (Ibrahimi et al., 2004) and to result in an increase of ligand-binding affinity, receptor autophosphorylation, and sustained downstream signaling responses to FGF. In addition to enzymatic activity, the intracellular domain harbors protein binding and phosphorylation sites (including PKC and FRS2 sites) as well as several auto-phosphorylation sites that interact with intracellular substrates (Böttcher and Niehrs, 2005).

3. Genomic organization of FGFRs In humans, FGFR1, 2, 3, and 4 are localized to chromosomes 8p12, 10q26, 4p16.3, and 5q33qter, respectively (Muenke and Schell, 1995). The cDNAs for all four FGFRs have been cloned from human, mouse, chicken, rat, and Xenopus. Amino acid sequence comparison shows the high conservation of the FGFR proteins among all vertebrate classes and the likelihood that individual FGFR genes arose from a common ancestral gene through duplication (Xu et al., 4

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1999). The four proteins in the human FGFR family share a 46% amino acid identity: the FGFRs 1/2 pair displays the highest level of amino acid identity (70% homology), with the least homology in FGFRs1/4 pair (53%) (Table 1).The most conserved regions are the tyrosine kinases II (80–92%) and I (75–88%) domains. High homologies are also observed in the IgII (61–80%) and IgIII (71–82%) regions among all of the FGFRs, with the IgI domain sharing much less homology (14-24%) (Table I). The amino acid sequences of the second half of the IgIII domain (the IIIb or IIIc; see section 4.1) sequence from FGFR1 and 2 are highly homologous whereas the sequence of IIIb from FGFR3 is markedly different from that of FGFR1 and 2, suggesting a possible different ligand binding affinity for FGFR3 (Avivi et al., 1993). A direct alignment of the four human FGFR genes demonstrates that the overall organization is relatively conserved (Zhang et al., 1999). The FGFR2 gene is the largest and spans a region of at least 120 kb and contains 20 exons (the murine Fgfr2 gene has only 19 exons), with a unique exon (exon 7) that encodes a secreted form of the receptor (Zhang et al., 1999), absent in FGFR1 and 3 (which have 19 exons each). The FGFR4 consists of 18 exons, rather than 19 or 20, and is unique amongst the four receptors in that it does not possess two alternative exons for the 3′ half of the IgIII domain (Vainikka et al., 1992). One or two large introns have been detected in all four genes at their 5′ ends. FGFR2, however, unlike the other three FGFR genes, has large introns throughout. Common splice donor and acceptor sequences have been identified for all exons in all four genes with the exception of the alternative splice donor site in the transmembrane region that is conserved in human FGFR1–3 genes. The mouse Fgfr2 gene has also been characterized to contain 19 exons that span a 120 kb segment of DNA (Twigg et al., 1998). Although the human FGFR2 gene has one additional exon compared with its mouse counterpart (Twigg et al., 1998), the overall genomic organization, e.g., pattern of exon grouping and the length of introns, is highly conserved between them and sequence alignment of the intron/exon boundaries revealed very limited variations in these two FGFR2 genes. Regulation of receptor function can occur in several ways (Rose-John and Heinrich, 1994). For example, the expression of receptor can be restricted to specific cell types, or the number of receptors on the plasma membrane can be modulated by varying receptor mRNA regulation or receptor internalization (degradation versus recycling). Also, their affinities to certain cells can be altered by the presence or absence of membrane proteins, which interact with the ligandbinding protein. The presence of soluble forms can also cause an alteration in downstream signaling pathway. A review of the isoforms of FGFR identified so far show, amazingly, that all the aforementioned strategies of receptor regulation are utilized by members of the FGFR families, mostly via the process of alternative splicing to create variants that possess varying functions. 4. Types of isoforms With the discovery that multiple transcripts can arise from the same RNA molecule (Gilbert, 1978), the “one gene, one protein” paradigm (Beadle and Tatum, 1941) was replaced with “one gene, many protein” paradigm (reviewed in Mittendorf et al., 2012). Evidence from expressed sequence tags, cDNAs, genome-wide tiling and splicing microarray datasets demonstrate that alternative splicing occurs in more than 90% of genes, thus generating a variety of exon combinations from a single primary transcript (Matlin et al., 2005; Modrek and Lee, 2002; Pan et al., 2008; Wang et al., 2008). Splicing of FGFR was first reported in the early 90’s (Johnson et 5

