Introduction:

Receptor tyrosine kinases (RTKs) influence the cell cycle, cell mitration, cell metabolism and cell proliferation –virtually all aspects of the cell are affected by signaling through these receptors. Some of the earliest example of cancer cuasing genes or ongogenes involved RTK function.

About two-thirds of the 90 tyrosine kinase (TK) genes described in the human genome encode for receptor tyrosine kinases (RTKs). (Bonfil, “Role and significance of c-KIT receptor tyrosine kinase in cancer: A review” Pathology, 2022).

Structure:

Receptor tyrosine kinases are one class of enzyme linked receptors that phosphorylate specific tyrosines on signaling proteins. The cascade of events for RTKs generally takes longer than those for GPCRs (minutes rather than seconds). Tyrosine kinase receptors bind an extracellular ligand and a rearrangement of the transmembrane receptor chains is induced so that the two kinase domains autophosporylate and create docking sites for docking proteins. Phosphorylated tyrosines serve as docking sites for a whole range of intracellular signaling proteins that usually share highly conserved phosphotyrosine binding domains such as  SH2 domains. Many signaling proteins also contain other protein modules that allow them to interact with other proteins as part of the signaling process such as the SH3 domain.

58 unique known RTKs are subdivided into 20 subfamilies. All have an extracellular region with ligand-binding domains, a single transmembrane alpha-helix, and a cytoplasmic region consisting of a juxtamembrane domain, a tyrosine kinase domain (TKD), and a C-terminal tail. The ligand-binding domains of the extracellular region vary based on the receptor subfamily. For the majority of RTKs, the ligand that is bound is a soluble growth factor peptide. Exceptions include the ephrin (Eph) family of receptors, whose ligands are membrane-bound ephrins on nearby cells, and discoidin receptors, activated by collagen fibers. (Silberstein, “Physiology, tyrosine kinase receptors” 2022).

Signalling Pathways of Receptor Tyrosine Kinases

On ligand binding to a specific TRK receptor, two of the recetpor ligand complexes associate togetehr (dimerization) and phosphorylate each other (autophosphorylation) which transmits across the membrane the signal that began with the binding of the lgiand to the creptor. Next, propagation of the signal in the cyoplasms can take a variety of different forms which include activaiton of the tyrosine kinase domain to phosporylate other intracellular targets or interaction of other proteins with the phosphorylated receptor. The cellular response after activation depends on the possible response proteins in the cell. Two different cells can have the same receptor eyt a different response, depnding on what response proteins are present in the cytoplasm. For example, fibroblast growth factor simulates cell division in fibroblasts but stimulates nerve cell to differentiate rather than to divide.

A whole range of intracellular signaling proteins can bind to the phosphotyrosines on activated receptor tyrosine kinases or on special docking proteins to help relay the signal onward. Some docked proteins are enzymes, such as phosphlipase C-y (PLC-y) which functions in the same way  as phospholipase C-B in connection with G protein linked receptors by activating the inositol phosphlipid signaling pathway thereby increasing cytosolic Ca2+ levels. Some signaling proteins are composed almost entirely of SH2 and SH3 domains and function as adaptors to couple tyrosine phosphorylated proteins to other proteins that do not have their own SH2 domains. For example, adaptor proteins such as Grb-2 help couple activated receptors to the downstream signaling protein ras. The Grb-2 protein binds through its SH2 domain to specific phosphotyosines on activated receptor tyrosine kinases and through its SH3 domains to a GEF called Sos. The assembly of the complex receptor-Grb-2-Sos brings Sos into position to activate neighboring Ras by stimulating it to exchange its bound GDP for GTP. Ras activates multi downstream pathways including the MAP-kinase pathway which is important in gene expression and for regulating protein activity.

Phosphatidylinositol 3-kinase (PI 3-kinase) is an intracellular signaling pathway leading to cell growth. This kinase principally phosphorylates inositol phospholipids rather than proteins and it can be activated by receptor tyrosine kinases. When activated PI3-kinase catalyzes the phosphrylation of inositol phospholipids at the 3 position of the inositol ring to generate lipids called PI(3,4)P2 or PI(3,4,5)P3. Intracellular signaling proteins bind to the PI(3,4)P2 and PI(3,4,5)P3 mainly through their pleckstrin homology (PH) domain. For example, PI3-kinase signals cells to survive by indirectly activatingprotein kinase B (PKB) (also called Akt) which is a kinase with a PH domain.

It is important to distinguish the inositol phospholipids PI(3,4)P2 and PI(3,4,5)P3 from PI(4,5)P2 which is cleaved by PLCB (in the case of G protein linked receptors) or PLC-y (in the case of receptor tyrosin kinases) to generate soluble IP3 and membrane bound diacylglycerol. PI(3,4)P2 and PI(3,4,5)P3 are not cleaved by PLC.

