Receptor Tyrosine Kinases and intracellular signal transduction Flashcards
Describe the Structure of insulin
Composed of two peptide chains: A and B.
Linked together by two disulfide bonds.
An additional disulphide bond within the A chain.
Several regions highly conserved across species:
- positions of disulphide bonds
- N- and C-terminal regions of A chain
- C-terminal region of B chain
Three dimensional structure is very similar across species.
Describe the structure of receptor tyrosine kinases (RTKs)
Most members of the receptor tyrosine kinase family exist as a single receptor molecule with the exception of isulin receptor and the IGF-1 receptor which sits as a dimer in the plasma membrane
What is the typical RTK Structure?
- Extracellular Domain:
‒characteristic motifs for each subfamily, responsible for bidning differnt ligands
‒ligand binding
- Transmembrane Domain
- Intracellular Domain:
‒most conserved between receptors
‒catalytic domain
‒kinase activity
Describe the roduction of the Insulin Receptor
- Single INSR gene produces two mRNA splice variants IR-A and IR-B .
- Translated proteins are then proteolytically cleaved into α and β chains.
- α and β chains of receptor form homo- or hetero-dimers.
- Receptor dimers held together single disulphide link between α and β chains and by two disulphide links extending from each α-chain.
Insulin Receptor: dimers
- The 2 α chains form the extracellular domains
- The 2 β chains form the transmembrane domain and the intracellular kinase domain
- 2 disulfide bonds between the 2 α chains
- 1 disulfide bond links each β chain with each alpha chain
Insulin Receptor: Describe ligand binding
•Two insulin binding sites on each monomer (4 binding sites in total): site 1 & site 2.
- Insulin binds to low affinity site – site 1 – on either of the α chains.
- Bound insulin then binds to site 2 on the other α chain.
Describe insulin receptor activation
- Insulin receptor already exists as dimer.
- Insulin binding induces conformational change to receptor structure.
- Juxtaposition/conformational change of cytoplasmic domains results in autophosphorylation of specific tyrosine residues.
- Activates kinase domain in receptor and generates binding sites for other signalling proteins.
Describe Protein Phosphorylation
- Reversible Post-Translational Modification (PTM) of proteins.
- Protein kinases mediate addition of phosphate group at serine, threonine and tyrosine side chains.
- Protein phosphatases reverse protein phosphorylation by hydrolyzing the phosphate group.
- Phosphorylation changes activity of target protein (both negatively and positively).
Insulin-induced tyrosine phosphorylation of Insulin Receptor: How can we monitor/visualise phosphorylation of the insulin receptor?
Western blot analysis of cell extracts after insulin stimulation using antibody that specifically recognises phospho-Tyr(1361) in insulin receptor
Signal transduction via PI3K/AKT or Ras/MAPK
Signal transduction: PI3K/AKT
- Insulin binds and activates the receptor
- Autophosphorylation of tyrosine residues within the β chains of the dimer
- Those phosphorylated tyrosine residues acts as bind sites in the cytoslic tail for insulin receptor substrates (IRS proteins)
- This brings the IRS proteins in close proximity to the kinase domains within the intracellular receptor tail
- This allows the IRS proteins to be phosphorylated through the kinase activity of the activated receptor
Describe SH2 domains
- Src Homology 2 domain (contained within Src oncoprotein).
- Found in wide range of signalling molecules.
- SH2 domains allow proteins to bind to phosphorylated tyrosine residues on other proteins.
- Commonly found in adaptor proteins (IRS proteins belong to this group) that aid in the signal transduction of receptor tyrosine kinases.
- They have no intrinsic activity but can localise (bring) signalling proteins together to generate signalling hubs
Signal transduction: PI3K/AKT
= phosphatidylinositol 4,5-biphosphate (PIP2)
= phosphatidylinositol 3,4,5-triphosphate (PIP3)
- Ligand binding to receptor activates receptor kinase activity.
- Autophosphorylation of specific tyrosine residues on cytoplasmic tail.
- Act as binding sites for insulin receptor substrate (IRS) proteins – via SH2 domains
- Kinase activity of receptor phosphorylates IRS protein.
- Phosphorylated IRS protein recruits PI3K to the membrane.
- PI3K is itself phosphorylated & activated and phosphorylates lipid phosphatidylinositol 4,5-biphosphate (PIP2 which is anchored in the plasma membrane) to form phosphatidylinositol 3,4,5-triphosphate (PIP3) .
- PIP3 then recruits AKT to the plasma membrane promoting the phosphorylation of AKT by PDK1.
- Phosphorylated AKT then dissociates from the membrane to phosphorylate other proteins at other subcellular localisations.
How does activated AKT mediates cellular changes?
- Promotes Glut4 (glucose transporters) translocation to the cell membrane - increases glucose uptake into target cell.
- Promotes nuclear exclusion of the FoxO transcription factor – inhibits expression of FoxO target genes (gluconeogenesis).
- Indirectly activates Glycogen Synthase – increases glycogen synthesis.
Signal transduction
Signal transduction also occurs through the Ras/MAPK pathway
The Ras GTPase
Ras is a GTPase
- It constantly cycles between the active GTP bound form and the inactive GDP bound form (Ras-GTP→Ras-GDP)
- Guanine nucleotide exchange factor (GEF) promotes the activation of Ras
- GTPase activating protein (GAP) promotes the deactivation of Ras
Signal transduction: Ras/MAPK
- Ligand binding to receptor activates receptor kinase activity.
- Autophosphorylation of specific tyrosine residues on cytoplasmic tail.
- Act as binding sites for insulin receptor substrate (IRS) proteins – via SH2 domains.
- IRS proteins recruit Grb2 adaptor protein to activated receptor.
- Grb2 then binds to the Ras-GEF, SOS.
- SOS stimulates the conversion of Ras-GDP to Ras-GTP.
- Activated Ras then initiates phosphorylation cascade through the sequential activation of a series of kinases - Raf→Mek→Erk.
- Activated Erk then phosphorylates target proteins – transcription factors, other kinases, it is the downstream effector.
Switching off insulin signalling
Intrinsic Negative Feedback can feedback to seitch off upstream events
- Activated ERK or AKT can inhbit the phosphorylation of IRS proteins
- Inhibition of IRS phosphorylation negatively feedsback to the pathway to prevent further downstream signalling events
- Activation of PTEN phophatase can hydrolyse phosphate groups on PIP3 (converts it back to PIP2) (no AKT activation)
Switching off insulin signalling: SOCS proteins (negative regulation of signalling)
- SOCS proteins contain an SH2 domain which can bind to the phosphorylated tyrosine domains on the RTK cytoplasmic tail to inhibit RTK activty directly
- They can also block the binding sites to prevent activated IRS proteins from reaching the activated receptor
- SOCS proteins contain the SOCS box which
recruits E3 ubiquitin ligase that targets IRS for degradation
