Phospholipids And Phospholipid Signalling

Question 1

What are phospholipids?

Answer 1

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

Question 2

What are some of the headgroups which exist?

Answer 2

Choline
Serine
Inositol 
Amine
Sugars

Question 3

How long is the FA tail?

Answer 3

Typically 16-18 carbons long

Question 4

What does PIP2 stand from?

Answer 4

Phosphatidylinositol 4,5 bisphosphate

Question 5

How are the number of carbon on a carbon ring counted?

Answer 5

Right hand of turtle = 1

Increases in anti-clockwise manner

Question 6

Where does PLC cleave?

Answer 6

Cleaves between the phosphate and the glycerol back bone

Question 7

What does PLC cleave of PIP2 form?

Answer 7

Diacylglycerol (DAG)

Inositol 1,4,5 trisphosphate (IP3)

Question 8

What can DAG go on to do?

Answer 8

Acts as a secondary messenger to bind/activate enzymes with a c1 domain (e.g. PKC)
May be converted into phosphatidic acid

Question 9

What can IP3 go on to do?

Answer 9

Increase calcium (actives PKC)
Stimulate multiple kinases, transcription and RNA processing

Question 10

What can PIP2 function as?

Answer 10

Can itself regulate cellular processes e.g. PKC
Bind to proteins which have a pleckstrin homology domain
Substrate for PI3K

Question 11

How many families and isoforms of PLC are there?

Answer 11

6 families with 13 isoforms

These different isoforms allows for different signalling processes are are regulate din different ways

Question 12

What is the most primitively PLC isoform.

Answer 12

PLC (Delta)

Question 13

What motifs does PLC (delta) have?

Answer 13

PH domain
4xEF motif 
Catalytic domain (X, x-y linker, Y)
C2 domain

Question 14

What does an EF motif allow forM

Answer 14

Binds calcium therefore calcium likely regulates function

Question 15

What is the percentage homology between the catalytic domains in PLC isoforms?

Answer 15

60%

Question 16

What does the X-Y linker do?

Answer 16

Blocks the active site of Plc (delta) when inactive

Question 17

What does the C2 domain allows for?

Answer 17

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

Question 18

What activates PLCB2

Answer 18

GBY (from Gi)
Gaq
Calcium

Question 19

Where are the different PLCB isoforms?

Answer 19

B1 +3 = widespread
B2 = immune/haemopoetic cells
B4 = retina and certain neurons

Question 20

What domains does the beta family have that the delta family does not?

Answer 20

Coiled coil domain
PDZ domain

On C terminus

Question 21

What isoforms will be activated by GBY subunit?

Answer 21

B2>B3>B1 (not B4)

Question 22

What is the effect of GBY and Gaq on PLCB3 activation?

Answer 22

Act synergistically to potentiate PLC function. (Coincidence detection)
This co activation allows cross talk of signalling pathways

Question 23

Can PLCB regulate G protein function?

Answer 23

Yes, evidence suggests the can act as GTPase activating protein (GAPS) for Gq/11 increasing GTPase activity by 1000 fold

Question 24

PLCY (gamma) is unique, why is this?

Answer 24

X-Y linkers contains 2xSH2 domains and 1xSH3 domain

Question 25

What does SH2 domains allow for?

Answer 25

Interaction with phospho tyrosine eg. On RTKs which can activate PLCY in a G protein independent fashion

Question 26

What does the SH3 domain allow for?

Answer 26

Interaction with proline rich sequences

Question 27

Ultimately, what does the scr homology domains allows for?

Answer 27

Protein protein interactions and mean PLCY can form part of a scaffolding complex

Question 28

What activates PLCY?

Answer 28

1. RTKs via the SH2 domain - role in development and cell cycle
2. PIP3 binds to PH and C terminal SH2
3. Non receptor tyrosine kinases (cytokine receptors which phosphorylated recruit effectors with SH2 domains e.g. PLCY)
4. GPCRs can cause tranactivation of RTKs

Question 29

Where on a RTK has to be phosphorylated for PLC-Y1 activation?

Answer 29

Y783 is necessary and sufficient

Question 30

What is the importance of the number of PLC activation mechanisms?

Answer 30

Cross talk between pathways

Question 31

What s the importance of the diverse rand eof isoforms?

Answer 31

Different activation mechanism and subcellular locations this different space time and amplitude of signal = signalling variety, specificity and intergration

Question 32

What are 4 ways we can monitor PLC activity?

Answer 32

Changes in calcium fluo-4
Accumulation of [3H]-labelled inositol phosphates
Mass IP3
Single cell imaging of PLC activity

Question 33

How can [3H] labelled inositol be used to measure IP3 activity?

Answer 33

[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

Question 34

What does an accumulation of [3H] ip3 mean for a cell with the addition on lithium?

Answer 34

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.

Question 35

Why might lithium be useful for treating bipolar manic phases?

Answer 35

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

Question 36

How can mass of IP3 be measured?

Answer 36

Use a radioligand binding assay

Question 37

How can single cell imaging of PLC activity be measured?

Answer 37

Use a biosensor for IP3/PIP2