2nd Nov - Phospholipids and Phospholipid Signalling Flashcards
What is the most abundant membrane lipid?
Phosphatidylcholine
What is the function of phospholipases?
To turn glycophospholipids into a variety of different second messengers
What reaction does phospholipase D catalyse?
Phosphatidylcholine –> phosphatidic acid
What reaction does phospholipase C catalyse?
PIP2 –> IP3 and DAG
What are the main downstream effectors of DAG?
Phosphatidic acid
Enzymes and structural proteins w/ CI domain e.g. PKC
What are the main downstream effectors of IP3?
IP3 receptor on ER
Multiple kinases, transcription and RNA processing factors
What are the mammalian isoforms of PLC?
PLC-beta, gamma, delta, epsilon, zeta, eta
What is believed to be the original isoform of PLC?
PLC-delta
What are the domains of PLC delta?
X + Y catalytic domains
PH domain
4 tandem EF hand domains
C2 domain
How is PLC delta activated?
By calcium - PLC delta responds to calcium 50-100x better than the beta and gamma isoforms
What is believed to be the function of PLC delta?
To amplify and prolong calcium signals
Outline the domain structure of PLC beta
PH EF X Y C2 Coiled-coil domain
What is the function of the X-Y linker in PLC beta?
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
What are the different isoforms of PLC beta and which tissues are they localised to?
1 and 3 are ubiquitous
2 Immune system/hematopoeitic cells
4 Retina and certain nervous
What domains does the GPCR g proteins beta gamma subunit bind to in PLC beta?
PH domain
Y region
What domains does the GPCR g proteins alpha subunit bind to in PLC beta?
EF hand
Y domain
C2 domain
What is the cellular localisation of PLC beta?
Primarily near the plasma membrane however also can be cytosolic or nuclear
What is the function of PLC beta 2?
To regulate the monomeric G proteins Rac1 and Rac2
What is the function of Cdc 42 in regard to PLC?
Cdc42 binds to the PH domain and stimulates PLC beta
How can PLC beta 1, 2 and 3 be inhibited?
By phosphorylation by PKA, PKC, PKG and Cam II
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?
Synergistic activation i.e. a greater activation than the addition of both stimulations
How can PLC beta isoforms regulate Galphaq/11?
They act as RGS proteins thus a GAP for G alpha q/11 allowing the signal to shut off rapidly
Outline the domain structure of PLC gamma
PH EF X Split PH domain with SH2, SH2 and SH3 inside Y C2 (calcium binding)
Which domain of PLC-gamma is key to it’s regulation?
The multi-domain X-Y linker
How can PLC gamma be activated?
By RTKs, PIP3, GPCRS or Non-receptor tyrosine kinases
How RTKs activate PLC gamma?
Activated RTKs recruit PLC gamma through its SH2 domain, phosphorylating it (Activating it) and then releasing it
What are the physiological functions of RTK recruitment of PLC gamma?
Development
Cell cycle control
Cell Death
How can PIP3 activate PLC gamma?
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
How can non-receptor tyrosine kinases activate PLC gamma?
E.g. Cytokine receptors
Recruit effectors with SH2 domain and then phosphorylate them, thus activating PLC gamma
How can GPCRs activate PLC gamma?
Can transactivate RTKs and activate PLC gamma
What is the largest member of the PLC family?
PLC epsilon
What regulates PLC epsilon?
Ras and Rho families, heterotrimeric G12 family and G beta gamma subunits
Which three proteins could potentially activate both PLC epsilon and PLC beta?
ET-1
LPA
Thrombin
What is the difference between PLC beta and PLC epsilons stimulation temporally?
PLC beta is activated acutely (peak at 15s, duration 1min)
PLC epsilon is activated during sustained PI hydrolysis (>30s)
How can PLC activity be monitored?
Changes in calcium
Accumulation of [3H] labelled inositol phosphates
Mass of IP3
Single cell imaging
How can PLC activity be monitored using calcium?
Monitoring changes in intracellular calcium
One way to do this would be using polycyclic chelators such as Fura-2 or Indo-1
How can PLC activity be monitored by accumulation of [3H]-labelled inositol phosphates?
Use Lithium to block inositol phosphatase, thus IP3 concentration will not be depleted, thus IP3 concentration is solely dependent on PLC
How can PLC activity be monitored by measuring the mass of IP3?
Using a radioreceptor assay in which IP3 competes with radioactively labelled IP3 for binding to a crude preparation of IP3 receptors
How can PLC activity be monitored by single cell imaging?
Biosensor for IP3/PIP2 which uses a PH domain tagged with a molecule of enhanced GFP. Biosensor fluorescence will increase with IP3.
How would a biosensor be prepared?
- Generate plasmid
- Prepare a sufficient quantity and transfect cells
- Incubate cells for 24-48h to express protein
- Image cells by confocal fluorescence microscopy
Why is the activity of PLC important?
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
How would PLC be targeted therapeutically?
Most commonly through Gq