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al., 1991). Previous documentation of differences in size of FGFRs from crosslinking studies had already hinted at the possibility of alternatively splicing of FGFR (reviewed in Powers et al., 2000). Since then, the number of alternative FGFR forms is one of the most frequently reported of any other growth factor receptor. Alternatively spliced variants of FGFRs could be the result of utilization of different coding regions for the same Ig-like domains, different exon usage to result in the translation of proteins lacking certain domains, or premature truncation to yield soluble receptors. The different isoforms that have been described in the literature are categorized and discussed in more details in the following subsections: 4.1 Use of alternate exons The specificity of binding between the FGF ligands and their receptors is achieved primarily through alternative splicing in the second half of IgIII in FGFRs, generating isoforms that utilize the use of alternate coding exons, or “switch” of exons, with no gain or loss of amino acids (reviewed in Holzmann et al., 2012). Classic examples are the IIIb and IIIc isoforms found in FGFR1-3 in which there are differences in the C-terminal half of IgIII (Cheon et al., 1994; Johnson and Williams, 1993; Orr-Urtreger et al., 1993; Werner et al., 1992) (Fig. 2). For FGFRs1-3, the IgIII is encoded by exons 7-9, producing the domains designated IIIa, IIIb, and IIIc (Fig. 2I). The N-terminal half of IgIII comprises the IIIa-sequence, encoded by exon 7, whereas the C-terminal half is formed by the alternative usage of either exons 8 or 9, creating the IIIb and IIIc isoforms of each receptor, respectively (Fig. 2). Alternative usage of the two separate exons leads to two different isoforms which possess different ligand-binding and celland tissue-expression specificities. The ligand-, cell- and tissue-specificities allow the regulation of specific responses to the ubiquitously expressed FGF family of growth factors (Beenken and Mohammadi, 2009). Thus, the IIIb and IIIc isoforms are predominantly epithelial and mesenchymal, respectively, with their corresponding ligands only activating either the epithelial or mesenchymal isoforms (except FGF1 which binds all receptor isoforms). The ligand-binding pattern of the FGFR2IIIb variants is much more restricted than that of the FGFR2IIIc variants FGFR2IIIb functions as the receptor for FGF1, 3, 7, 10, and 22 and FGFR2IIIc functions as the receptor for FGF1, 2, 4, 6, 9, 16, 17, and 18 (Eswarakumar et al., 2005; Ornitz et al., 1996) (Fig. 2I). Indeed, the FGFR2IIIb/FGFR2IIIc system continues to serve as the paradigm by which alternative splicing modulates FGF binding specificity (Mohammadi et al., 2005). The tissue-specific expression of the IIIb and IIIc isoforms and their ligands allows the generation of a directional paracrine regulatory axis in multiple organs (Thesleff, 2003; Thomson, 2001). For instance, during tooth development, there exists a tight temporal and spatial specificities of expression for Fgf3 and 10, both of which are exclusively expressed in dental mesenchymal cells whereas their cognate receptors, the IIIb isoform of FGFR1 and FGFR2, respectively, are expressed in the dental epithelium (Kettunen et al., 1998). The interactions between the ligands and receptors promote proliferation of dental epithelial cells and stem cells (Harada et al., 1999; Kettunen et al., 2000; Lin et al., 2009; Wang et al., 2007). The reciprocal nature of the signaling mechanism is also well illustrated in the developing limb where Fgf10 is the required mesenchymal signal that induces formation of the apical ectodermal ridge (AER, Fig. 2II) via its epithelial-expressing receptor FGFR2IIIb, and Fgf8 is an epithelial factor that signals via FGFR2IIIc in the distal mesenchyme (DM, Fig.2II). The binding affinity of IIIb/IIIc splice variants to certain proteins may differ. For example, the IIIb splice variants bind with greater affinity with NCAM, the cell adhesion molecule, compared 6

