Phospholipids And Phospholipid Signalling Flashcards
What are phospholipids?
Amiphipatic molecules which have 2 fatty acid tails which may or may not contain a double bond, a glycerol back bone and a polar head group
What are some of the headgroups which exist?
Choline Serine Inositol Amine Sugars
How long is the FA tail?
Typically 16-18 carbons long
What does PIP2 stand from?
Phosphatidylinositol 4,5 bisphosphate
How are the number of carbon on a carbon ring counted?
Right hand of turtle = 1
Increases in anti-clockwise manner
Where does PLC cleave?
Cleaves between the phosphate and the glycerol back bone
What does PLC cleave of PIP2 form?
Diacylglycerol (DAG)
Inositol 1,4,5 trisphosphate (IP3)
What can DAG go on to do?
Acts as a secondary messenger to bind/activate enzymes with a c1 domain (e.g. PKC)
May be converted into phosphatidic acid
What can IP3 go on to do?
Increase calcium (actives PKC) Stimulate multiple kinases, transcription and RNA processing
What can PIP2 function as?
Can itself regulate cellular processes e.g. PKC
Bind to proteins which have a pleckstrin homology domain
Substrate for PI3K
How many families and isoforms of PLC are there?
6 families with 13 isoforms
These different isoforms allows for different signalling processes are are regulate din different ways
What is the most primitively PLC isoform.
PLC (Delta)
What motifs does PLC (delta) have?
PH domain 4xEF motif Catalytic domain (X, x-y linker, Y) C2 domain
What does an EF motif allow forM
Binds calcium therefore calcium likely regulates function
What is the percentage homology between the catalytic domains in PLC isoforms?
60%
What does the X-Y linker do?
Blocks the active site of Plc (delta) when inactive
What does the C2 domain allows for?
Binds 4 calciums which increase enzyme activity 50-100 fold more than the beta and gamma isoforms
It may amplify and prolong rather than nitrate calcium signal
What activates PLCB2
GBY (from Gi)
Gaq
Calcium
Where are the different PLCB isoforms?
B1 +3 = widespread
B2 = immune/haemopoetic cells
B4 = retina and certain neurons
What domains does the beta family have that the delta family does not?
Coiled coil domain
PDZ domain
On C terminus
What isoforms will be activated by GBY subunit?
B2>B3>B1 (not B4)
What is the effect of GBY and Gaq on PLCB3 activation?
Act synergistically to potentiate PLC function. (Coincidence detection)
This co activation allows cross talk of signalling pathways
Can PLCB regulate G protein function?
Yes, evidence suggests the can act as GTPase activating protein (GAPS) for Gq/11 increasing GTPase activity by 1000 fold
PLCY (gamma) is unique, why is this?
X-Y linkers contains 2xSH2 domains and 1xSH3 domain
What does SH2 domains allow for?
Interaction with phospho tyrosine eg. On RTKs which can activate PLCY in a G protein independent fashion
What does the SH3 domain allow for?
Interaction with proline rich sequences
Ultimately, what does the scr homology domains allows for?
Protein protein interactions and mean PLCY can form part of a scaffolding complex
What activates PLCY?
- RTKs via the SH2 domain - role in development and cell cycle
- PIP3 binds to PH and C terminal SH2
- Non receptor tyrosine kinases (cytokine receptors which phosphorylated recruit effectors with SH2 domains e.g. PLCY)
- GPCRs can cause tranactivation of RTKs
Where on a RTK has to be phosphorylated for PLC-Y1 activation?
Y783 is necessary and sufficient
What is the importance of the number of PLC activation mechanisms?
Cross talk between pathways
What s the importance of the diverse rand eof isoforms?
Different activation mechanism and subcellular locations this different space time and amplitude of signal = signalling variety, specificity and intergration
What are 4 ways we can monitor PLC activity?
Changes in calcium fluo-4
Accumulation of [3H]-labelled inositol phosphates
Mass IP3
Single cell imaging of PLC activity
How can [3H] labelled inositol be used to measure IP3 activity?
[3H] integrated into PIP2 head group
Cleaved by PLC which forms radio labelled IP3 (and DAG)
IP3 is then dephosphorylated in steps until IP1 is converted into inositol by inositol monophosphatase to be recycled to make PIP2.
Lithium inhibits inositol monophosphatase
Therefore radio label IP3 will build up in cell
What does an accumulation of [3H] ip3 mean for a cell with the addition on lithium?
Amount of [3H] increase compared to time will indicate how much PLC activity is occuring.
These steeper the graph the more PLc s being activated.
Why might lithium be useful for treating bipolar manic phases?
The more inositol monophosphatase activity the ore lithium will uncompetitively inhibit. Manic phases may be caused by overact pathways
Thus it can act as a stabiliser for over active pathways
How can mass of IP3 be measured?
Use a radioligand binding assay
How can single cell imaging of PLC activity be measured?
Use a biosensor for IP3/PIP2
