2nd Nov - Phospholipids and Phospholipid Signalling

Question 1

What is the most abundant membrane lipid?

Answer 1

Phosphatidylcholine

Question 2

What is the function of phospholipases?

Answer 2

To turn glycophospholipids into a variety of different second messengers

Question 3

What reaction does phospholipase D catalyse?

Answer 3

Phosphatidylcholine –> phosphatidic acid

Question 4

What reaction does phospholipase C catalyse?

Answer 4

PIP2 –> IP3 and DAG

Question 5

What are the main downstream effectors of DAG?

Answer 5

Phosphatidic acid

Enzymes and structural proteins w/ CI domain e.g. PKC

Question 6

What are the main downstream effectors of IP3?

Answer 6

IP3 receptor on ER

Multiple kinases, transcription and RNA processing factors

Question 7

What are the mammalian isoforms of PLC?

Answer 7

PLC-beta, gamma, delta, epsilon, zeta, eta

Question 8

What is believed to be the original isoform of PLC?

Answer 8

PLC-delta

Question 9

What are the domains of PLC delta?

Answer 9

X + Y catalytic domains
PH domain
4 tandem EF hand domains
C2 domain

Question 10

How is PLC delta activated?

Answer 10

By calcium - PLC delta responds to calcium 50-100x better than the beta and gamma isoforms

Question 11

What is believed to be the function of PLC delta?

Answer 11

To amplify and prolong calcium signals

Question 12

Outline the domain structure of PLC beta

Answer 12

PH
EF
X
Y
C2 
Coiled-coil domain

Question 13

What is the function of the X-Y linker in PLC beta?

Answer 13

To keep the X and Y domains together creating inhibition of PLC beta. When activated the linker moves away allowing X and Y to become completely active

Question 14

What are the different isoforms of PLC beta and which tissues are they localised to?

Answer 14

1 and 3 are ubiquitous
2 Immune system/hematopoeitic cells
4 Retina and certain nervous

Question 15

What domains does the GPCR g proteins beta gamma subunit bind to in PLC beta?

Answer 15

PH domain

Y region

Question 16

What domains does the GPCR g proteins alpha subunit bind to in PLC beta?

Answer 16

EF hand
Y domain
C2 domain

Question 17

What is the cellular localisation of PLC beta?

Answer 17

Primarily near the plasma membrane however also can be cytosolic or nuclear

Question 18

What is the function of PLC beta 2?

Answer 18

To regulate the monomeric G proteins Rac1 and Rac2

Question 19

What is the function of Cdc 42 in regard to PLC?

Answer 19

Cdc42 binds to the PH domain and stimulates PLC beta

Question 20

How can PLC beta 1, 2 and 3 be inhibited?

Answer 20

By phosphorylation by PKA, PKC, PKG and Cam II

Question 21

What happens to PLCBeta 3 stimulation when two signalling pathways e.g. G alpha q and G beta gamma stimulate it at the same time?

Answer 21

Synergistic activation i.e. a greater activation than the addition of both stimulations

Question 22

How can PLC beta isoforms regulate Galphaq/11?

Answer 22

They act as RGS proteins thus a GAP for G alpha q/11 allowing the signal to shut off rapidly

Question 23

Outline the domain structure of PLC gamma

Answer 23

PH
EF
X
Split PH domain with SH2, SH2 and SH3 inside
Y 
C2 (calcium binding)

Question 24

Which domain of PLC-gamma is key to it’s regulation?

Answer 24

The multi-domain X-Y linker

Question 25

How can PLC gamma be activated?

Answer 25

By RTKs, PIP3, GPCRS or Non-receptor tyrosine kinases

Question 26

How RTKs activate PLC gamma?

Answer 26

Activated RTKs recruit PLC gamma through its SH2 domain, phosphorylating it (Activating it) and then releasing it

Question 27

What are the physiological functions of RTK recruitment of PLC gamma?

Answer 27

Development Cell cycle control Cell Death

Question 28

How can PIP3 activate PLC gamma?

Answer 28

It binds to PLC gamma through its PH domain and C-terminal SH2 domain causing it to be localised to the membrane where it can be activated by a TRK or non-receptor tyrosine kinase

Question 29

How can non-receptor tyrosine kinases activate PLC gamma?

Answer 29

E.g. Cytokine receptors | Recruit effectors with SH2 domain and then phosphorylate them, thus activating PLC gamma

Question 30

How can GPCRs activate PLC gamma?

Answer 30

Can transactivate RTKs and activate PLC gamma

Question 31

What is the largest member of the PLC family?

Answer 31

PLC epsilon

Question 32

What regulates PLC epsilon?

Answer 32

Ras and Rho families, heterotrimeric G12 family and G beta gamma subunits

Question 33

Which three proteins could potentially activate both PLC epsilon and PLC beta?

Answer 33

ET-1 LPA Thrombin

Question 34

What is the difference between PLC beta and PLC epsilons stimulation temporally?

Answer 34

PLC beta is activated acutely (peak at 15s, duration 1min) | PLC epsilon is activated during sustained PI hydrolysis (>30s)

Question 35

How can PLC activity be monitored?

Answer 35

Changes in calcium Accumulation of [3H] labelled inositol phosphates Mass of IP3 Single cell imaging

Question 36

How can PLC activity be monitored using calcium?

Answer 36

Monitoring changes in intracellular calcium | One way to do this would be using polycyclic chelators such as Fura-2 or Indo-1

Question 37

How can PLC activity be monitored by accumulation of [3H]-labelled inositol phosphates?

Answer 37

Use Lithium to block inositol phosphatase, thus IP3 concentration will not be depleted, thus IP3 concentration is solely dependent on PLC

Question 38

How can PLC activity be monitored by measuring the mass of IP3?

Answer 38

Using a radioreceptor assay in which IP3 competes with radioactively labelled IP3 for binding to a crude preparation of IP3 receptors

Question 39

How can PLC activity be monitored by single cell imaging?

Answer 39

Biosensor for IP3/PIP2 which uses a PH domain tagged with a molecule of enhanced GFP. Biosensor fluorescence will increase with IP3.

Question 40

How would a biosensor be prepared?

Answer 40

1. Generate plasmid 2. Prepare a sufficient quantity and transfect cells 3. Incubate cells for 24-48h to express protein 4. Image cells by confocal fluorescence microscopy

Question 41

Why is the activity of PLC important?

Answer 41

PLC activity has the ability to regulate many cellular processes, alterations in which could underlies disease and/or pathways that could provide targets to treat disease

Question 42

How would PLC be targeted therapeutically?

Answer 42

Most commonly through Gq