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with the IIIc variant (Christensen et al., 2011), suggesting that the expression pattern of various FGFR isoforms determines the cell context-specific effects of NCAM signaling through FGFR. Analogous splice variants of different FGFRs also exhibit differential ligand binding specificity. FGF2 binds FGFR1IIIc and FGFR2IIIc with comparable affinity but binds weakly to FGFR3IIIc (Chellaiah et al., 1999). Unlike FGFR1-3, alternative splicing cannot occur in FGFR4 as it contains only one exon encoding the C-terminal half of IgIII and therefore presents with only one isoform, the IIIb (Kostrzewa and Müller, 1998). The regulation of splicing has been best studied in the FGFR1 and FGFR2 proteins. In general, alternative RNA splicing decisions frequently require a balance of positive and negative selection and is a process that is tightly linked with transcription (Pandya-Jones and Black, 2009). Regulatory RNA-binding proteins (RBP) with splicing enhancer and silencer properties bind to elements (Matlin et al., 2005) which are either positive and negative regulatory short conserved RNA sequences located either in exons or introns of the genes (Chasin, 2007). A number of RBP that regulate the splice event of the IIIb and IIIc isoforms of FGFRs have been identified and, in general, were shown to be essential for activation of exon inclusion (Hovhannisyan and Carstens, 2007 and references therein; Mauger et al., 2008). In the case of FGFR2 exon IIIc, however, these proteins have been identified as silencers that repress exon inclusion (Mauger et al., 2008). Additionally for FGFR2, two paralogous epithelial cell-typespecific RBPs, termed epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2), have been shown to be essential for promoting splicing of the FGFR2 exon IIIb expression (Warzecha et al., 2009a; Warzecha et al., 2009b). ESRP1/2 not only regulate FGFR2 splicing but a whole set of genes involved in epithelial-mesenchymal transition (EMT), thus acting as a master splice regulator, with additional gene regulatory control over the EMT process (Warzecha and Carstens, 2012; Warzecha et al., 2010). 4.2 Deletions/changes in specific exons in the extracellular domains (inclusions or exclusions of exons) 4.2.1 Absence of IgI and/or Acid box domains: One major alternative splicing event results in FGFR isoforms differing in the presence or absence of IgI and/or the AB-containing linker domains, each encoded by one exon (ChampionArnaud et al., 1991; Eisemann et al., 1991; Johnson et al., 1990). Often, both IgI and/or the AB/linker between IgI and IgII are spliced to generate isoforms lacking IgI or AB/linker, or both IgI and AB/linker (Hou et al., 1992; Shimizu et al., 2001; Xu et al., 1992) (Fig. 3I). For example, IgI can be utilized or excised by alternate splicing to produce a 3(FGFRα)- or 2(FGFRß)-Ig loop extracellular domain (McKeehan and Kan, 1994). Presence or absence of IgI does not affect ligand binding activity (Johnson et al., 1990). Rather, this alternative splicing event controls receptor autoinhibition (Barnard et al., 2005; Sakaguchi et al., 1999). Isoforms without IgI or AB/linker contain HS proteoglycan attachments that enhance the affinity of FGFR for FGF and increase the signaling capacity of FGFR (Roghani and Moscatelli, 2007; Shi et al., 1993; Shimizu et al., 2001; Wang et al., 1995; Xu et al., 1992). There are numerous examples of isoforms of FGFR1-3 lacking IgI and AB. A FGFR1 isoform lacking IgI, termed FGFR1β (compared toFGFR1α that contains all three Ig-like domains), has been reported (reviewed in Groth and Lardelli, 2002) (Table I). As expected, the FGFR-1β isoform has a higher affinity for some FGF ligands compared to the FGFR-1α isoform (Barnard et al., 2005; Johnson et al., 1990). Although no differences in signaling were observed (in terms of cell proliferation and survival and ligand specificity), the level of activation differed. FGFR1β 7

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showed higher affinity for low concentrations of FGF1, leading to enhanced signaling and increased proliferation. FGFR1βalso showed altered nuclear localization when compared with FGFR1α. Tissue-specific alternative splicing involving IgI has also been found. FGFR1α is the predominant form of receptor expressed in brain and during mouse embryogenesis (Bernard et al., 1991; Werner et al., 1992). In contrast, FGFR1β, the two Ig domain forms, are only detected after birth. Postnatally, the 3 Ig and 2 Ig forms are simultaneously expressed at nearly equal levels in a number of different tissues, including heart, lung, and muscle. In brain and kidney, however, the 3 Ig domain forms continue to be the predominant, if not the exclusive, form expressed. There is also evidence that ligand activation of FGFR-1β, but not FGFR-1α, modulates endothelial cell growth during neovascular formation via the Src family of nonreceptor tyrosine kinases (Zhang et al., 2006). Isoforms of FGFR3 lacking IgI and/or AB/linker have been implicated during chondrogenesis. Normally, FGFR3 acts as a negative regulator during chondrogenesis (Kannan and Givol, 2000; Shimizu et al., 2001) and signals through the Ras-MAPK pathway, which leads to cell proliferation, and STAT1, which induces cell cycle inhibitors (Sahni et al., 1999) (Table I). In an undifferentiated chondroprogenitor cell line, the FGFR3 isoform lacking the AB domain is present and mediates a higher mitogenic response to FGF2 and FGF1 (compared to the full length FGFR3), suggesting therefore that the effects of the FGFR3 isoform are no longer inhibitory. In vitro studies also showed that the FGFR3 missing the AB domain does not induce the STAT1 signaling pathway and has lost the ability to activate signals involved in cell rounding (Shimizu et al., 2002)(Fig. 3). So far, only a short form of FGFR4, which lacks a hydrophobic signal sequence, the first Ig domain and the AB, has been identified in rat (Horlick et al., 1992) and no such isoform has been found in humans. For FGFR1, the serine/arginine (SR) and members of the heterogeneous nuclear ribonucleoprotein (hnRNP) families of proteins have been demonstrated to be mediators and inhibitors of exon inclusion, respectively (reviewed in Jin and Cote, 2004). Production of FGFR1α requires the inclusion of one additional exon via an exonic splicing enhancer (ESE) sequence (Jin et al., 1998). Two intronic splicing silencer (ISS) sequences that flank this exon play a critical role in excluding the exon to produce FGFR1β (Jin et al., 1999). 4.2.2 Lacking exon 8/9 and 10 There are a number of variants of FGFR2 and FGFR3 with exon 7 (encoding the N-terminal half of IgIII) spliced directly to exon 11, therefore lacking the transmembrane region (Fig. 3II). These receptors, being devoid of the TM domain, are soluble and will be discussed in the next subsection. 4.3 Soluble receptors Several soluble FGFRs have been described in the literature. Some are truncated receptors that harbor IgI, IgII and IgIIIa, with or without fusion to the first three amino acids of the TM domain or non-coding intronic sequences (Hanneken, 2001; McKeehan et al., 1998) (Fig. 3II). Some of these truncated receptors are probably eliminated by the nonsense-mediated decay pathway machinery (Maquat, 2005). In a mass spectrometric study, a number of cleaved and alternatively spliced FGFRs were found in multiple biological fluids, including blood (Hanneken, 2001). For example, soluble FGFR1s were mapped and found to be the two and three Ig-like domain 8

