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BS3054 > 2nd Nov - Phospholipids and Phospholipid Signalling > Flashcards

2nd Nov - Phospholipids and Phospholipid Signalling Flashcards

1
Q

What is the most abundant membrane lipid?

A

Phosphatidylcholine

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2
Q

What is the function of phospholipases?

A

To turn glycophospholipids into a variety of different second messengers

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3
Q

What reaction does phospholipase D catalyse?

A

Phosphatidylcholine –> phosphatidic acid

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4
Q

What reaction does phospholipase C catalyse?

A

PIP2 –> IP3 and DAG

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5
Q

What are the main downstream effectors of DAG?

A

Phosphatidic acid

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

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6
Q

What are the main downstream effectors of IP3?

A

IP3 receptor on ER

Multiple kinases, transcription and RNA processing factors

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7
Q

What are the mammalian isoforms of PLC?

A

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

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8
Q

What is believed to be the original isoform of PLC?

A

PLC-delta

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9
Q

What are the domains of PLC delta?

A

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

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10
Q

How is PLC delta activated?

A

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

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11
Q

What is believed to be the function of PLC delta?

A

To amplify and prolong calcium signals

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12
Q

Outline the domain structure of PLC beta

A 
PH
EF
X
Y
C2 
Coiled-coil domain
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13
Q

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

A

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

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14
Q

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

A

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

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15
Q

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

A

PH domain

Y region

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16
Q

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

A

EF hand
Y domain
C2 domain

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17
Q

What is the cellular localisation of PLC beta?

A

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

18
Q

What is the function of PLC beta 2?

A

To regulate the monomeric G proteins Rac1 and Rac2

19
Q

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

A

Cdc42 binds to the PH domain and stimulates PLC beta

20
Q

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

A

By phosphorylation by PKA, PKC, PKG and Cam II

21
Q

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?

A

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

22
Q

How can PLC beta isoforms regulate Galphaq/11?

A

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

23
Q

Outline the domain structure of PLC gamma

A 
PH
EF
X
Split PH domain with SH2, SH2 and SH3 inside
Y 
C2 (calcium binding)
24
Q

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

A

The multi-domain X-Y linker

25
Q

How can PLC gamma be activated?

A

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

26
Q

How RTKs activate PLC gamma?

A

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

27
Q

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

A

Development
Cell cycle control
Cell Death

28
Q

How can PIP3 activate PLC gamma?

A

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

29
Q

How can non-receptor tyrosine kinases activate PLC gamma?

A

E.g. Cytokine receptors

Recruit effectors with SH2 domain and then phosphorylate them, thus activating PLC gamma

30
Q

How can GPCRs activate PLC gamma?

A

Can transactivate RTKs and activate PLC gamma

31
Q

What is the largest member of the PLC family?

A

PLC epsilon

32
Q

What regulates PLC epsilon?

A

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

33
Q

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

A

ET-1
LPA
Thrombin

34
Q

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

A

PLC beta is activated acutely (peak at 15s, duration 1min)

PLC epsilon is activated during sustained PI hydrolysis (>30s)

35
Q

How can PLC activity be monitored?

A

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

36
Q

How can PLC activity be monitored using calcium?

A

Monitoring changes in intracellular calcium

One way to do this would be using polycyclic chelators such as Fura-2 or Indo-1

37
Q

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

A

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

38
Q

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

A

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

39
Q

How can PLC activity be monitored by single cell imaging?

A

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

40
Q

How would a biosensor be prepared?

A
  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
41
Q

Why is the activity of PLC important?

A

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

42
Q

How would PLC be targeted therapeutically?

A

Most commonly through Gq

BS3054 (25 decks)

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