What are receptors that have intrinsic enzymatic activity?
Receptor Tyrosine Kinases.
What is activation of RTKs usually associated to?
Long term changes in the cell function, where gene expression is required.
Give 3 examples of RTKs and functions regulated by them.
- EGF (Proliferation): Vascular remodelling/Cancer
- VEGF (Angiogenesis): Cancer
- Insulin (Glucose Homeostasis): Diabetes
What organisms are RTKs found in?
All multicellular organisms
What do RTKs possess in their structure in terms of the membrane/domains?
- Ligand binding domain - Extracellular
- Single hydrophobic transmembrane (TM) a-helix
- Cytoplasmic domain
What do RTKs possess in their cytoplasmic domain?
A protein tyrosine kinase enzyme in their cytoplasmic domain (intrinsic part of the receptor).
How is kinase activity stimulated in RTKs?
By binding of ligands to the extracellular domain.
What processes can kinase activity induce?
- Cell proliferation
What are the ligands for RTKs?
What 2 types of proteins can be ligands for RTKs? Give examples of each.
Secreted/Soluble Proteins - e.g Insulin, Epidermal Growth Factor (EGF), Platelet-Derived Growth Factor (PDGF)
Membrane/Extracellular Matrix-Bound Proteins - e.g Fibroblast Growth Factor (FGF)
In the first stage (1/3) of RTK Activation, describe the level of kinase activity and the state of the Activation Lip.
- Low or no kinase activity
- Activation lip is unphosphorylated, blocking conformation.
- RTK Inactive
In the second stage of RTK Activation (2/3), describe what happens when a ligand binds.
- Ligand binding promotes receptor dimerisation
- Dimerisation-induced conformational change has 2 effects
- RTK Active
Describe the 2 effects that dimer-induced conformational change has.
- Allows Mg-ATP to bind to each tyrosine kinase domain
- Allows one of the ATP-bound tyrosine kinases to phosphorylate tyrosines on the other partner in the dimeric complex i.e "Transphosphorylation"
In the third stage of RTK Activation (3/3), what do the phosphorylated tyrosined function as?
- Docking sites for intracellular proteins which relay & amplify downstream signal
- Signal relayed by activated signalling proteins into the cell's interior
Why is the loop on EGF binding domains important?
Important for a functional receptor dimer
How many Human EGF Receptors (HER) are there and what are they called?
There are 4:
Describe the ligands associated with HER1 and what type of dimerisation is needed for activation?
- EGF, HB-EGF, TGFa
- Homo or Heterodimerisation
What conditions are required for activation of HER2 and what does it facilitate?
- No ligand as always active
- Dimerisation with HER1, HER3 and HER4
- Facilitates EGF signalling
When is HER3 active and what does it lack in structure?
- Lacks functional kinase domain (weak)
- Only active when complexed with HER2
What ligands activate HER4?
- HB-EGF, Neuregulins
What does PDGF exist as?
- PDGF exists as a preformed dimer i.e 2 receptor binding sites, so the agonist directly cross-links 2 PDGF receptor chains
What parts of the RTK does FGF bind to?
- The extracellular domain of the receptor
- Heparan sulfate on receptor (essential for efficient activation of the receptor)
What type of polypeptides are Insulin/IGF-1 and how are their receptors expressed?
- Insulin/IGF-1 are soluble monomeric polypeptides
- Receptors are expressed as disulphide bond-linked preformed heteromers
What does insulin/IGF-1 bind to on the receptor and what happens when they bind?
- Bind to the alpha subunits
- Induces a conformational change that is transmitted to the beta subunits which then transphosphorylate each other
In RTK Signalling, what pathways can occur when a docking/adaptor protein attaches to the RTK dimer?
1. Docking/Adaptor protein → PI3K → Phospholipids
→ Biological Effects
2. Docking/Adaptor protein → Small GTPases → MAPKs
→ Gene Transcription → Biological Effects
In RTKs, what do P-Tyr's serve as docking sites for?
- Src Homology 2 (SH2) and Phospho-Tyrosine Binding (PTB) domains present on specific intracellular signalling molecules e.g insulin signalling
What family is Ras part of and what does it control?
- Part of GTPase superfamily that control many cellular processes e.g migration, cell shape, contraction
- Cycles between GTP-bound (active) and GDP-bound (inactive) states
What makes Ras GDP become active (Ras GTP) and vice versa?
Ras GDP → Guanine-nucleotide echange factors → Ras GTP
Ras GTP → GTPase-activating proteins (GAPs) → Ras GDP
What pathways does Ras control and what do these pathways lead to?