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isoforms of FGFR1 IIIc and a carboxyl-terminal cleavage peptide from the two and three Ig-like domain isoforms of FGFR1IIIb. Secreted receptors truncated after either IgI or IgIII by the introduction of early stop or premature termination codons have also been identified in other studies (Johnson and Williams, 1993; Wheldon et al., 2011). For example, a secreted receptor, FGFR1IIIa, is the IIIa splice form that contains intronic sequence upstream of exons 8 and 9, terminating within the IgIII domain and with no known signaling ability (Duan et al., 1992). There are also variants of FGFR2 and FGFR3 lacking the transmembrane region, with exon 7 spliced directly to exon 11, and which may be targeted to the nucleus (Johnston et al., 1995). An intracellular non-transmembrane type isoform of FGFR3 lacking signal peptides has been reported (Johnston et al., 1995). The FGFR3 isoform missing exons 8 to 10 binds to FGF1 and FGF2 (Terada et al., 2001), indicating relaxed ligand-binding specificity. A C-terminally truncated isoform of FGFR4, containing only the signal peptide and the first Iglike and AB domains, has also been identified in human breast cancer MCF-7 cells. This soluble isoform is capable of attenuating FGF1 signaling. A FGFR4 transcript where exon 9 is displaced by intron 9 leading to loss of the transmembrane domain is also found in human intestinal epithelial cells (Takaishi et al., 2000). There are several possible functions for a soluble or secreted receptor (Root and Shipley, 2000). For example, soluble receptors can compete with ligands for binding to membrane-bound counterparts, having dominant-negative suppressive effect by prevention of receptor dimerization. Soluble receptors can also be involved in transportation of ligand to aid in the availability of the ligand to membrane-bound counterparts extracellularly. 4.4 Deletions in C-terminal end The long C-termini of FGFRs are known to contain regulatory domains. Three isoforms containing progressive reduction in their cytoplasmic carboxyl termini have been reported in the literature. These three variants appear to have a common splice donor site 30 bp downstream of the tyrosine kinase domain and are spliced alternatively to produce three COOH-termini: C3, C2, C1 (Fig. 4 I, top). The C2-type carboxyl terminus is 34 aa shorter than the C1-type carboxyl terminus. The C3-type carboxyl terminus is 19 aa shorter than C2-type carboxyl terminus and therefore lacks 5 tyrosine residues present in C1. The presence of a consensus sequence of intron-exon junction “AG” and two polyadenylation signals suggests that the C1-type and C2type carboxyl termini are encoded by the same exon with two different splice acceptor sites, whereas the C3-type carboxyl terminus is encoded by a separate exon (Itoh et al., 1994) (Fig. 4II). Different combinations of C-terminus with presence or absence of IgI exist, e.g., K-sam-II which is FGFR2IIIb that lacks IgI and with its carboxyl terminus truncated (Fig.5 I, bottom). The sequence differences of the three variants result in differential retention of tyrosine residues which serve as docking and receptor autophosphorylation sites for cytoplasmic signaling proteins (Fig. 4II). Their truncation leads to disordered phosphorylation of the target proteins and enhanced transforming activity on the NIH3T3 cells. The C-terminal FGFR2 truncation interferes with receptor internalization (Cha et al., 2009), thereby preventing a potential mechanism of signaling and contributing to constitutive activation of the receptor. Indeed, it was found that FGFR2 C1 contains a putative YXXɸ motif that, if disrupted or missing, may impair receptor internalization and contribute to the enhanced transforming activity of the C3 variant. 4.5 Presence of specific amino acids

