Receptor Tyrosine kinases PDF

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Receptor Tyrosine kinases...

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Receptor Tyrosine kinases (RTKs): Tyrosine kinases are enzymes which phosphorylate tyrosine residues (makes tyrosine negatively charged as oppose to neutral. The human genome encodes for 90 tyrosine kinases. There are two major classes of tyrosine kinases:  Non-receptor (cytoplasmic) – intracellular proteins which relay intracellular signals.  Receptor (plasma membrane associated) transduce extracellular signals to the cytoplasm. There are 58 RTKs. RTKs:  Single pass growth factor receptors.  Implicated in diverse cellular responses; cell division, differentiation, motility.  Oncogenic RTK mutations exist e.g. erbB gene encodes an N-terminal truncated, constitutively active form of EGF receptor.  Four common structural features of RTKs:  Kinase domain.  Regulatory domain.  Single TM domain.  Extracellular ligand binding domain (cysteine rich)  Cysteine residues are important for maintaining the shape of the receptor.  Ligand binding:  Induces dimerization.  Where the receptor exists in different isoforms, the dimers can be made up of the same polypeptide or may be a mixture of different isoforms.  An example of this is the platelet-derived growth factor receptor where you can have dimers of the same subunit αα or ββ or one of each subunit as a heterodimer αβ with each conformation having a different specificity towards the precise ligand that can bind.  Leads to activation of the kinase domain.  Enzymes that are turned on by this type of kinase include PI3K, GAPs and MAP kinases.  Leads to receptor transphosphorylation (autophosphorylation) of tyrosine residues in the cytoplasmic domain.  This further stimulates kinase activity.  Leads to phosphorylation of additional proteins.  Provides ‘docking sites’ for downstream signalling proteins e.g. Grb2, PLCγ  Proteins with SH2 domains are able to recognise and bind to phosphotyrosine residues. The transforming growth factor β (TGFβ):  A family of related extracellular signalling molecules that play a widespread role in development.  May also play a role in some cancer cells.  TGFβ members:  Originally identified as inducing transformation of normal cells to tumour cells in culture. Three human isoforms:  Activins and inhibin’s involved in inductive gradients in morphogenesis.  Bone morphogenetic protein (BMP7) used clinically to strengthen bone after fractures.

Transmembrane domain of an RTK:  Constitute an essential part of the dimeric RTK.  Single amino acid mutations in these TM domains affect signalling through different mechanisms.  Achondroplasia is a common cause of dwarfism and is caused by a mutation in the fibroblast growth factor receptor 3 (FGFR3) in normal development FGFR3 has a negative regulatory effect on bone growth. However, in achondroplasia, the mutated form of the receptor is constitutively active and leads to severely shortened bones.  Changes within this domain are so severe as they cause changes in:  Dimer stability  Membrane-embedded segment  Catalytic domain interactions  Extracellular contacts  Receptor down-regulation.  Ligand-receptor interactions Structure and function of the intracellular tyrosine kinase domain, and how it is regulated:  Structure looks similar between different kinase domains  specificity is encoded in specific amino acids in the domain.  Autoregulation of the catalytic pocket of some RTKs  Inactivating loop present in the cleft.  Dimerization allows trans-autophosphorylation – switches on kinase activity – phosphorylation moves the inhibitory loop out of the left.  EGFR activation requires the formation of an asymmetric dimer in which an activator kinase domain allosterically activates a receiver kinase. Juxtamembrane segments:  Found in-between transmembrane segment and kinase domain.  Involved in regulation.  Only around 40 amino acids (in EGFR)  Has two regions: JM-A and JM-B  JM-B binds to the kinase domain, causing a conformational change that results in the activation loop occluding the kinase site – inhibits kinase activation. Human EGFRs:  Four RTKs participate in signalling by EGF family of signalling molecules.  In humans they are called HER1-4 (Human EGFR).  HER3 is a ‘dead’ kinase, has no signalling ability itself – instead interacts (dimerises) with others to produce signals.  HER2 = cancer target.  HER2 does not directly bind ligand; already in a pre-activated conformation, it does not form homodimers; signals by forming complexes with ligand bound HER1, HER3 and HER4.  HER2 enhances signalling by all EGF members – less ligand required for activation.  The protein tyrosine phosphotase PTP1B dephosphorylates the EGFR – this switches off the EGFR kinase. There are 107 members of the PTP family – compared to only 90 TKs. HER2 in breast cancer:  Normal epithelial cells express very little amount of HER2 – do not grow abnormally.  Amplification of HER2 gene occurs in ~25% of breast cancers.