- MAP Kinase Pathway - Proliferation
- PI3 Kinase Pathway - Survival, Cell growth
Describe the events that cause the activation of the small G-Protein Ras.
- Inactive Ras in in GDP-bound OFF state at plasma membrane
- Phosphorylated Tyr's act as docking sites for SH2 domain on an adaptor protein Grb2
- Grb2 interacts with a Ras guanine nucleotide exchange factor Sos
- Membrane-localised Sos promotes release of GDP, allowing GTP to bind active GTP-bound Ras protein
Describe how Ras activation then leads to MAPK activation and what action MAPK has.
- GTP-bound Ras recruits a MAP-kinase-kinase called Raf to the plasma membrane
- Raf phosphorylates/activates a MAP-kinase-kinase called MEK which phosphorylates/activates MAP Kinase
- MAP Kinase phosphorylates and alters the activity of multiple enzymes and transcription factors to stimulate cell proliferation
What do RTKs also signal through that is derived from phosphatidyl inositol and what does this signalling include?
Enzymes that synthesise different phospholipids & proteins with domains that can bind to these molecules.
How does Ras activation lead to PI3K activation?
- Phosphorylated receptor & GTP-Ras recruit PI3-kinase to the membrane
How does PI3K activation lead to PKB/Akt activation?
- p110 (catalytic subunit) phosphorylates membane lipid PIP2 to PIP3
- PIP3 acts as a second messenger - binds and activates protein kinase B (PKB a.k.a Akt) which is a Ser/Thr-directed protein kinase
- PKB/Akt phosphorylates & alters the activity of multiple enzymes & transcription factors to stimulate cell survival, cell growth & migration
What do PDK1 and PDK2 do in the activation of PKB/Akt?
- PDK1 binds to PIP3 & phosphorylates the Thr residue of PKB.
- PDK2 phosphorylates the Ser residue of PKB
How is PIP3 inactivated?
- By conversion back to PIP2 by a lipid phosphatase PTEN = 3-phosphatase & tensin homologue.
How can distinct R-Tyr residues interact with specific SH2 domains?
- P-Tyr binding site - common to all SH2 domains
- Binding site for side chain of amino acid 3 positions down from P-Tyr variable between SH2 domains from different proteins - Confers specificity
What does simultaneous binding of multiple proteins to an RTK receptor ensure?
That multiple signalling pathways are activated simultaneously - essential to initiate complex biological responses.
What can overexpression of RTKs trigger?
What is overexpressed in 20-30% of breast cancers?
The HER family member HER2.
What does overexpression of HER2 cause to happen?
- Constitutively active
- Inappropriate amplification of mitogenic signalling
- Aggressive tumour growth
- Increased risk of metastasis
What treatments are now available for HER2+ cancers?
Herceptin/Trastuzumab = Monoclonal Ab which binds & inactivates HER2
Lapatinib = HER-targetted tyrosine kinase inhibitor molecule
What do oncogenic mutations illustrate the importance of?
Turning cell signalling OFF
Describe the action of the HER1 mutation on an EGF receptor in tumours.
- e.g Glioblastomas-mutant HER1 in which much of the EGF binding domain is deleted
- Causes the receptor to be incapable of binding EGF
- Constitutive dimerisation independent of ligand binding
- Unrestricted trans-phosphorylation & downstream signalling
Describe the action of mutations in tumours that cause inactivation of PTEN.
- PTEN dephosphorylation of PIP3 essential for appropriate control of cell growth/survival/proliferation
- PTEN gene mutated/inactivated in large number of cancers at high frequency
- Inactivation of PTEN in cancers results in unrestricted PIP3-directed signalling
Describe the action of mutations in tumours that cause activation of Ras.
- Mutated Ras proteins occur in 30% of all cancers (90% of pancreatic cancers)
- Ras mutations at codons 12,13,59 and 61 block interaction with GAPs
- Generates constitutively active GTP-bound active Ras that functions independently from growth factor stimulation
Describe the action of mutations in tumours that cause activation of Raf.
- There are 3 Raf isoforms: c-Raf, A-Raf & B-Raf
- Mutated Val600 → Glu B-Raf is present in 70% of melanomas
- Mutation mimics effect of GTP-Ras binding
- Generates constitutively active B-Raf that cannot be turned off
- B-Raf can be activated single point mutations (unlike c-Raf and A-Raf)
What is a potential target for treating melanoma and how does it work?
Vemurafenib - Orally available ATP-binding site inhibitor
- Selectively inhibits Val600Glu B-Raf
- Recently approved for treatment of metastatic melanoma
What can VEGF inhibitors cause?