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For FGFR1-FGFR3 (but not FGFR4), two competing 5’ splice sites are present at the 3’ end of exon 10 (Hou et al., 1992; Root and Shipley, 2000). These splice sites direct inclusion or exclusion of 6 nucleotides (GTAACA) encoding the valine and threonine (VT) motif which is required for interaction of the receptor with the FRS2 signaling adapter protein (Burgar et al., 2002) (Fig. 3III).The residue in the FGFR1 VT+ isoform is phosphorylated by protein kinase C, with the VT+ isoform competent of initiating signaling via the MAPK signaling pathway (Twigg et al., 1998). Thus a relatively minor change in the sequence encoded by the mRNA has a dramatic effect on the function of the resulting protein. 5. FGFR isoforms and cancers FGF signaling pathways have been implicated in tumor development and progression (reviewed in Ahmad et al., 2012). Several reports have indicated different oncogenic potential of the various isoforms of the FGFRs. Groups of FGFR isoforms shown to be involved in cancers are: 5.1 Switch in expression between alternatively spliced isoforms Strong evidence exists for a switch between alternatively spliced isoforms in various cancers (reviewed in Ahmad et al., 2012; Grose and Dickson, 2005; Oltean and Bates, 2013). This is best exemplified by exon switching from the epithelial IIIb to the mesenchymal IIIc isoform, leading to abnormality of paracrine signaling interactions between the epithelium and the mesenchyme. The disruption of the delicate homeostasis between epithelial and mesenchymal tissues maintained by the tissue-specific expression of the IIIb and IIIc isoforms and their ligands has been described in animal models of prostate and bladder cancer and epithelial cell tumor progression (Oltean et al., 2006; Savagner et al., 1994; Yan et al., 1993). Up-regulation of FGFR1IIIc isoform is also associated with high-grade astrocytomas (Yamaguchi et al., 1994), ovarian cancer (Valve et al., 2000), oral squamous cell carcinoma (Drugan et al., 1998), bladder cancer (Chaffer et al., 2006), and non-small cell lung cancer cells (Marek et al., 2009). For example, analysis of the expression of FGFR2 isoforms during malignant progression of epithelial cells in a rat prostate tumor model revealed a switch from expression of exclusively exon IIIb to exclusively exon IIIc in malignant epithelial cells (Yan et al., 1993). In poorly differentiated prostate cancers, several FGFs, including FGF2 (Giri et al., 1999) and FGF6 (Ropiquet et al., 2000), are up-regulated, suggesting the potential existence of a paracrine loop with the up-regulated FGFR1IIIc isoform. In rat bladder cancer cells, FGFR2IIIc expression correlates with EMT, a process associated with tumor progression and invasion (Baum et al., 2008) In humans, the link between exon switching and cancers is not as clear. Studies of 16 human samples of prostate cancers showed that only a subgroup of these patients showed an increase of the FGFR2IIIc isoform, with the majority containing the FGFR2IIIb isoform (as found in normal epithelial cells) (Kwabi-Addo et al., 2001). Seventy-one percent of patients with pancreatic cancer, however, showed an abundance of FGFRIIIc isoform (Matsuda et al., 2012). Expression levels of FGFR2IIIc were also found to be positively correlated with increasing stage of colorectal carcinoma, metastasis and poor prognosis (Matsuda et al., 2012). The expression levels of some isoforms have also been found to be different in cancers. FGFR2IIIb over-expression is correlated with well-differentiated histologic type of colorectal carcinoma (Yoshino et al., 2005). Down-regulation of FGFR2 IIIb as well as FGFR2 IIIc has been reported in several human cancers, suggesting that FGFR2 in some cases might function as 10

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a tumor suppressor (Ricol et al., 1999). It has also been shown that down-regulation of FGFR3 IIIb expression in colorectal carcinoma cells is mediated by aberrant splicing that produces outof-frame nonsense transcripts (Jang et al., 2000). 5.2 Expression of distinct isoforms: Loss of IgI and AB/linker domains is implicated in numerous types of cancer (Kobrin et al., 1993). An increase in the FGFR1β:FGFR1α ratio has been associated with tumor progression, reduced relapse-free survival, and malignancy in astrocytomas (Yamaguchi et al., 1994), breast cancers (Luqmani et al., 1992), pancreatic cancers (Vickers et al., 2002), multiple myelomas (Onwuazor et al., 2003), urothelial carcinoma cell lines and tumors (Tomlinson and Knowles, 2010). Since FGFR1β shows a higher affinity for low concentrations of FGF1, a FGFR1α-toFGFR1β isoform switch and increased FGF1-induced activation of FGFR1β may result in a proliferative advantage that plays a key role during bladder tumor progression (Tomlinson and Knowles, 2010). A soluble splice variant lacking exons encoding the COOH-terminal half of IgIII and TM domain of the IIIb form was shown to be present in cultured normal human urothelial cells and inhibits proliferation; however, this variant was repressed in urothelial carcinoma cell lines (Tomlinson and Knowles, 2010). Other examples in the literature include deletion of exons 9 and 10, found in breast epithelial tumor cell line (MCF-7) (Johnston et al., 1995), human osteosarcoma cell line SaOS-2, squamous carcinoma line DJM-1, all soluble and capable of binding both FGF1 and FGF2 (Jang and Chung, 2003; Terada et al., 2001). There is preferential expression of the C2 and C3 isoforms in human cancers. For example, expression of isoform C3 has been found in gastric cancer cell line (Itoh et al., 1994), of C2 and C3 in human breast carcinoma cell line (Cha et al., 2008), with loss of Tyr-770 alone enhancing FGFR2 IIIb C1 transforming activity (Cha et al., 2009). K-sam was first identified in a human cancer line (Hattori et al., 1990; Nakatani et al., 1990). 6. FGFR isoforms and human birth defects Aberrant regulation of alternative splicing in some proteins has been implicated in several human diseases (Faustino and Cooper, 2003; Lopez, 1998). The FGF pathway was one of the first signaling pathways found to be directly involved in human skeletal dysplasias and it affects both limb development and suture closure of the skull (reviewed in Baldridge et al., 2010; Wilkie, 2005). A soluble truncated FGFR2 molecule encoded by a premature termination codoncontaining transcript is up-regulated and persists in tissues of the Apert Syndrome (AS) craniosynostosis mouse model (Wheldon et al., 2011). This isoform arises from aberrant splicing of FGFR2 exon 7 (IIIa) into exon 10 (the transmembrane domain) and negatively regulate FGF signaling in vitro and in vivo. Interestingly, the presence of this soluble isoform in patients with AS is suggested to result in loss-of-function changes, in contrast to the gain-ofFGFR2 signaling frequently ascribed to FGFR mutations in craniosynostosis syndromes. 7. Concluding remarks and perspectives The existence of many isoforms of FGFR is clearly documented, with the functions of some of these isoforms known and others not as clearly documented. Most of these isoforms were identified or characterized in the 90’s, with relatively slow progress in the last twenty years or so The characterization and elucidation of the specific roles of the FGFR isoforms is challenging due primarily to technical problems, e.g., difficulty in differentiating isoforms within a specific 11