Specific Types of Receptor Tyrosine Kinases

In humans, the 58 RTKs described so far are classified into 20 subfamilies or classes based on the structure of their amino (N) terminal ligand binding ectodomains, which consist of one or mroe defined motifs including cysteine rich regions, fibronectin type III like domains, immunoglobulin (Ig)-like domains, kringe-like domains, epidermal growth factor like domains, cadherin-like domains, discoidin-like domains, and leucine-rich regions. Among the different classes of human RTKs, class III RTKs which are characterized by the presence  of five Ig-like EC domains, including platelet-derived growth factor alpha and beta receptors (PDGFR alpha/beta), colony-stimulating factor 1 receptor, fjm-like RTK 3, and c-KIT. These RTKs play a pivotal role in several aspects of normal cell physiology, and different mutaitons that affect them can cause aberrant downstream signaling that is often linked to many disorders, including cancer. (Bonfil, “Role and significance of c-KIT receptor tyrosine kinase in cancer: A review” Pathology, 2022).

c-Kit: 

The proto-oncogene c-kit, mapped to chromosome 4q11-12 in humans and chromosome 5 in mice was discovered in 1986 as the cellular homolog of the trans-forming viral ocogne v-kit in the Hardy-Zuckerman 4 feline sarcoma virus. Wild-type c-kit encodes for a 145 kDa, 976 amino acid type IIIa RTK protein known as c-KIT, which is often referred to as CD117 or stem cell factor receptor due to its association with its ligand SCF. The -KIT protein resides in the cell membrane and is comprised of EC, TM, and intracellular (IC) regions. Like all class III RTKs. the EC porition of c-KIT comprises 5 Ig-like domains (D1-D5). The frist three domains are essential for c-KIT binding to SCF, whereas D4 and D5 are involved in dimerizing adjacent c-KIT monomers. Different c-KIT isoforms generated by alternative mRNA splicing have been described, including two that differ by the presence or absence of the tetrapeptide sequence lycine-asparagine-asparagine-lysine (GNNK) in the EC domain. Upon physiological conditions and when it is not bound to SCF, c-KIT resides in the cell membrane as a monomer. In this resting state, c-KIT is xcis-autoinhibited by the JM domain that inserts between the TK1 (N-lob) and TK2 (C-lobe) domains. This leads to a static configuration that sterically blocks the activaiton loop that resides in the catalytic cleft between the lobes form assuming an extended and active conformation (JM autoinhibition). The binding of dimeric SCF to D1-D3 regions bridges two adjacent c-KIT molcules together and leads to a D4 and D5 reorientation taht results in c-KIT homodimerization. This conformation change leads to trans-autophosphorylation of selected tyrosine residues events that appear to occur in a specific order. Many of the tyrosine residues serve as substrate docking sites after transphorsphorylation, activating downstream transduciton pathways that lead to various cellular resposnes. Various cancers present an aberrant activaiton of C-KIT kinase, caused eitehr by overexpression or mutations of c-kit. Most of the 500 c-kit mutations identified so far in human cancer are passenger thater than driver mutations. To target and inhibit dysregulated c-KIT, small molecule inhibitors and monoclonal antibodies have been two main approaches. Among small molecule inhibiors, the frist one developed was imatinib mesylate (Gleevec) which are originally found to inhibit the TK activity of the chimeric BCR-ABL fusion ocoprotein resulting from the translocation of t(9;22) in chronic myelogenous leukemia and was approved for teh treatment of this hematologic cancer in 2001. Serendipitously, imatinic was also found to inhibit the autophosphorylation and activaiton of some RTKs, such as c-KIT and PDGFR and was approved as standard firt line treatment fo metastatic GIST. (Bonfil, “Role and significance of c-KIT receptor tyrosine kinase in cancer: A review” Pathology, 2022).

Epidermal Growth Factor Receptor (EGFR, ErbB-1 and HER1 in humans): is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF) of extracellular protein ligands.

Epidermal growth factor receptor (EGFR) is the first discovered and prototypical member of receptor tyrosine kinase family (RTK) of receptors. It is activated by various ligands in extracellular milieu and transmits the cellular response to mediate various cellular activities, including cell proliferation, cell survival, growth, and development.

Growth factors belong to a family of polypeptides which have been shown to stimulate proliferation and/or differentiation in both normal and malignant cells, with EGF being one of the frist growth factors discovered. Later studies have shown that this protein binds to EGFR. The binding of a ligand to the EGFR indcues conformational changes within the receptor which increases the catalytic activity of its intrinsic tyrosine kinase, resulting in autophosphorylation which is necessary for the biological activity. The ligands binding to EGFR are, besides EGF, transforming growth factor-alpha, amphiregulin, Heparin-binding EGF-like growth factor and betacellulin.  (Among the ligands taht bind to EGFR, a generalised motif containing six conserved cysteines is found, which via disulphide bonds creates three peptide loops. Poulsen, Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials” Annals of Oncology 8, 1197-1206, 1997).

