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Molecular Signalling > AM lecture 2 > Flashcards

AM lecture 2 Flashcards

1
Q

P2X receptors

A

Ionotropic

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2
Q

P2Y receptors

A

GPCRs

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3
Q

How is endogenous ATP released from cells into the external environment?

A

Pannexin 1
CFTR
Vesicular transport/secretion
Necrotic cell death (ATP floods into environment after cell membrane lysis)

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4
Q

Concentration of ATP in cells

A

mM range

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5
Q

What happens to ATP once it is extracellular?

A

Can be degraded by cell surface enzymes to control levels

Adenosine binds to adenosine GPCRs

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6
Q

Pannexins

A

Glycoproteins that form hexameric channels
Cytosolic N- and C-terminals, 4 transmembrane domains
Three pannexins have been described so far: Panx1, Panx2 and Panx3

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7
Q

Pannexin 1

A

Ubiquitously expressed, including in immune cells, platelets and endothelial cells
Implicated in the release of ATP, propagation of calcium waves, regulation of vascular tone, mucociliary lung clearance and taste bud function
Mouse Pannexin 1 forms an anion-selective ion channels when expressed in HEK293 cells (makes sense given that ATP is anionic)

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8
Q

Activation of Pannexin 1

A

Can be receptor-dependent or independent

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9
Q

Receptor-independent activation of Pannexin 1

A 
Ischemia
Caspase cleavage
Low O2 tension
Hypotonicity
Mechanical stimulation
Physiological depolarisation
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10
Q

Receptor-dependent activation of Pannexin 1

A

Ionotropic: P2X4, P2X7, NMDAR
Metabotropic: P2Y1/2/6, PAR-1
(a lot of these receptors are activated by ATP which is what Pannexin releases - positive feedback mechanism at work?)

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11
Q

Investigating the role of Pannexin 1 channels in the inflammatory response

A

Assessed ATP release in response to activation by the pro-inflammatory cytokine TNF-a
Ex-vivo vascular perfusion assay - murine mesenteric venules were cannulated on glass micropipettes in a temperature-controlled bath
Perfusion of recombinant murine TNF-a through the lumen of the venules produced a time- and dose-dependent increase in ATP accumulation in the perfusate, assessed bioluminescence using luciferase

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12
Q

Method for generating Panx1 knockout mice

A

Genetically engineered mice that lacked Panx1 expression specifically in endothelial cells
Crossed mice carrying loxP sites flanking exon 3 of the murine Panx1 gene with transgenic mice carrying a tamoxifen-sensitive Cre recombinase driven by an EC-specific promoter
Cre bascially inactive until tamoxifen treatment which allowed mice to develop normally

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13
Q

Pannexin 1 knockout mice

A

Used to directly investigate the contribution of endothelial Panx1 channels to TNF-a-induced ATP release in the intact venous circulation
After 10 days of tamoxifen administration, the transgenic mice show a substantial reduction on Panx1 expression
Luminal perfusion of TNF-a in isolated mesenteric venules from these mice showed a dramatic inhibition of ATP release compared with controls

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14
Q

Elucidation of the mechanism by which activation of TNF receptors in endothelial cells translates to Panx1 opening

A

PP2 = Src family Tyr kinase inhibitor
PP3 = inactive analogue of PP2
Inhibition of SFKs with PP2 significantly reduced TNF-a-induced ATP release, while PP3 showed no significant effect
Can conclude that Src kinase must be involved in the downstream signalling linking TNF receptors to Panx1

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15
Q

Release of ATP from platelets

A

‘Electron dense’ secretory granules in platelets contain ATP, ADP and serotonin

Platelets express P2X and P2Y receptors
Endothelial cells express CD39 on their surface to control the levels of ATP and affect how much ATP the platelet is exposed to as it passes through the endothelium

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16
Q

How does extracellular ATP exert its effects on cells?

A

By binding to type 2 purinergic receptors (P2X / P2Y)
P2X = cation-permeable ion channel
P2Y receptors can be Gs, Gi and Gq coupled

17
Q

CD39

A

Plasma-membrane-bound ectoenzyme that catalyses the hydrolysis of ATP into ADP/AMP

18
Q

CD73

A

Plasma-membrane-bound ectoenzyme that catalyses the hydrolysis of AMP into adenosine
Adenosine can then engage purinergic P1 receptors (adenosine receptors)

19
Q

Activity of CD39 and CD73

A

Contributes to the removal of ATP from the extracellular space, thus limiting the duration of signalling

20
Q

CD39 knockout mice

A

No platelet plug formation upon injury of the arterioles
Indicating platelets aren’t being activated and attaching onto blood vessels
Treatment of CD39–/– platelets with a soluble ATPase (apyrase) consistently restored aggregatory potential in platelets
These results indicate that purinergic receptors on platelets from CD39–/– mice had undergone desensitization in vivo

21
Q

Discovery of the P2Y1 receptor

A

Hybridisation screening with the partial cDNA of an identified GPCR from a guinea pig against a chick brain cDNA library
Identified a sequence for a new GPCR
Injected RNA into Xenopus Oocyte to evaluate phenotype
ATP, 2-MeS-ATP and ADP activated slow inward current (via CACCs)
UTP and ab-me-ATP did not activate a current
Current blocked by suramin

22
Q

Why was chick brain chosen for the hybridisation screening for the P2Y1 receptor?

A

Chick brains show high levels of ATP binding so must be a good source of this receptor
Chick brains are in a phase of highly active expression of many receptor mRNAs around the time of hatching

23
Q

Discovery of the P2Y2 receptor

A

Used an expression cloning strategy to isolate a cDNA clone from the NG108-15 cell line
Injected RNA into Xenopus Oocyte to evaluate phenotype
Saw slow current responses to ATP, UTP and ATPyS
Saw no response to ADP, 2-MeS-ATP or adenosine

24
Q

How many human P2Y receptor subtypes are there?

A

8

Divided into the P2Y1-like and P2Y12-like subfamilies

25
Q

Expression profile of P2Y receptors

A

Throughout the body

26
Q

P2Y1-like subfamily

A

P2Y1/2/4/6/11
All Gaq coupled (P2Y11 also Gas coupled)
Activated by a mixture of ATP, ADP, UTP and UDP

27
Q

P2Y12-like subfamily

A

P2Y12/13/14
All Gai coupled
Activated by a mixture of ADP, UDP and UDP-glucose

28
Q

ADP activates

A

P2Y1/12/13

29
Q

ATP activates

A

P2Y2/4(rat not human)/11

30
Q

UDP activates

A

P2Y6/14

31
Q

UTP activates

A

P2Y2/4

32
Q

UDP-glucose and other UDP-sugars activate

A

P2Y14

33
Q

FRET approaches have demonstrated that…

A

…GPCRs can form functional dimers

FRET enables protein-protein interactions to be studied in live cells

34
Q

P2Y1 KO mice

A

Decreased platelet aggregation
Increased bleeding time

Interesting that platelets could still respond to ATP - was later realised that this was because P2Y12 was also present

35
Q

P2Y2 KO mice

A

Lacked flow-induced vasodilation
Developed hypertension

P2Y2 controls blood pressure by mediating endothelial mechanotransduction

36
Q

P2Y6

A

Cytokine release by macrophages

37
Q

P2Y13

A

Skeletal development

38
Q

P2Y14

A

Regulates insulin secretion

Molecular Signalling (6 decks)

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