Receptors and Tyrosine Kinases
By Thomas G. Brock, Ph.D.
Central to cell signaling are two types of proteins, the receptors and the kinases. The former perceive and respond to the extracellular cues that arrive at a cell’s surface, while the latter propagate the response within the cell by phosphorylation and altering the activity of assorted specific targets. The receptor tyrosine kinases (RTK) integrate these functions, responding to extracellular cues by phosphorylating intracellular targets. Closely related are a group of receptors which, before or after activation, bind TKs to initiate signaling. Many RTKs and receptor-associated TKs direct events essential to cell division, cellular differentiation, and morphogenesis. As a result, their actions are central to ontogeny and development, as well as diseases like cancer. This article gives a brief overview of RTKs.
The Human Tyrosine Kinases
The human kinome was characterized almost a decade ago.1 Human TKs are a large family with some 90 members which can be organized phylogenetically (Figure 1). Functionally, the TKs can be separated into RTKs and non-receptor TKs (also called cytoplasmic TKs, CTK). RTK, but not CTK, have a transmembrane helix separating the kinase domain from a ligand binding domain. Interestingly, the RTKs, shown in blue in Figure 1, are not neatly segregated from CTKs (in red) when organized phylogenetically. As a group, the TKs are newer than their functional analogs, the Ser/Thr kinases. The TKs are absent from plants and unicellular organisms like Dictyostelium and yeast. Moreover, certain families have shown substantial expansion in humans: there are 14 genes for the Eph family RTKs in humans but only 1 in flies. Such expansion is thought to relate to a role for these RTKs in processes that are more advanced in humans, such as angiogenesis, hematopoiesis, and functioning of the nervous and immune systems.1
Diversity in Structure and Substrate
The 50-plus human RTKs have been divided into some 20 families.2 As the Eph family has 14 members, the remaining families are very small. The splintering of the RTKs into numerous families reflects diversity of structure, which in turn relates to the variety of agonists which act specifically at each type of receptor. An examination of individual members of each of the branches of the TK tree reveals certain notable findings (Figure 2). First, many of the CTKs contain either Src homology 2 (SH2) or SH3 domains, or both. These domains are involved in binding the TK to other proteins. The SH2 domain specifically recognizes phosphorylated tyrosine residues. Thus, a CTK may bind an activated (and autophosphorylated) RTK via an SH2 domain, only to then tyrosine phosphorylate its own substrate. Other CTKs contain additional domains which move the kinase to specific positions within the cell and in this way control the subcellular targeting of kinase activity. For example, JAKs contain a FERM domain, which positions these CTKs on cytoplasmic tails of receptors for cytokines and polypeptide hormones.3 Another point of interest centers on the kinase domains. While most TKs have highly similar kinase domains, many growth factor RTKs (e.g., FGF receptors) have large inserts in the conserved sequence. Similarly, the TRKC kinase domain is 20% larger than the classic domain because of numerous small inserts. The JAKs contain 2 kinase-like domains, with the C-terminal one displaying activity and the other serving a regulatory role. Finally, the RTKs have many types of ligand binding domains (LBD), hinting at the diversity of potential ligands. Remarkably, even similarities in LBDs are misleading. For example, the ephrin LBDs of the EphA receptors generally bind a different class of ligand from those of the EphB RTKs. Similarly, both CSF1R and PDGFRα have clusters of five immunoglobulin-like domains for binding ligands, yet they have distinct agonists. The apparent absence of LBDs on HER2 (EGFR2) invites further consideration of the related receptor, EGFR.