 Results in overexpression of HER2 protein – makes tumour cell susceptible to growth stimulation by low levels of any member of the EGFT family, so HER2 positive breast cancers grow more quickly – worse prognosis.  Monoclonal antibodies targeted to HER2 = treatment.  Herceptin = used only to treat HER2 positive breast cancer. Regulation of receptor PTP function by dimerization:  The activity of receptor protein tyrosine kinases (RPTPs) may be controlled by ligand-regulated dimerization.  Ligand binding inhibits RPTP activity by inducing dimerization. In the dimeric state there is a reciprocal inhibition of the catalytically competent D1 PTP domain.  The wedge motif of one D1 domain occludes the active site of the opposing D1 domain in the dimer.  This is the converse of the situation for receptor protein tyrosine kinases (RPTKs) in which ligandinduced dimerization stimulates autophosphorylation and activation.  Mutations in phosphotases linked to many hereditary diseases e.g. diabetes, schizophrenia, rheumatoid arthritis.  Very important in regulating RTKs. The EGFR gene is mutated in human brain tumours:  The EGFR gene is distributed in 26 exons over 200kb of genomic DNA.  In 50% of gliomas (a type of brain tumour) the gene is amplified, leading to over-expression.  In half of these cases exons 2-7 have been deleted (called a type III mutant). This leads to expression of a truncated receptor.  A type III EGFR is catalytically active in the absence of ligand (this shouldn’t happen) – tyrosine phosphorylation should be controlled by the binding of ligand. N-terminal deletions of the EGFR are activating:  Big chunk of the extracellular region is therefore missing.  The autoinhibition action (maintained by this domain) is lost.  This means without ligand being present there is still activation (though this is weak and moderate constitutive activation.  Due to constraint promotion of cell division this results in malignancy. C-terminal deletions are also activating in EGFR:  Type IV mutation: lack a bit of regulation on the intracellular face. Similarly to the type III mutation they are always a bit active – however differently to type 3 when EGF is bound to the receptor it becomes hyperactive – very EGF sensitive – leads to malignant transformation.

EGF signalling and Ras:

 Ras is at the top of three signalling pathways.  Ras belongs to a class of protein called small GTPases and is embedded in the plasma membrane.  Ras activation is triggered by GEFs e.g. SoS – these promote the exchange of GDP for GTP.  Ras inactivation is triggered by GAPs (GTPase activating protein) – enhances the intrinsic GTPase activity of Ras proteins.

GRB2:  Is the linker protein that connects the GFR to the signalling pathways. The protein links receptors to SoS (A Ras GEF – activates Ras).  Growth factor binds to the RTK, initiating its autophosphorylation – Grb2 binds to phosphotyrosines via its SH2 domain.  The Grb2 SH3 domain subsequently binds SoS:  SoS usually resides in the cytosol, away from Ras (which is embedded in the membrane)  SoS is recruited to the membrane by Grb2 where it is now in proximity of Ras. SoS can now activate Ras  this triggers downstream signalling pathways. Immediate vs delayed early genes:  Immediate early genes: Activation of pre-existing inactive transcription factor:  Very fast (within minutes of GF stimulation).  Examples of genes include – β-actin, γ-actin, tropomyosin, fibronectin, glucose transporter.  Delayed early genes: Requires the synthesis of a new transcription factor.  Slower....