RECEPTORS WITH INTRINSIC PROTEIN KINASE

ACTIVITYM.Prasad Naidu

MSc Medical Biochemistry, Ph.D,.

Receptor tyrosine kinases (RTK)s are the high-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones.

Of the 90 unique tyrosine kinase genes 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 extracellular N-terminal region, and an intracellular C-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 termedtyrosine kinases.

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 cytoplasmic kinase domains, the first substrate for these domains being the receptor itself.

The activated receptor as a result then becomes autophosphorylated on multiple specific intracellular tyrosine residues

 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 phospholipase Cγ. 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 kinase signalling cascade.

Receptors that are kinase or bind kinases.

Growth factor receptor

Insulin receptor

Tyrosine kinase domains

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 dimer phosphorylates 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

The insulin receptor PKB signalling pathway

Membrane rafts and caveolae sequester groups of signaling proteins in small regions of the plasma membrane, enhancing their interactions and making signaling more efficient.

Receptor serine/threonine kinases

Proteins in the transforming growth factor superfamily use receptors that have serine/ threonine kinase 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.

Jak – stat receptors

Jak – stat transduction mechanism

The JAK-STAT transduction mechanism for the erythropoietinReceptor

Binding of erythropoietin (EPO) causes dimerization of 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)

Signalling mechanism in bacterial chemotaxis

Similarities between the signaling pathways that trigger immune responses in plants and

mammalsimmune responses in plants and

animals.

Signal termination

Oncogene encoded defective EGF receptor

Oncogene-encoded defective EGF receptor. The product of the erbB oncogene (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 intrinsicTyr kinase 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 ras oncogene 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.

Proto oncogene sites in growth factor signalling path

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;: sunitinib and sorafenib target several protein kinases including VEGR-R and PDGF-R used for treatment of GI stromal tumors and advanced renal cell carcinoma.

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