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cellular context. The development of newer strategies and techniques will help accelerate the discovery of new isoforms of FGFR. The utilization of modern proteomics and high throughput technologies such as high-density microarray data of diseased tissues (de la Grange et al., 2010), exon-based clustering transcriptome analysis (Dutertre et al., 2010), high-throughput sequencing for analysis of variation in transcription, splicing and allele-specific expression across different tissues may help in the identification of the expression of new isoforms of FGFR. It can be imagined that the timing, duration and location of the expression of the many possible isoforms of FGFRs must be precisely regulated, both at the transcriptional and post-translational levels. The challenge therefore lies in identifying more precisely these regulators together with the feedback loops (both positive and negative) in which they are involved and the downstream signaling pathways activated by specific isoforms. There is already evidence, for instance, that ligand activation of alternatively spliced FGFR1 isoforms may induce differential signal transduction pathways (Jiao et al., 2003; Zhang et al., 2006). To be able to understand the specific signaling downstream pathways activated by the different isoforms will be especially challenging in light of the fact that most tissues, cell lines and diploid cell strains express more than one type of FGFR in vitro. Added to the complexity of FGFR function is the fact that ligand activation of FGFRs involves dimerization and subsequent formation of both homodimers and heterodimers between different structural isoforms. Future experiments geared towards a better understanding of the functions and signaling pathways of FGFs and their receptors have to take into consideration all the challenges involved in the study of such a diverse and complex group of growth factor receptor family. Studies aimed at characterizing the isoforms and their functional and signaling characteristics will undoubtedly contribute to our understanding of how such a ubiquitously expressed group of receptors and their ligands are able to generate the diversity of functions described in the literature. Conflict of interest: The author has no conflict of interest and has received no payment in preparation of this manuscript.

LEGENDS:

Figure 4.C-terminal truncated isoforms. I. Presence of truncations at the C-terminal ends (top), with the most common being the C1, C2, and C3. In addition to the truncations at the Cterminal end, some of these isoforms are missing certain exons at the extracellular region, e.g., IgI and/or AB (bottom). II. Schematic representation of predicted locations of C-terminus in the FGFR2 gene. Exons and introns are shown by boxes and thin lines, respectively (not drawn to scale). The putative splicing patterns are shown by a black bar (coding region),white bar (noncoding region) and dashed line (spliced region).Adapted, and reproduced with permission from Itoh et al., 1994 (Itoh et al., 1994).

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prostate epithelial cells accompany stromal independence and malignancy. Mol Cell Biol 13(8):4513-4522. Yayon A, Klagsbrun M, Esko JD, Leder P, Ornitz DM. 1991. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 64(4):841-848. Yoshino M, Ishiwata T, Watanabe M, Komine O, Shibuya T, Tokunaga A, Naito Z. 2005. Keratinocyte growth factor receptor expression in normal colorectal epithelial cells and differentiated type of colorectal cancer. Oncol Rep 13(2):247-252. Yu K, Ornitz DM. 2001. Uncoupling fibroblast growth factor receptor 2 ligand binding specificity leads to Apert syndrome-like phenotypes. Proc Natl Acad Sci U S A 98(7):36413643. Zhang P, Greendorfer JS, Jiao J, Kelpke SC, Thompson JA. 2006. Alternatively spliced FGFR-1 isoforms differentially modulate endothelial cell activation of c-YES. Archives of Biochemistry and Biophysics 450(1):50-62. Zhang Y, Gorry MC, Post JC, Ehrlich GD. 1999. Genomic organization of the human fibroblast growth factor receptor 2 (FGFR2) gene and comparative analysis of the human FGFR gene family. Gene 230(1):69-79.

