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Receptors with intrinsic protein kinase activity M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
Receptor tyrosine kinases (RTK)s are the high-affinitycell surface receptors for many polypeptide growth factors, cytokines, and hormones. • Of the 90 unique tyrosine kinasegenes identified in the human genome, 58 encode receptor tyrosine kinase proteins. • Receptor tyrosine kinases have been shown not only to be key regulators of normal cellular processes but also to have a critical role in the development and progression of many types of cancer.
Receptor tyrosine kinases classes • RTK Approximately 20 different RTK classes have been identified.[3] • class I (EGF receptor family)(ErbB family) • RTK class II (Insulin receptor family) • RTK class III (PDGF receptor family) • RTK class IV (FGF receptor family) • RTK class V (VEGF receptors family) • RTK class VI (HGF receptor family) • RTK class VII (Trk receptor family) • RTK class VIII (Eph receptor family) • RTK class IX (AXL receptor family • RTK class IX (AXL receptor family) • RTK class X (LTK receptor family) • RTK class XI (TIE receptor family) • RTK class XII (ROR receptor family) • RTK class XIII (DDR receptor family) • RTK class XIV (RET receptor family) • RTK class XV (KLG receptor family) • RTK class XVI (RYK receptor family) • RTK class XVII (MuSK receptor family)
RTKs Most are single subunit receptors but some exist as multimeric complexes, e.g., the insulin receptor that forms disulfide-linked dimers in the absence of hormone; moreover, ligand binding to the extracellular domain induces formation of receptor dimers. • Each monomer has a single hydrophobic transmembrane-spanning domain composed of 25-38 amino acids, an extracellularN-terminal region, and an intracellularC-terminal region.
The extracellular N-terminal region exhibits a variety of conserved elements including immunoglobulin (Ig)-like or epidermal growth factor (EGF)-like domains, fibronectin type III repeats, or cysteine-rich regions that are characteristic for each subfamily of RTKs; • these domains contain primarily a ligand-binding site, which binds extracellular ligands, e.g., a particular growth factor or hormone. • The intracellular C-terminal region displays the highest level of conservation and comprises catalytic domains responsible for the kinase activity of these receptors, which catalyses receptor autophosphorylation and tyrosine phosphorylation of RTK substrates.
Kinase activity • In biochemistry, a kinase is a type of enzyme that transfers phosphate groups (see below) from high-energy donor molecules, such as ATP to specific target molecules (substrates); the process is termed phosphorylation. • The opposite, an enzyme that removes phosphate groups from targets, is known as a phosphatase. • Kinase enzymes that specifically phosphorylate tyrosine amino acids are termedtyrosinekinases.
When a growth factor binds to the extracellular domain of an RTK, its dimerization is triggered with other adjacent RTKs. • Dimerization leads to a rapid activation of the protein's cytoplasmickinase domains, the first substrate for these domains being the receptor itself. • The activated receptor as a result then becomes autophosphorylated on multiple specific intracellular tyrosineresidues
Signal transduction • The phosphorylation of specific tyrosine residues within the activated receptor creates binding sites for Src homology 2 (SH2) domain- and phosphotyrosine binding (PTB) domain-containing proteins. • Specific proteins containing these domains include Src and phospholipaseCγ. Phosphorylation and activation of these two proteins on receptor binding lead to the initiation of signal transduction pathways. • Other proteins that interact with the activated receptor act as adaptor proteins and have no intrinsic enzymatic activity of their own. • These adaptor proteins link RTK activation to downstream signal transduction pathways, such as the MAP kinasesignalling cascade.
Activation of the insulin-receptor Tyr kinase by autophosphorylation. • (a) In the inactive form of the Tyr kinase domain • (PDB ID 1IRK), the activation loop (blue) sits in the active site, and • none of the critical Tyr residues (black and red ball-and-stick structures) • are phosphorylated. This conformation is stabilized by hydrogen • bonding between Tyr1162 and Asp1132. (b) When insulin binds to • the chains of insulin receptors, the Tyr kinase of each subunit of • the dimerphosphorylates three Tyr residues (Tyr1158, Tyr1162, and • Tyr1163) on the other subunit (shown here; PDB ID 1IR3). (Phosphoryl • groups are depicted here as an orange space-filling phosphorus • atom and red ball-and-stick oxygen atoms.) The effect of introducing • three highly charged P –Tyr residues is to force a 30 Å change • in the position of the activation loop, away from the substrate-binding • site, which becomes available to bind to and phosphorylate a target • protein, shown here as a red arrow
Membrane rafts and caveolae sequester groups of signaling proteins in small regions of the plasma membrane, enhancing their interactions and making signaling more efficient.