EGFR, also known as HER1 or erbB1, is one of four related RTKs which can homo- or heterodimerize following ligand binding. EGFR binds EGF, TGF-α, amphiregulin, or epigen and, after dimerization, undergoes autophosphorylation (Figure 3). These tyrosine phosphorylated sites are targeted by the SH2 domains on a variety of proteins, including Grb2, Shc1, p85α and β, PLCγ, and JAK1.4 Grb2 and Shc1, with SOS, signal to activate the Ras/Raf/MEK/ERK1,2 pathway, altering gene expression and promoting cell proliferation. Through p85α and β, PI3K converts phosphatidylinositol (4,5)-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and directs PKD1 to activate Akt, which promotes cell survival through several mechanisms. PLCγ mediates hydrolysis of PIP2 to give inositol (1,4,5)-trisphosphate (IP3) and DAG, leading to the release of calcium from intracellular stores and the activation of certain PKC isoforms, which can alter gene expression, cell mobility, and, in cancer, metastasis. JAK1, through STAT3, alters gene expression related to cell survival and proliferation.
There are four genes encoding EGFR family products, with the four full length products named HER1-4 (erbB1-4). Importantly, the different monomers can homo- or heterodimerize, with profound implications. As noted earlier, HER2 has no recognized LBD, but it can dimerize with EGFR (HER1) and signal in response to ligands that activate EGFR. Or, HER2 can dimerize with HER3 and facilitate its signaling through its ligands, the neuregulins. HER3 itself has a neuregulin binding domain but lacks an active kinase domain, and thus requires heterodimerization for signaling. Amplification or overexpression of either HER2 or HER3 occurs in numerous cancers. HER4 is also activated by neuregulins. Alternatively-spliced transcriptional variants as well as proteolytic cleavage products of these receptors act outside the cell or within the nucleus to evoke a wide range of effects.
Signaling initiated by interferon-γ (IFN-γ)
IFN-γ is a cytokine with prominent anti-viral, immunoregulatory, and anti-tumor roles and is a potent activator of macrophages. Produced primarily by activated T-cells, natural killer (NK), and NKT-cells, IFN-γ acts as a dimer when binding to its multimeric receptor, IFNGR (Figure 4). The IFNGR receptor consists of two α subunits (IFNGR1), which combine to form an LBD and have long cytoplasmic tails, and two β subunits (IFNGR2), which lack an LBD and have relatively short cytoplasmic tails.5,6 Each subunit has an inactive form of JAK constitutively bound to its intracellular domain, with JAK1 on α subunits and JAK2 on β subunits. The initial interaction of a single IFN-γ dimer with the LBD of an α subunit triggers receptor assembly followed by JAK1-JAK2 transactivation. Tyrosine phosphorylation of the tails of both α subunits by JAKs provides docking sites for two Stat1 molecules, which are then phosphorylated by JAKs. This releases the Stat1 pair, which dimerize and translocate to the nucleus to alter gene expression.
A large selection of TK inhibitors have been developed for research applications and are available from Cayman Chemical. Inhibitors of both RTKs and CTKs are also headlining the pharmaceutical news. For example, currently available multi-targeted VEGF tyrosine kinase inhibitors (TKI) have been approved for treating renal cell carcinoma. These TKI, which include sunitinib, sorafenib, and pazopanib, are effective but have off-target effects. An oral inhibitor of SYK, PRT062607, shows potential for the treatment of rheumatoid arthritis as well as other autoimmune and inflammatory diseases. These examples suggest the importance of TKs in pathogenesis.
1. Manning, G., Whyte, D.B., Martinez, R., et al. Science 298, 1912-1934 (2002).
2. Robinson, D.R., Wu, Y.-M., and Lin, S.-F. Oncogene 19, 5548-5557 (2000).
3. Yamaoka, K., Saharinen, P., Pesu, M., et al. Genome Biol. 5(12), 1-6 (2004).
4. Ono, M. and Kuwano, M. Clin. Cancer Res. 12, 7242-7251 (2006).
5. Kotenko, S.V., Izotova, L.S., Pollack, B.P., et al. J. Biol. Chem. 270(36), 20915-20921 (1995).
6. Bach, E.A., Tanner, J.W., Marsters, S., et al. Mol. Cell. Biol. 16(6), 3214-3221 (1996).
Figure 1. Phylogenetic relationships of the human tyrosine kinases
(receptor tyrosine kinases: blue; non-
Figure 2. Structural features of representative TKs from different branches of the TK phylogenetic tree
Figure 3. Signaling through EGFR
Figure 4. IFN-
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