TABLE I. Percentage homologies of human FGFR amino sequences. At the overall levels (top) and in the three immunoglobulin domains (IgI, II and III) , tyrosine kinase domains I and II (TK1 and TKII) and C-terminal tail. Adapted from Zhang et al, 1999.

R1 R1 R2 R3 R4

R2

R3

R4

Overall

Overall

Overa

70

61

53 55 59

65

38 80 77

87

92

62

20 64 82

82

90

49

21 72 81

88

91

54

14 61 73

75

84

39

22 69 71

77

80

41

Ig I

Ig II

Ig TK TK CIII I II tail

Ig I

Ig II

R1

Ig TK TK CIII I II tail

R2

24 77 74 Ig I

Ig II

79

Alterations in

Exons 20

Observed in

38

Ig TK TK CIII I II tail

R3

TABLE II. Groups of Identified FGFR Isoforms Types of

85

References

R4

Isoforms

affected

switching

Specific Function Ligand binding specificity

Switch of exons 8/9

• Tissue specific expression • Cancers

Missing exons

Inhibitory properties

Missing exons: Exons 1, 3

• Chondrogenesis • Cancers

Interferes with autoregulatory activities as well as receptor internalization Membrane tethering

C-terminus end

• Cancers

Missing exon: Exons 10, 8/9 Additional

• Cancers • Birth defects

Accepted Article

A Exon

B

C C-terminal truncations

D Soluble receptors

E Additional amino acids

Altered signaling

• Cheon et al., 1994; Johnson and Williams al., 1993; Werner et al., 1992; Thesleff, 2 Kettunen et al., 1998; Harada et al., 1999; Lin et al, 2009; Wang et al., 2007 • Reviewed in Ahmad et al., 2012 and Gros Savagner et al., 1994; Yan et al., 1993; M Yamaguchi et al., 1994; Giri et al., 1999; 2001; Guo et al., 2012; Ropiquet et al., 20 • Champion-Arnaud et al., 1991; Eisemann al., 1990; McKeehan and Kan, 1994; Hou al., 2001; Xu et al., 1992 • Yamaguchi et al., 1994; Luqmani et al., 1 2002; Tomlinson and Knowles, 2010 • Hattori et al., 1990; Nakatani et al., 1990; al., 2008; Cha et al., 2009

• Tomlinson and Knowles, 2010; Johnston Chung, 2003; Terada et al., 2001 • Wheldon et al., 2011

• Hou et al., 1992; Root and Shipley, 2000;

TABLE III. Comparative function and expression pattern of isoforms missing domains IgI and/or AB.

FGFR1 Full Length FGFR1α Missing IgI FGFR1β

Auto-inhibitory; expressed in brain and embryonic mouse, predominant form after birth Inhibitory function abolished; higher affinity for ligands, altered nuclear localization; expressed only after birth

Reviewed in Groth & lardelli, 2002; Bernard et al., 1991; Werner et al., 1992; Barnard et al., 2005; Johnson, 1990; Zhang et al., 2006

FGFR3 Full length

Missing IgI and/or AB

Negative regulator of chondrogenesis; induces growth arrest; signals through both MAPK and STAT1 pathways No longer inhibitory; unable to signal through STAT pathway

21

Kannan and Givol, 2000; Shimizu et al., 2001; Sahni et al., 1999; Shimizu et al., 2002

Accepted Article

TABLE I. Percentage homologies of human FGFR amino sequences. At the overall levels (top) and in the three immunoglobulin domains (IgI, II and III) , tyrosine kinase domains I and II (TK1 and TKII) and C-terminal tail. Adapted from Zhang et al, 1999.

