Lecture 6 - RTK signalling Flashcards
Describe how enzyme-linked receptors work
Second very important class of cell surface signalling molecule - generally act through being dimerised
Enzymes on the intracellular side only activated by receptor activation - can be part of receptor or second protein that is recruited
The ligands induce receptor dimerization, which activates the enzymatic activity of the receptor on the cytosolic side.
This can be through:
Mutual trans-phosphorylation of the two subunits
Recruitment of a catalytic subunit from the cytosol, which then becomes activated.
The active enzyme will in most cases phosphorylate and thereby activate targets to initiate signalling cascades.
How do RTKs work?
Have an intracellular kinase domain that is activated when the receptor dimerises
What is phospholipase C?
Phospholipases (PLCs) can transduce both GPCR and RTK initiated signals
PLC:
is activated by either a suitable Gα protein or an activated RTK
is an enzyme, so amplifies the signal
generates second messengers from an important signalling lipid
provides a platform for cross-talk between GPCRs (environmental cues) and RTK (growth hormones)
Phospholipase C can be activated by either a suitable G@ of a trimeric G protein or an activated receptor tyrosine kinase - different subtypes activated by each but it is the same enzyme so downstream signalling is the same no matter what it is activated by
Describe the enzymatic function of PLC
Once activated PLC hydrolyses PI4,5P2 to diacylglycerol (DAG) and inositol trisphosphate (IP3).
Both DAG and IP3 act independently as second messengers, although PKC is a common downstream target.
Hydrolyses phospholipid
(phosphoinositide 4,5 bisphosphate) Phosphoinositides are very minor component of phospholipid membranes but are very important for various aspects of signalling.
Inositol is a sugar carbohydrate derivative - doesn’t have an aldehyde group it only has hydroxyl groups so is a reduced form of the carbohydrates we normally see - has 6 hydroxyl groups so each carbon has one - can be phosphorylated in multiple places. When it forms the phospholipid it always uses the 1 hydroxyl group to attach to the phosphate of the phospholipid.
Describe Ca2+ release through IP3-gated channels from the ER
Ca2+ released into the cytosol and acts as a second messenger as well
Pathway generates 3 different second messengers - IP3, glycerol and Ca2+
The second messenger IP3 activates a channel in the ER to release Ca2+ ions
Ca2+ release is transient
It increases Ca2+ concentration about 10- to 100-fold, mainly locally around the channel
IP3 “degradation” is either by phosphatases removing the 4’ or 5’ phosphate, or a kinase adding a 3’ phosphate
What are PKCs?
PKCs are serine/threonine kinases that contain a regulatory domain attached to the catalytic domain.
Classified historically into 3 subgroups:
- conventional (a, b, g) activated by DAG and Ca2+
- novel (d, e, h, q) activated by DAG only
- atypical (z) activated by ceramide
The main target for the Ca2+ and diacylglycerol are PKCs
Calcium ions not always needed to activate PKC
All have a kinase domain, a calcium binding domain and a glycerol binding domain
Atypical - differs from novel in that it is activated by a different lipid but is now quite different from conventional subgroup
What does PKC target?
Targets phosphorylated by PKCs modulate:
Metabolism (GSK3b – glycogen synthase kinase 3b)
Other signalling pathways (MAP kinases)
cytoskeleton, movement (CPI-17 inhibits a myosin phosphatase)
How are RTKs activated?
These activate e.g phospholipase C - dimerisation involved
Normally there are ligand binding sites on both of the receptors halves and it forms a homodimer
Once the homodimer is formed on the intracellular side there is a conformational change which allows transphosphorylation
One side phosphorylates activation lip on the other side of the receptor - that side then phosphorylates the other receptor half. The phosphorylation is then propagated and the receptor is activated. The receptor is now ready to form a platform for more intracellular signalling molecules.
Describe how RTK signalling involves a lot of ‘domains’
The activated insulin receptor binds the multidocking protein insulin receptor substrate 1 (IRS-1).
The still active receptor phosphorylates IRS-1, which provides docking sites for further signalling components.
This provides a further site for regulation – phosphatases have one more opportunity to remove the phosphate and interfere with the propagation of signalling.
IRS-1 can also recruit multiple factors that would individually be inactive but form an active signalling complex in each others presence.
PTB and SH2 domains can bind phosphotyrosine.
Describe ligand Recognition of the IRS-1 PTB domain
The phosphotyrosine binding domain (PTB) of IRS-1 binds specifically to Tyr-P of the insulin receptor (IR) and interleukin-4 receptor (IL-4R).
Binding specificity is determined by the amino acid preceding the Tyr.
Alanine (in IL-4R) is best. The glutamate in IR reduces the affinity 50-fold, whereas aspartate and glutamine found in EGF receptor, or the neurotrophin receptor TrkA, show very weak to no binding.
Changing the glutamate in IR to alanine converts the affinity of IR to that of IL-4R.
Describe phosphoinositide kinase mediated signalling
One of the signalling molecules recruited by IRS-1 is phosphoinositol-3-kinase (PI3K). The p85 subunit of PI3K contains an SH2 domain that binds phosphorylated IRS-1.
The catalytic subunit of PI3K – p110 – phosphorylates PI4P or PI(4,5)P2 at the 3-position. This creates binding sites for yet other domains, for example the PH domain.
Phosphoinositide kinase changes phosphoinositides not by hydrolysing them but by adding another phosphate group to the phosphoinositide’s inositol head group
Can use either PI(4,5)P2 or PI4P. At the plasma membrane, where this is activation usually happens, the majority will be PI(4.5)P2 so majority of the time PI(3,4,5)P3 is generated. Adds a phosphate in position 3 of the phosphoinositol head group. Although phosphoinositides are very small component of the membrane they provide binding site for certain domains and molecules.
Describe how local generation of PI(3,4)P2 activates Protein kinase B (PKB/Akt)
PI3,4P2 provides a binding site for the PH domain of PKB/Akt. Binding of the PH domain to the phospholipid partially activates PKB.
The activator of PKB – PDK1 – also contains a PH domain. Thus PDK1 is recruited to the same site as PKB.
The close proximity of PKB and PKD1 results in the activation of PKB.
PKB activated by PI(3,4)P2- binds via PH domain. This brings it to the membrane for its second activator PDK1. The PH domain would normally cover activation lip - binding to phosphoinositide head group frees up activation lip, partially activating PKB. PDK1 phosphorylates activation lip of PKB. PDK1 also has a PH domain that binds phosphoinositide head group.
