Phospholipase C Flashcards
What is PLC’s catalytic reaction?
How would you describe the two products?
How are PIP2 levels increased?
Where are the 2 products found?
Where is the first product formed?
What are phosphatidic acid and DAG used for?
Hydrolyses PIP2 into IP3 and DAG at the plasma membrane
IP3 = cytosolic phophatidylinositol with phosphate at 1,4,5 of inositol ring
DAG = membrane associated diglyceride made up of glycerol with C18 fatty acid chain and C20:C4D fatty acid chain
PI phosphorylated by PI-4 kinase (to PI4P) phosphorylated by PI4P-5 kinase to PI(4,5)P2
PI4P at golgi & plasma membrane and PIP2 at plasma membrane
Golgi
Synthesis of phospholipids at the ERt
How is PLC activated?
What are the outcomes of DAG?
What are the outcomes of IP3?
What are the 4 domains of PKC?
What is PMA and what is its problem with PKC regulation?
- GPCR activated on ligand-binding such that G-alpha subunit dissociates
- Alpha subunit binds & activates PLC (phospholipase C) which hydrolyses PIP2 into DAG and IP3
Activates PKC and converted into phosphatidic acid by phosphorylation
Binds to IP3 receptors on ER to release Ca 2+ and then degraded back to inositol
C1 DAG-binding, C2 Ca2+ & phospholipid binding, C3 ATP binding, and C4 substrate binding (for phosphorylation)
Phorbol ester carcinogen - binds to C1 domain but no mechanism of its removal so PKC is activated & no signalling
In 4 steps, how does PIP2 regulate clathrin mediated endocytosis?
How does PIP2 regulate ERM proteins in 2 steps?
Why is ERM essential for cells?
How does PIP2 regulate N-WASP? 2 steps
How does PIP2 regulate cofilin? 3 steps
- A & ß2 subunits of AP2 complex samples the membrane if there’s lots of PIP2 present
- Results in their change of conformation & release µ2 subunit at plasma membrane
- Change in conformation allows for clathrin to bind for endocytosis
- If phospholipase C & lots of PIP2 is hydrolysed, then endocytosis downregulated
kk
- ERM has a high affinity to PIP2 so binds to PIP2 firm domain on plasma membrane
- ERM is then activated and can then interact with actin filaments
Maintains rigidity of plasma membrane
- Binds to PIP2 of the plasma membrane in order for its open conformation for its activation
- Recruits ARP-2 and ARP-3 for their activation allowing for actin filament synthesis & actin remodelling
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- Has a low affinity for PIP2 (so requires high amounts of PIP2 to bind to the membrane) but also is inactive when it is bound
- Therefore, on a decrease of PIP2 due to its hydrolysis, it is released from the membrane & becomes active
- In its active state it disassembles actin filaments into monomers
How does PIP2 regulate activation M-current (KCNQ2/3 channels) at the plasma membrane? 2 steps
How does Oxo-M inactive the M-current? 5 steps
What does the M-current regulate in cells?
What are the 4 domains of PLC delta?
- Ion channels maintained in open state by binding to PIP2 through -ve lipids binding to +ve charged residues
- Allows for K+ to move into cells
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1. Oxo-M agonist binds to the receptor for activation
2. Results in G-protein activation
3. G-alpha subunit activates PLC
4. PLC hydrolyses PIP2
5. Therefore the ion channel closes
Neuronal excitability such that its inhibition increases excitability
PH, EF hands, X-Y (with linker), C2
What does the PH domain do in PLC delta?
What is the EF hands structural role?
What is EF hands functional role?
From this, how is PLC delta regulated?
What is the structure & function of the XY domain?
What is the feature of its linker?
What is the function of the C2 domain?
What are the 3 steps for the activation of PLC delta?