EGFR consists of a single polypeptide chain of 1186 amino acids, M, 170 KDaltons (kDa) and is expressed on the surface of the majority of normal cells. The extracellular aminoterminal end can be divided into four domains with domain III responsible for ligand binding. The cytoplasmic carboxy-terminal region of the EGFR is the region responsible for the tyrosine kinase activity and carboxyterminal regulatory functions. Tyrosine phosphorylation is a key element in the signal transduction mediated by EGFR. The stimulation of PLC-y by the EGFR mediated tyrosine posphorylation casues the release of Ca2+ from intracellular compartments and the generation of diacylglycerol, the activator of protein kinase C (PKC). PKC is a serine/threonine kinase that possibly is responsible for teh phosphorylation of the serine/threonine residues involved in teh desensitisation of EGFR. Another protein that is activated by the EGFR tyrosine kinase domain is Ras, which leads to DNA synthesis and cell prolfieraiton, through a pathway leading from teh cell surface to the nucleus. This pathway involves a large number of protein factors besides Ras, including Raf, MAPK, cytosolic kinases and nuclear transcription factors. (Poulsen, Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials” Annals of Oncology 8, 1197-1206, 1997)

EGFR tyrosine kinase is also involved in the progression of cells through G1 phase and into S phase. This progression is mediated by a family of protein kinases, the cyclin dependent kinases, (CDK) and their corresponding activating partners, the cyclins. Progression through G1 phase requries activaiton of the various cyclin-CDK kinase complexes. One of the critical substrates of G1 CDKs is the retinoblastoma protein, (RB), whose phosphorylation and subsequent release of RG-bound transcription factors are required for G1 to S phase transition. The blocking of EGFR ligand binding by a monoclonal antibody has been shown to reduce G1 phase CDK activities, causing G1 cell cycle arrest. (Poulsen, Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials” Annals of Oncology 8, 1197-1206, 1997)

EGFR has been causally implicated in human malignancy such as breast, bladder, lung, head, neck and stomach cancer. (US2009/0202546). Interruption of EGFR signalling, either by blocking EGFR binding sites on the extracellular domain of the receptor or by inhibiting intracellular tyrosine kinase activity, can prevent the growth of EGFR-expressing tumours and improve the patient’s condition. EGFR has been used as a prognostic marker for a number of years, as the overexpression of eGFR is correlated to a poor prognosis in a number of acner forms, cinluding breast cancer, gliomas, squamous carcinoma and larngeal cancer. Poulsen, Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials” Annals of Oncology 8, 1197-1206, 1997)

The avian erythroblastosis virus carries an altered form of the epidermal growth factor recetpor that lacks most of its extracellular domain. When this virus infects a cell the altered receptors produced are stuck in the on state. This continuous signaling from this receptor leads to cells that have lost the normal controls over growth.

A large number of deletions of the EGFR mRNA has been observed in a number of neoplasia, frist in glioblastoma, but also in non-small cell lung carcinomoas, breast cancer, paediatric gliomas, medullobastomas and ovarian carcinomas. These deletions are found both in the part of the mRNA that encodes the extracellular region of EGFR and in the part that encodes the intracellular region of the EGFR. A large number of these deletions are the result of genomic rearrangements, resulting in alternative splicing of the mRNA. Poulsen, Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials” Annals of Oncology 8, 1197-1206, 1997)

The down-regulation of EGFR is partly accomplished by internalisation of the activated EGFR, followed by degradation in the lysosomes, and partly by the desensitisation induced by phosphorylation of serine and threonine residues in the intracellular domain. Poulsen, Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials” Annals of Oncology 8, 1197-1206, 1997)

–HER Family: of receptor tyrosine kineases are important mediators of cell growth, differentation and survival. The receptor family includes four distnct members including epidermal growth factor receptor (EGFR or HER1), HER2, HER3 and HER4.

The ErbB family comprises of four receptors, which include EGFR (ErbB-1/HER1), ErbB-2 (Neu, HER2), ErbB-3 (HER3), and ErbB-4 (HER4). This receptor tyrosine kinase family (RTK) of proteins has an extracellular ligand-binding domain, hydrophobic transmembrane domain, and a cytoplasmic tyrosine kinase domain. ErbB receptors are activated by growth factors of EGF-family characterized by three disulphide-bonds that confer binding specificity. Additional structural motifs include immunoglobulin-like domains, heparin-binding sites and glycosylation sites.

All four members of the human epidermal growth factor (EGF) receptor (HER) family are implicated in human cancers. Although efficacious in a subset of patients, resistance to single-targeted anti-HER therapy [i.e., cetuximab (Erbitux) and trastuzumab (Herceptin)] is often associated with coexpression of other HER family members.

Fibroblast Growth Factor: are a family of RTKs expressed on the cell membrane tha that play crucial roles in both development and adult cells.

Two different cells can have the same receptor yet a different response, depnding on what response proteins are present in the cytoplasm. For example, Fibroblast growth factor simulates cell division in fibroblasts but stimulates nerve cells to differentiate rather than to divide.

Insulin Receptor: is a RTK. Insulin, which binds to this RTK, lowers blood glucose.

–Insulin Response Protein is an RTK that passes the signal on by binding to additional proteins that elad to the activiton of the enzyme glycogen synthase, which converts glucose to glycogen, thereby lowering blood glucose. Other proteins activated by the insulin receptor act to inhibit the enzythesis of enzymes involved in making glucose, and to crease the number of glucose transporter prtoeins in the plasma membrane.