Lecture 5 Flashcards
What are the major sites of glucose storage and usage?
Brain –> obligate glucose utiliser (can’t store it), Liver –> major site of gluconeogenesis (also uses glucose)
What is the majority of dietary glucose used by?
Used by skeletal muscle –> Taken up and stored as glycogen, ~10% is taken up by adipocytes for storage as triglycerides
Describe the structure of insulin receptors
Extracellular domain: Have characteristic motifs for each subfamily, Involved in ligand binding –> very different between each receptor, Transmembrane domain (hydrophobic region), Intracellular domain: Most conserved between receptors, Has a catalytic domain, Has kinase activity
What family is the insulin receptor a part of?
Receptor tyrosine kinase family
Describe the structure of the insulin receptor on the level of genes and chains
Essentially 4 components to the receptor, They are all part of a single INSR gene –> This produces 2 mRNA splice variants IR-A and IR-B, The translated proteins are then proteolytically cleaved into alpha and beta chains, The alpha and beta chains of the receptor form homo- or hetero-dimers, These receptor dimers are held together by disulphide bonds –> Single bond link between alpha and beta chains –> 2 bonds extending from each alpha chain
What is produced by alternative splicing of the INSR gene?
Alternative splicing produces 2 InR isoforms –> A, B, Each isoform is then enzymatically cleaved into chains –> Alpha, Beta
What are the 3 different combinations of the alpha and beta chains of the different insulin receptor isoforms?
A homodimer (all alpha and beta chains from A isoform), B homodimer (all alpha and beta chains from B isoform), A/B heterodimer (mix of alpha and beta chains from both isoforms)
Compare the 2 insulin receptor homodimers
Show a similar affinity for insulin, Differ in their sites of expression –> IR-A is ubiquitous, IR-B is highly expressed in the liver, They differ in their rate of receptor internalisation and recycling –> Rate in the A homodimer is more than the rate in the B isoform, Quite hard to isolate the homodimers from the heterodimers so difficult to study them
How does insulin bind to insulin receptors? What effect does this have on the receptor?
Only 1 insulin molecule binds with high affinity to each receptor dimer, Insulin binding to the 2 sites on different monomers cross-links them, Insulin binding induces conformational change to the receptor structure, Juxtaposition/conformational change of cytoplasmic domains results in autophosphorylation of specific tyrosine residues, Further activates kinase domain in receptor
Describe negative cooperativity in insulin receptor binding
Kinetics of insulin-receptor binding shows that they demonstrate negative cooperativity, At high insulin concentrations, the affinity of the receptor for a second insulin molecule is much lower than for the 1st insulin molecule
Describe positive cooperativity in insulin receptor binding
At low insulin concentrations, the affinity of the receptor for a second insulin molecule is higher
What do ligand-bound insulin receptors do?
They cluster and aggregate within the membrane
Describe phosphorylation of proteins. What effect does it have on the protein?
Protein phosphorylation = reversible post-translational modification (PTM), Protein kinases mediate addition of a phosphate group at the side chains of specific amino acids, Protein phosphatases reverse protein phosphorylation by hydrolysing the phosphate group, Phosphorylation changes activity/localisation of target protein (both negatively and positively) –> Can act as a targeting signal to send the protein to specific parts of the cell
Which amino acids can be phosphorylated? Why?
Serine, Threonine, Tyrosine, They have side chains containing nucleophilic (-OH) groups that attack the terminal phosphate group on the ATP resulting in the transfer of that phosphate group to the amino acid side chain
Which technique can we use to study insulin-induced auto-phosphorylation of the insulin receptor?
Western blotting, Can analyse cell extracts after insulin stimulation using antibodies that specifically recognise phospo-Tyr (1361) in the insulin receptor
What does activated AKT do?
Mediates important cellular changes, Promotes GLUT4 translocation to the cell membrane –> increases glucose uptake into the target cell, Indirectly activates glycogen synthase –> increases glycogen synthesis, Promotes nuclear exclusion of the FoxO transcription factor –> inhibits expression of the FoxO target genes (gluconeogenesis) –> If FoxO can’t get into the nucleus, it can’t express its target genes which are involved in gluconeogenesis
How is AKT activated?
Insulin binds to receptor, Causes receptor to change shape and the kinase domain changes shape and is phosphorylated, Phosphorylation occurs at specific tyrosines on cytoplasmic tail or activated receptor act as binding sites for other signalling proteins, Insulin receptor substrate (IRS) proteins are attracted, Close proximity of IRS proteins to the receptor kinase domain leads to the IRS proteins being phosphorylated, Phosphorylated proteins bind to lipid kinase PI3K which brings it to the inner surface of the cell membrane, PI3K is then itself phosphorylated by the receptor tyrosine kinase which activates it, PI3K then phosphorylates the membrane anchored lipid called PIP2 –> this converts it to PIP3, PIP3 then binds to both PDK1 and AKT and brings them together, PDK1 phosphorylates AKT which activates it, Phospho-AKT then dissociates from the membrane to phosphorylate other proteins at other subcellular localisations
What do IRS proteins do?
IRS proteins = insulin receptor substrate proteins, Bind to activated insulin receptors, Can then be phosphorylated
What are SH2 domains?
Found in a wide range of signalling molecules, including IRS proteins, SH2 domains allow proteins to bind to phosphorylated tyrosine residues on other proteins, Commonly found in adaptor proteins that aid in the signal transduction of the receptor tyrosine kinases, They localise signalling proteins together to generate signalling hubs
How does AKT activation affect glucose uptake in skeletal muscles?
AKT activation leads to AKT phosphorylating and inhibiting AS160, Inhibition of AS160 promotes Rab-dependent vesicle formation, This induces GLUT4 translocation and promotes glucose uptake into the skeletal muscles cells from the blood stream
How does AKT activation affect glycogen synthesis?
Activated AKT phosphorylates and inactivates glycogen synthase kinase-3 (GSK-3) –> GSK-3 is an INHIBITOR of glycogen synthase so this prevents inhibition of glycogen synthase, When GSK-3 is active, it targets and inactivates glycogen synthase, preventing glycogen formation, AKT prevents this negative regulation of glycogen synthase promoting the conversion of intracellular glucose to glycogen
How does AKT activation affect FoxO transcription factors?
FoxO proteins are insulin-responsive transcription factors –> In absence of insulin (inactive receptor), FoxO residues in nucleus and coordinates expression of range of target genes –> In presence of insulin (active receptor), activates AKT, Phosphorylation of FoxO by AKT changes the subcellular localisation of these FoxO proteins (moves them from the nucleus to the cytoplasm), This prevents FoxO-dependent gene expression, Many thousands of genes are directly regulated by FoxO TFs –> E.g genes involved in gluconeogenesis
How do we switch off AKT signalling?
Need negative feedback to regulate this process, 2 ways: –> AKT negatively regulates signal transduction through this pathway by preventing IRS proteins from binding to activated insulin receptors –> This is INTRINSIC negative feedback –> Insulin signalling induces expression of SOCs proteins
What do SOCs proteins do?
SOCs: –> Can stop IRS proteins binding to insulin receptor –> Can bind directly to kinase domain in intracellular domain to prevent kinase activity –> Can bind to and degrade IRS proteins through ES ubiquitin ligase
