L10- Intracellular Signalling 2 Flashcards
Where is the binding site for a water soluble hormone?
On the extra-cellular part of the receptor, which spans the plasma membrane.
How many membrane-spanning domains does a G-coupled protein receptor have?
7 transmembrane alpha helices
What is the process a G-coupled protein receptor undergoes after the binding of a hormone, to end up altering the activity of an effector protein?
Hormone binds to extracellular domain, inducing an intracellular conformational change, allowing the receptor to bind to the trimeric G-protein. A GDP molecule is bound to the alpha subunit of this protein. On binding to the receptor, a conformational change is induced, which causes GDP to dissociate from the alpha subunit - GTP now binds to alpha.
The binding of GTP causes the alpha subunit to dissociate from the rest of the protein, and allows it to bind to an effector protein, changing the effector proteins function.
The alpha subunit has hydrolytic properties and is slowly hydrolysing the GTP. Once hydrolysed and reverted back to GDP, the alpha subunit cannot bind the effector protein, and returns back to the rest of the G-protein.
Once hormone levels have dropped to an appropriate level, there will be no hormone on the extracellular domain of the receptor, and the conformational change will be reversed, allowing GDP to remain bound to the alpha subunit, returning the receptor back to resting state
What is the effector protein that produces the second messenger cAMP?
Adenylate cyclase
Explain G-coupled protein signal amplification
Because the receptor does not return to resting state until hormone levels have dropped, it remains bound to the G-protein, which prevents GDP from remaining bound to the alpha subunit. Therefore, there is continuous activation of the effector proteins, which provides the level of signal amplification by activating several G-proteins with one hormone molecule
How does the Cholera Toxin produce it’s effect?
The toxin destroys the hydrolytic ability of thh alpha subunit, as it is an enzyme that catalyses the ADP-ribosylation of a tyrosine residue in the alpha subunit. This means that GTP remains bound to the alpha subunit, whcih keeps adenylate cyclase active, resulting in massive concentrations of cAMP in the cell. This results in a rush of electrolytes from the gut epithelial cells - water follows, producing the diarrhoea that can dehydrate a patient to the extent that they die
How does cAMP activate protein kinase A
4 cAMP molecules bind to the regulatory subunits of phosphate kinase A, which induces a conformational change and allows the catalytic subunits to dissociate. These are now active
What do phosphate kinases do, and what are the two main types in eukaryotes?
Phosphorylate other proteins, which can modify the target proteins function
Serine/threonine kinases
Tyrosine kinases
What do protein phosphatases do?
These remove a phosphoryl group from a protein, adding it to water
How does glucagon induce glycogen break down in the liver/gluconeogenesis?
- Glucagon binds to receptor, allowing it to bind to G-coupled protein, which causes dissociation of GDP from alpha subunit, allowing GTP to bind and alpha subunit to dissociate
- Alpha subunit binds to adenylate cyclase, activating it, and allowing it to produce cAMP. cAMP molecules bind to regulatory subunits of protein kinase A, allowing catalytic subunits to dissociate.
- Protein kinase A (PkA) phosphorylates Inhibitor 1, which in turn phosphorylates phosphatase 1, deactivating it.
- PkA phosphorylates glycogen synthase A, converting it to low activity glycogen synthase B.
- PkA phosphorylates Phosphorylase Kinase (low activity) t high activity Phosphorylase Kinase.
- Phosphorylase Kinase goes on to activate phosphorylase B to phosphorylase A - phosphorylase A converts glycogen and phosphate to glucose-1-phosphate
- PkA is also able to enter the nucleus and activate CREB, which binds to DNA and upregulates transcription of a key regulatory enzyme in gluconeogenesis
What is the general signalling pathway of growth factor induced cell division?
Growth factor binds to extracellular receptor, inducing an intracellular conformational change. Tyrosine kinases autophosphorylate tyrosine residues, which are recognised by adjacent proteins Grb2/Sos.
Sos domain is associated with another protein, Ras, which normally has GDP bound to it. The formation of a complex between Grb2/Sos and Ras displaces GDP, allowing GTP to bind to Ras, which goes on to dissociate from the complex, and act on effector molecules, the main one of which is Raf.
Raf acts on Mek/MAPCK, which phosphorylates MAPK, activating it.
MAPK can go on to phosphorylate a variety of transcription factors, involved with a number of genes in cell division.
S6 kinases is also activated, which activates ribosomes to enhance translation rates
Describe interleukin/cytokine receptors
Plasma membrane proteins, 1-3 protein components
Binding of interleukins/cytokines induces dimerisation/polymerisation. They are NOT tyrosine kinases
On a IFNy dimer binding to the alpha component of the receptor, the alpha and beta components form a dimer, and the tyrosine kinases JAK1 and JAK 2 are phosphorylated and bind to the receptor dimer
STAT1 molecules are now able to bind to the receptor, as their -SH2 domains are attracted to the phosphates. STAT 1 alpha molecules are phosphorylated on a tyrosine residue, dissociate from the receptor and dimerise with another phosphorylated STAT 1 alpha molecule, with a phosphate of one binding to the -SH2 of the other.
This dimer can then move to the nucleus, where it binds to GAS, a DNA response element in the promoter regions of the genes controlled by IFNy. This activates transcription of those genes
Describe the function of the two Zinc fingers of steroid hormone receptor DNA-binding domains
- 1 zinc finger is involved in binding to DNA phosphate backbone, usuing specific backbone binding residues - this one is therefore involved with the specificity of where the receptor binds to DNA
- The other zinc finger contains residues that participate in receptor dimerisation
Describe hormone receptor responsive elements
Comprised of two identical sequences of 6 bases, either both on the same DNA strand, or one on each strand. The hormone-receptor complexes act as dimers so that each sequence of 6 bases is bound by one of the hormone-receptor complexes in the dimer.
Are thyroid hormone and retinoic acid receptors able to silence gene expression without their hormone bound to them?
Yes. Binding of the hormone results in release of the repression of expression and activation
