Cell signaling by receptor tyrosine kinases

Abstract

Recent structural studies of receptor tyrosine kinases (RTKs) have revealed unexpected diversity in the mechanisms of their activation by growth factor ligands. Strategies for inducing dimerization by ligand binding are surprisingly diverse, as are mechanisms that couple this event to activation of the intracellular tyrosine kinase domains. As our understanding of these details becomes increasingly sophisticated, it provides an important context for therapeutically countering the effects of pathogenic RTK mutations in cancer and other diseases. Much remains to be learned, however, about the complex signaling networks downstream from RTKs and how alterations in these networks are translated into cellular responses.

Figure 1. Receptor tyrosine kinase families

Human receptor tyrosine kinases (RTKs) contain 20 subfamilies, shown here schematically with the family members listed beneath each receptor. Structural domains in the extracellular regions, identified by structure determination or sequence analysis, are marked according to the key presented in Supplementary Figure 1, where all 58 RTKs in the human proteome are listed. The intracellular domains are shown as red rectangles.

Figure 2. Receptor Tyrosine Kinase Dimerization and Kinase Activation

Top: In general, receptor tyrosine kinases (RTKs) associate into dimers when ligand (red) binds to their extracellular regions. The bound ligand, which can form all, a portion, or none of the dimer interface, activates the receptors by stabilizing a specific relationship between two individual receptor molecules. A. A nerve growth factor dimer (red) cross-links two TrkA molecules without any direct contact between the two receptors (Wehrman et al. 2007). B. A stem cell factor dimer (red) also cross links two KIT molecules. In addition, two Ig-like domains (D4 and D5), which reorient upon receptor activation, interact across the dimer interface (Yuzawa et al., 2007). Thus, KIT combines ligand-mediated and receptor mediated dimerization modes. C. Two fibroblast growth factor receptor (FGFR) molecules contact one another through the Ig-like domain D2, and the accessory molecule heparin or heparin sulfate proteoglycans (white sticks) also contacts this domain (Schlessinger et al., 2000). In addition, each fibroblast growth factor molecule (red) contacts Ig-like domains D2 and D3 of both FGFR molecules. D. Dimerization of ErbB receptors is mediated entirely by the receptor. Binding simultaneously to two sites (DI and DIII) within the same receptor molecule, the ligand drives conformational changes in epidermal growth factor receptor (EGFR) that expose a previously-occluded dimerization site in Domain II Bottom: Dimerization of the extracellular regions of RTKs activates the intracellular tyrosine kinase domains (TKDs), which contain a C-lobe (light purple or yellow), N-lobe (dark purple or yellow in the inactive and active states), and an activation loop (dark purple or yellow in the inactive and active states, respectively). Although the crystal structures of the activated TKDs are very similar (Huse and Kuriyan, 2002), structures of inactive TKDs differ substantially among the receptors (top row), reflecting the diversity in their regulatory mechanisms. However, many receptors are inhibited by a set of intramolecular (or cis) interactions: E. Insulin receptor-like (activation loop inhibition). In FGFR, insulin receptor, and IGF1-receptor the activation loop interacts directly with the active site of the kinase and blocks access to protein substrates (in FGFR) or to both ATP and protein substrates (in insulin and IGF1 receptors). Phosphorylation of key tyrosines (‘Y’) disrupts these autoinhibitory interactions and allows the kinase to ‘relax’ to the Active state. KIT-like (juxtamembrane inhibition). In KIT, PDFGR, and Eph receptors the juxtamembrane region (red) interacts with elements within the active site of the kinase (including the αC helix and the activation loop) to stabilize an inactive conformation. Phosphorylation of key tyrosines in the juxtamembrane region destabilizes these autoinhibitory interactions and allows the TKD to assume an active conformation. Tie1-like (C-terminal tail inhibition). In Tie1 and Tie2(and possibly Met and Ron), the C-terminal tail (red) interacts with the active site of the TKD to stabilize an inactive conformation(Shewchuk et al., 2000). F. The EGFR TKD is allosterically activated by direct contacts between the C-lobe of one TKD, the ‘Activator,’ and the N-lobe of another TKD, ‘Receiver’ (Zhang et al., 2006). The Activator TKD destabilizes autoinhibitory interactions that involve the activation loop of the Receiver TKD. No activation loop phosphorylation is required in this mechanism (Jura et al., 2009; Red Brewer et al., 2009).

Figure 3. Coincidence detection and network branching in RTK Signaling

A. Coordinated assembly of multiprotein complexes in receptor tyrosine kinase (RTK) signaling provides branching points in a signaling network. The docking protein FGF receptor substrate-2 (FRS2α forms a complex with activated fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors via its phosphotyrosine-binding domain (PTB). The activated RTK phosphorylates FRS2α on multiple tyrosines, and the resulting phosphotyrosines recruit multiple Grb2 and Shp2 molecules, which bring a second docking protein, Gab1, into the complex. Gab1 is tyrosine phosphorylated and recruits additional signaling proteins, including phosphoinositide 3-kinase (PI3K). PI3K initiates a positive feedback loop in which PtdIns(3,4,5)P₃ (PIP3), generated by PI3K, recruits more Gab1, leading to further PI3K activation. B. The multiple domains of phospholipase C-γ (PLCγ cooperate to integrate multiple signals at the plasma membrane. The N-terminal SH2 domain is responsible for complex formation with activated receptor tyrosine kinases (RTKs). The C2 and PH domains cooperate with the SH2 domain to target PLCγ to the plasma membrane. One or both of the PH domains may also specifically recognize products of RTK-activated PI3K. RTK-mediated tyrosine phosphorylation of PLCγ leads to intramolecular binding of the C-terminal SH2 domain to phosphotyrosine 783. This stimulates enzymatic activity of PLCγ, leading to hydrolysis of PtdIns(4,5)P₂ (PIP2) and consequently leads to the formation of Ins(1,4,5)P₃ (IP3) and diacylglycerol (DG).

Figure 4. Intracellular signaling networks activated by EGFR

A. A subset of intracellular signaling components influenced by epidermal growth factor receptor (EGFR) activation are intertwined in a complex network. Through a combination of stimulatory (black arrows) or inhibitory (red lines) signals, several key positive feedback loops (blue circular arrows) and negative feedback loops (red circular arrows) emerge in the network and exert significant influence on its behavior. For example, inhibition of Ras by Ras-GAP or EGFR by protein kinase C (PKC) serve a negative feedback function. On the other hand, H₂O₂ inhibits protein tyrosine phosphatases (PTPs) and thus, prolongs or increases activity of EGFR by a positive-feedback mechanism. B. A conceptual representation of a ‘bow tie’ or ‘hourglass’ network, as described by Kitano (2004). A wide ‘input layer’ (green) includes multiple RTKs that all influence a relatively small number of ‘core processes’ (magenta), including phosphoinositide 3-kinase (PI3K) signaling, MAPK signaling, and Ca²⁺ signaling. Feedback processes within the core define specific emergent properties of the system. The behavior of the core processes is ‘read out’ by a wide ‘output layer’ (orange) that consists of diverse transcriptional responses and cytoskeletal changes. Extensive negative and positive feedback loops exists between the core processes and the input layer. Similar feedback exists between the output layer and the core processes, in addition to ‘feed forward’ regulation by core processes (e.g., MAPK signaling) of immediate early gene products described by Murphy and Blenis (2006). An additional layer of ‘system control’ also occurs between the input and output layers.