R1 R1 R2 R3 R4

R2

R3

R4

Overall

Overall

Overa

70

61

53 55 59

65

38 80 77

87

92

62

20 64 82

82

90

49

21 72 81

88

91

54

14 61 73

75

84

39

22 69 71

77

80

41

Ig I

Ig II

Ig TK TK CIII I II tail

Ig I

Ig II

R1

Ig TK TK CIII I II tail

R2

24 77 74 Ig I

Ig II

79

85

38

Ig TK TK CIII I II tail

R3

R4

TABLE II. Groups of Identified FGFR Isoforms Types of Isoforms

switching

Alterations in Specific Function Ligand binding specificity

Missing exons

Inhibitory properties

Missing exons: Exons 1, 3

• Chondrogenesis • Cancers

Interferes with autoregulatory activities as well as receptor internalization

C-terminus end

• Cancers

A Exon

B

C C-terminal truncations

Exons affected

Observed in

References

Switch of exons 8/9

• Tissue specific expression • Cancers

• Cheon et al., 1994; Johnson and Williams al., 1993; Werner et al., 1992; Thesleff, 2 Kettunen et al., 1998; Harada et al., 1999; Lin et al, 2009; Wang et al., 2007 • Reviewed in Ahmad et al., 2012 and Gros Savagner et al., 1994; Yan et al., 1993; M Yamaguchi et al., 1994; Giri et al., 1999; 2001; Guo et al., 2012; Ropiquet et al., 20 • Champion-Arnaud et al., 1991; Eisemann al., 1990; McKeehan and Kan, 1994; Hou al., 2001; Xu et al., 1992 • Yamaguchi et al., 1994; Luqmani et al., 1 2002; Tomlinson and Knowles, 2010 • Hattori et al., 1990; Nakatani et al., 1990; al., 2008; Cha et al., 2009

22

D Soluble

Accepted Article

receptors

E Additional amino acids

Membrane tethering

Altered signaling

Missing exon: Exons 10, 8/9 Additional

• Cancers • Birth defects

• Tomlinson and Knowles, 2010; Johnston Chung, 2003; Terada et al., 2001 • Wheldon et al., 2011

• Hou et al., 1992; Root and Shipley, 2000;

TABLE III. Comparative function and expression pattern of isoforms missing domains IgI and/or AB.

FGFR1 Full Length FGFR1α Missing IgI FGFR1β

Auto-inhibitory; expressed in brain and embryonic mouse, predominant form after birth Inhibitory function abolished; higher affinity for ligands, altered nuclear localization; expressed only after birth

Reviewed in Groth & lardelli, 2002; Bernard et al., 1991; Werner et al., 1992; Barnard et al., 2005; Johnson, 1990; Zhang et al., 2006

FGFR3 Full length

Missing IgI and/or AB

Negative regulator of chondrogenesis; induces growth arrest; signals through both MAPK and STAT1 pathways No longer inhibitory; unable to signal through STAT pathway

Kannan and Givol, 2000; Shimizu et al., 2001; Sahni et al., 1999; Shimizu et al., 2002

Figure 1.Structure of mouse Fgfr gene. The murine Fgfr1–3 genes each contain 19 exons (denoted by black boxes, top), encoding specific domains (bottom schematic). The extracellular portion of the protein contains a signal peptide and three immunoglobulin-like domains (IgI to III). Between IgI and IgII domains lies the acid box (AB). The C-terminal half of IgIII is encoded by either exon 8 or 9. The intracellular portion contains a split tyrosine kinase domain (TK). Adapted from Barnard et al., 2005 (Barnard et al., 2005).SP=signal peptide, IgI23

Accepted Article

III=immunoglobulin I-III, AB=acid box, TM=transmembrane, TK= tyrosine kinase, a=Nterminal half of IgIII, b/c = C-terminal half and can be represented by either isoform b or c, coded by exon 8 or 9, respectively. Figure 2.Isoforms with exon switching. I. Most common example of this group of isoform is the possession of either exons 8 (IIIb) or 9 (IIIc) to encode the C-terminal half of IgIII. The IIIb isoform is expressed in the epithelia and binds to FGF ligands that are mesenchymal based. The IIIc isoform is expressed in mesenchymal tissues and binds to epithelial-based ligands. II. Schematic diagram of a developing limb showing the apical ectodermal ridge (AER), distal mesenchyme (DM), mesenchyme, and mesenchymal condensation. Limb bud initiation and outgrowth is regulated by a reciprocal signaling loop in which FGF10 signals to the IIIb isoform of FGFR and FGFs 4, 8, 9, and 17 signal to the IIIc isoform of FGFR1. Adapted, and reproduced with permission, from Yu and Ornitz, 2001(Yu and Ornitz, 2001).

Figure 3.Three groups of isoforms with I. Missing exons- exons 3 (IgI) and/or 4 (AB) are the most commonly missing; II. Soluble receptors - with missing transmembrane domain combined with different missing domains e.g., IgI, AB, intracellular portion; III. Extra amino acids – valine and threonine present on the juxtamembrane portion of the intracellular domain.

24

Accepted Article

Fig 1 Domain structure - horizontal .

25

Accepted Article

Fig 2 Exon switch .

26

Accepted Article

Fig 3 - deletion, soluble AA .

27

Accepted Article

Fig 4 C-terminus and exons . TABLES: Table I: Table II: Table III: and/or AB

Percentage homologies of human FGFR amino sequences Groups of identified FGFR isoforms Comparative function and expression pattern of isoforms missing domains IgI

28

Isoforms of receptors of fibroblast growth factors.

The breadth and scope of Fibroblast Growth Factor signaling is immense, with documentation of its role in almost every organism and system studied so ...
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