Receptor tyrosine kinases (RTKs) are a family of trans-membrane proteins that play essential roles in many biological procedures. Protein tyrosine kinase receptors are transmembrane-spanning receptors located in the cell membrane that have an intrinsic protein tyrosine kinase activity that is normally dependent on binding of the cognate ligand. There are 58 known RTKs distributed within 20 different families. Each member has a specific biological function; however the different families of RTKs are morphologically related and share a common structure consisting of a large ligand-binding extracellular domain, a lipophilic transmembrane-spanning region, and a cytoplasmic domain.
RTKs regulate crucial cellular processes such as proliferation, differentiation, cell metabolism, migration and cell-cycle control. They are the entry point to the complicated signaling networks inside cells. Aberrant regulation, amplification, and/or mutations of RTKs result in numerous diseases, including cancers, diabetes, inflammations, cardiac diseases and development disorders. RTKs are also important drug targets.
The morphology of the extracellular domain varies between the different type of receptors, and determines the proper recognition and binding to a wide variety of specific ligands. The plasticity of this region is a result of combinations of cysteine-rich motifs, immunoglobulin-like repeats (Ig-like), fibronectin type III repeats (FNIII), and EGF motifs.
The transmembrane domain anchors and stabilizes the receptor in the plasma membrane. It functions as a communication bridge between the extracellular environment and the internal compartments of the cell.
The structure of the cytoplasmic portion is composed of a catalytic tyrosine kinase domain, a juxtamembrane region and a C-terminal tail. Tyrosine kinase domain has the highest level of conservation among tyrosine kinase receptors, and its integrity is essential for adequate receptor signaling. This domain contains an ATP-binding site that catalyzes autophosphorylation of tyrosine residues of the receptor. The juxtamembrane sequence that separates the transmembrane domain and the cytoplasmic domain is well conserved between members of the same receptor family, although it diverges between different families. This domain acts by modulating the receptor activity under stimuli originating outside the receptor itself, heterologous stimuli (transmodulation) and in some instances, acts as a negative regulator. The most variable region between tyrosine kinase receptors is the C-terminal tail, which contains numerous tyrosine residues that are phosphorylated by the activated kinase.
RTKs and diseases/drug targets
Not only do RTKs regulate many biological procedures, but they are also involved, and even responsible for, many human diseases. For example, the ErbB/EGFR family RTKs are well known for their oncogenic relevance. Point mutations, deletions and insertions in EGF receptor have been linked to non-small cell lung cancer (NSCLC) and to the sensitivity to kinase inhibitors. Structural studies have rationalized some of the effects of these mutations and alterations. For example, the L834R (L858R) EGFR mutation destabilizes a conserved set of autoinhibitory hydrophobic interactions in the inactive kinase and thus effectively activates EGFR. In another form, about 200 residues are deleted form the extracellular region, resulting in an EGFR variant (variant III, or vIII), which appears to show constitutive kinase activity despite the lack of dimerization arm. Other activating alterations are found in the juxtamembrane regions of EGFR, which presumably affect the asymmetric dimerization of the receptor.
ALK is another RTK that is strongly associated with various cancers. ALK was originally discovered in anaplastic large-cell non-Hodgkin’s lymphoma (ALCL) in the form of fusion protein. Nucleophosmin (NPM) becomes fused to the ALK kinase domain by chromosomal translocation, resulting in production of a soluble dimeric (and thus constitutively activated) form of ALK’s tyrosine kinase. Other ALK fusion proteins are also found, such as with echinoderm microtubule-associated protein-like 4 (EML4) in EML4-ALK in other tumors. In addition to fusion proteins, activating point mutations in full-length ALK have been found to cause neuroblastoma, by activating the kinase domain in a manner similar to that seen for EGFR in lung cancer.
In addition to ErbB family and ALK, many other RTKs are involved in cancers and other diseases. The realization that this is the case has led to RTKs becoming important drug targets – with the goal of limiting cancer by reducing aberrant signaling activity of oncogenic RTKs. Two main forms of therapeutics have been specifically developed for RTKs - antibodies targeting the extracellular regions and kinase inhibitors targeting the kinase domains. Antibodies can either compete with ligand binding or directly bind to a different region in ECR to inactivate receptors and promote immune responses. Kinase inhibitors are small molecules that are usually ATP analogues that compete with ATP for binding to the active site of the kinase.
Gefitinib, an EGFR-specific kinase inhibitor, was initially shown to be not effective in the general population of lung adenocarcinoma patients, but was found to be efficacious in a small subset of patients classified as Asian female never-smokers (in the early stage of the cancer, Gefitinib has a superior efficacy compared to chemotherapy). Sequencing results showed that tumors from these responding patients harbor mutations in the kinase domain of EGFR, which we now know to be activating. Crizotinib, a kinase inhibitor originally developed at Sugen as an inhibitor of the Met tyrosine kinase, has turned out to be useful as an ALK inhibitor in ALK-translocated NSCLC, for which it has Food and Drug Administration approval – as well as other cancers including neuroblastoma, where it is helpful for certain patients.
Kinase inhibitors have shown promising effects by inhibiting several TKDs. It is worth noting, however, that acquired resistance to these drugs almost always develops, leading to relapse of the cancer. Biochemical and structural analyses of the kinase domain in those resistant tumors have shown that new mutations can emerge that decrease inhibitor binding, or increase (competing) ATP binding, resulting in an overall reduced inhibitor efficacy. Fully understanding the mechanisms of drug resistance is a key step towards personalized treatment of cancers involving TKD mutations in RTKs and other kinases.