Anchors PLC to the plasma membrane through binding of PIP2
Links C2 and XY with the PH domain
Binds Ca2+ which promotes binding of PH to PIP2
Ca2+
TIM barrel catalytic domain
Negative so repulsed by the membrane
Calcium phospholipid binding
- PH docks PLC to PIP2 when [Ca2+] is high on the membrane but the rest of the complex (EF C2 TimXY) remains inactive
- Linker region sits at catalytic site where PIP2 needs to bind (substrate) for hydrolysis but is repulsed by the membrane due to negative charge
- When the linker region moves such that the catalytic site isn’t occupied, it can then interact with PIP2 at the membrane & hydrolyse it
How does the domain structure of PLC beta differ to delta?
In what 3 ways BRIEFLY is the activation of PLC beta regulated?
How does Gq activate it and which isoforms are activated?
What about Gi?
Even though Gq can use the same mechanism as Gi, why is this insufficient?
What are the 3 c-terminal domain features of PLC beta 3?
Has a C-terminal extension after the C2 domain
Gi, Gq and Rac1
GPCRs activate Gq & alpha subunit activates ß1/3
Beta-gamma subunits activate ß2/3
Gi is 10x as Gq so more beta-subunits released by Gq
Proximal CTD, distal CTD, linker
What 2 features of the X-Y linker in PLC beta 3 keep it in its autoinhibited state?
What 2 features of the distal CTD in PLC beta 3 keep it in its autoinhibited state?
How would you describe the structure of the distal CTD?
In 3 steps how is PLC beta activated?
- Acidic stretch
- ## Helix blocks active site
- The Distal CTD interacts with part of the catalytic domain sequestering its membrane interaction sites
- ~25 amino acids of the proximal CTD form a helical hairpin helix-alpha-2’ which binds between the catalytic TIM barrel and the C2 domain - inhibiting PLC activity
coil-coil structure
- G-alpha-Q-GTP binds to Distal CTD such that it can then interact with alpha helix 1 & 2 to allow the subsequent movement of helix-alpha-2’ (autoinhibitory segment)
- As a result of the helix-alpha-2’ movement, the distal CTD can now bind to PIP2 on the membrane & brings PLC beta into close proximity of the membrane
- The negative charge of the membrane displaces the acidic stretch of the X-Y linker by electrostatic repulsion allowing exposure of XY’s active site to hydrolyse PIP2
How is PLC beta 1 activated? 2 steps
In what 2 ways is PLC beta 2 activated? 2 steps each
In what cell is PLC beta 2 expressed?
What are the following mutations & what do they cause:
1. PLC beta 1
2. PLC beta 3
3. PLC beta 4
- Agonist binds to GPCR and activates G-alpha-q-GTP
- G-alpha-q activates PLC beta 1
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- Gi proteins & their beta gamma subunits binding near ha2’
- Rac1 binds to PH domain moving X-Y linker from barrel for activation
Neutrophils
- homozygous deletion & epilepsy
- loss of function & milder cystic fibrosis
- gain of function & eye tumour
How is PLC gamma’s domain structure different to PLC delta?
In 6 steps, how is PLC gamma activated?
What are the following mutations & what do they cause:
1. PLC gamma 1
2. PLC gamma 2
What mutations generally impact PLC gamma activity?
How is PLC epsilon activated and why?
What domain is not present in PLC zeta and how is it present in sperm?
Between X and Y domains is a split PH domain, n-SH2, c-SH2 and SH3 on the linker
- Receptor tyrosine kinase receptor is phosphorylated by tyrosine kinase
- Receptor recruits N-SH2 domain to bind to the phosphotyrosine residue of the active tyrosine kinase receptor
- Tyrosine kinase receptor phosphorylates tyrosine 783 of a loop due to closer proximity
- c-SH2 domain now bound to pY783 so both n-SH2 and c-SH2 are removed from the PLC-gamma core
- PLC-gamma core with membrane-interacting regions dock to the membrane
- PLC-gamma is now activated & hydrolyses PIP2
- Gain of function - Angiosarcoma in T cell lymphocytes
- Gain of function - Alzheimer’s in microglia cells
Regulatory domains n-SH2 and c-SH2
Ras due to Ras-GEF domain
PH domain - releases IP3 for increased calcium conc leading to oocyte activation