Proteins in the transforming growth factor superfamily use receptors that have serine/ threoninekinase activity and associate with proteins from the Smad family, which are gene-specific transcription factors . • This superfamily includes transforming growth factor (TGF-), a cytokine/hormone involved in tissue repair, immune regulation, and cell proliferation, and bone morphogenetic proteins (BMPs), which control proliferation, differentiation, and cell death during development.
The JAK-STAT transduction mechanism for the erythropoietinReceptor • Binding of erythropoietin (EPO) causes dimerizationof the EPO receptor, which allows the soluble Tyr kinase JAK to bind to the internal domain of the receptor and phosphorylate it on several Tyr residues. • The STAT protein STAT5 contains an SH2 domain and binds to the P –Tyr residues on the receptor, bringing the receptor into proximity with JAK. • Phosphorylation of STAT5 by JAK allows two STAT molecules to dimerize, each binding the other’s P –Tyrresidue. • Dimerization of STAT5 exposes a nuclear localization sequence (NLS) that targets STAT5 for transport into the nucleus. • In the nucleus, STAT causes the expression of genes controlled by EPO. • A second signaling pathway is also triggered by autophosphorylation of JAK that is associated with EPO binding to its receptor. • The adaptor protein Grb2 binds P –Tyr in JAK and triggers the MAPK cascade, as in the insulin system .
Epidermal growth factor receptor family • The ErbB protein family or epidermal growth factor receptor (EGFR) family is a family of four structurally related receptor tyrosine kinases. Insufficient ErbB signaling in humans is associated with the development of neurodegenerative diseases, such as multiple sclerosis and Alzheimer's Disease • Excessive ErbB signaling is associated with the development of a wide variety of types of solid tumor. ErbB-1 and ErbB-2 are found in many human cancersand their excessive signaling may be critical factors in the development and malignancy of these tumors
VEGF Receptor Family • Vascular endothelial growth factor (VEGF) is one of the main inducers of endothelial cell proliferation and permeability of blood vessels. Two RTKs bind to VEGF at the cell surface, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1)
Similarities between the signaling pathways that trigger immune responses in plants and mammalsimmune responses in plants and animals.
Oncogene-encoded defective EGF receptor. The product of the erbBoncogene (the ErbB protein) is a truncated version of the normal receptor for epidermal growth factor (EGF). • Its intracellular domain has the structure normally induced by EGF binding but the protein lacks the extracellular binding site for EGF. • Unregulated by EGF, ErbB continuously signals cell division
Some oncogenes encode surface receptors with defective or missing signal-binding sites such that their intrinsicTyrkinase activity is unregulated. • For example, the protein ErbB is essentially identical to the normal receptor for epidermal growth factor, except that ErbB lacks the amino-terminal domain that normally binds EGF and as a result sends the “divide” signal whether EGF is present or not. • Mutations in erbB2, the gene for a receptor Tyr kinase related to ErbB, are commonly associated with cancers of the glandular epithelium in breast, stomach, and ovary.
Mutant forms of the G protein Ras are common in tumor cells. • The rasoncogene encodes a protein with normal GTP binding but no GTPase activity. • The mutant Ras protein is therefore always in its activated (GTP-bound) form, regardless of the signals arriving through normal receptors. • The result can be unregulated growth. • Mutations in ras are associated with 30% to 50% of lung and colon carcinomas and more than 90% of pancreatic carcinomas.
Development of protein kinase inhibitors for cancer treatment • Drugs that target the inactive conformation of a specific protein kinase and prevent its conversion to the active form may have a higher specifity of action. • For eg; monoclonal antibodies . They eliminate receptor kinase activity by preventing dimerisation or by causing their removal from cell surface.
erlotinib: targets (non small cell lung cancer) small molecule kinase inhibitor. • Imatinibmesylate: 100% effective in early stage CML. • TRASTUZUMAB, CETUXIMAB are monoclonal antibodies that target HER2/neu, EGF- R, and VEGF-R that are in clinical use for certain types of cancer.(lung cancer, large intestinal cancers)
Because many cell division signalling systems involve more than one protein kinase , inhibitors that act on several protein kinases may be useful in the treatment of cancer • eg;: sunitiniband sorafenib target several protein kinases including VEGR-R and PDGF-R used for treatment of GI stromal tumors and advanced renal cell carcinoma.
