FGF signalling pathways

The binding of the FGF ligand to its receptor causes the FGFR to dimerise. This in turn enables phosphorylation of tyrosine residues in the kinase activation loop and then of tyrosines that are docking sites for signalling proteins (Goetz and Mohammadi, 2013). These phosphorylations cause the activation of many intracellular signalling pathways, e.g., RAS-RAF-MAPK, PI3K-AKT and phospholipase-IP3 (Fig. 1.10), which regulate cell fate and specific cell activities (Fig. 1.10) (Turner and Grose, 2010, Dorey and Amaya, 2010). Previous studies suggest HS/heparin is required for most, but not all signalling (Izvolsky et al., 2003, Delehedde et al., 2000, Delehedde et al., 2002b). FGF signalling can be negatively regulated by internalisation and degradation of the ligand-receptor complex, as well as by transmembrane regulators, such as FGFRL1 and intracellular ones, e.g., MAPK phosphatase 3, sprouty and spred proteins (Turner and Grose, 2010, Casci et al., 1999, Eblaghie et al., 2003) . Since there is a great diversity of FGF ligands, FGFR isoforms, HS structure and feedback loops, the understanding of FGF signalling is still far from complete, though the link between the activation of the RAS-RAF- MAPK pathway and the stimulation of cell division is well established, as least in cultured cells (Dorey and Amaya, 2010).

Figure 1.10 FGFR signalling. The interaction of FGF with the FGFR and HS co- receptor leads to the dimerization of the FGFR and the subsequent transphosphorylation of tyrosine residues on the intracellular tyrosine kinase domain. PLC and GRB2 can dock to the phosphorylated tyrosines, while FRS2 docks to the juxta-membrane domain. The bound GRB2 activates the RAS-RAF-MAPK and the PI3K-AKT signalling pathways, while phosphorylation of PLC induces the phospholipase-IP3 pathway. The signalling pathways regulate many cell activities, e.g., cell fate, cell proliferation, migration and differentiation. FGF, fibroblast growth factor; FGFR, FGF receptor; D1-D3, FGFR immunoglobulin domains 1-3; ‘To core protein’, indicates that the HS chain (blue line) will be linked to a proteoglycan core

protein; FRS2, FGF receptor substrate 2; GRB2, growth factor receptor-bound protein 2; GAB1, GRB2-associated protein 1; PLC, phospholipase C; AKT, Protein kinase B; SOS, son of sevenless; RAF: raf-leukemia viral oncogene homologue 1; RAS, rat sarcoma; IP3, inositol trisphosphate; MEK, mitogen- activated protein kinase kinase; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; PIP2, phosphatidylinositol 4, 5-bisphosphate; red dots, phosphorylation of signaling proteins.

1.8 Binding and transport of FGFs in ECM

In development, one important feature of paracrine FGFs mediating cell-cell communication is that they will often have to diffuse from the source to the target cells. In addition, this diffusion can set up a gradient (morphogen gradient, Section 1.2). These gradients are a key part of how the fate of cells is specified and how the cell activities are regulated. In the processes of homeostasis and tissue maintenance, FGFs also play important roles. Again, they signal between tissue compartments and so ensure, for example, repair.

Abundant studies have already suggested that the ECMs in different tissues and tissues at different ages or different status are very diverse (Section 1.3) (Frantz et al., 2010, Davies, 2001, Olczyk et al., 2014, Mouw et al., 2014). These ECMs contain different components or same components with different structures, which regulates the binding and diffusion of FGFs. HS chains are the main FGF binding components and by binding to HS chains many spatial and temporal FGF morphogen gradients are formed in development (Duchesne et al., 2012, Dowd et al., 1999, Makarenkova et al., 2009, Shute et al., 2004). Binding to HS also regulates the biological activities stimulated by FGFs in many situations (Zhu et al., 2010,

Delehedde et al., 2000, Delehedde et al., 2002a, Izvolsky et al., 2003, Patel et al., 2008).

The expression of HS chains produces many different tissue-specific structures, which selectively bind to the secreted FGFs and control their diffusion (Izvolsky et al., 2003, Shute et al., 2004). Originally, it was found that FGF2 was stored in endothelial cell ECM and it could be transferred to its cellular receptors to induce cell signals (Vlodavsky et al., 1987). At the tissue level, specific distributions of many FGFs have been observed (Izvolsky et al., 2003, Harada et al., 1999, Gonzalez et al., 1996). A study with FGF2 revealed that FGF2 in many tissues (skin, heart, lung, kidney and intestine) is distributed to defined cells (Gonzalez et al., 1996). Similarly, FGF10 has been found to preferentially bind to urothelium rather than lamina propria in bladder and ureter (Zhang et al., 2006a). Even on one cell, the binding of FGF2 in the pericellular matrix is not homogeneous (Duchesne et al., 2012, Nieves et al., 2015). These studies suggest FGFs selectively bind to HS structures in the ECM, which is consistent with the in vitro studies with heparin and its derivatives (Section 1.8) and that the distribution of these structures is spatially controlled (Xu et al., 2012, Ori et al., 2009).

In embryonic development, it seems that FGFs usually diffuse through ECM, rather become trapped and stored there, as in the example of the development of vertebrate limb, in which FGF10 is expressed mesenchymally to regulate the development of the overlying apical ectodermal ridge (Zeller et al., 2009). So, how do the paracrine FGFs move to their target in the ECM, after they are secreted? HS in the ECM was found to control the diffusion of FGF2, which indicates that HS proteoglycans in the ECM might regulate morphogen gradient formation (Dowd et al., 1999). FGF10

bound to heparin acrylic beads could hardly disassociate from heparin and diffuse into either a collagen gel or Matrigel, because of its high affinity for heparin, whereas a mutant FGF10 (R178V), which bound more weakly, diffused out of the heparin bead (Makarenkova et al., 2009). FGF9 diffused more slowly in its targeting ECM than its mutant FGF9Eks which is unable to homodimerise to efficiently interact with HS (Kalinina et al., 2009). These data suggest that HS should selectively bind to FGFs if the FGF is to be allowed to diffuse, or if it is to be stored. The study of the movement of gold nanoparticle-FGF2 in the pericellular matrix of rat mammary (Rama) 27 fibroblasts (cells described in Section 2.6) suggests most FGF2 diffuses slowly by reversible binding to HS chains (Duchesne et al., 2012). Since the binding sites for FGF2 in the HS of pericellular matrix were not evenly distributed, this is likely to contribute to the regulation of the diffusion of FGF2 (Duchesne et al., 2012). However, it is still unknown whether these properties of the binding and transport of FGF2 were a general phenomenon or only true for FGF2. Heparanase, a beta glucuronidase, which cleaves HS in NA and NAS domains, was also identified as an accelerator for the diffusion of FGFs by releasing the S domains to which FGFs will generally be bound (Shute et al., 2004).