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psc2002 > lecture 21 > Flashcards

lecture 21 Flashcards

1
Q

what is the ON mechanism for cAMP production?

A

-cAMP made by adenyl cyclase (AC)
-protein-protein interaction between alpha s and AC = conformational change in AC (2 catalytic subunits come together)

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2
Q

when is adenyl cyclase off?

A

when alpha s isn’t bound

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3
Q

what is type 1 adenyl cyclase?

A

-found in plasma membrane (TMDs and catalytic domains)
-activated by forskolin (plant alkaloid that bypasses GPCR)
-9 isoforms (all activated by G alpha s)-binding causes C1/C2 domains to interact allowing production of cAMP from ATP

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4
Q

what does forskolin do?

A

-bypasses GPCR
-directly induces the conformational change of AC

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5
Q

what is AC 8 activated by?

A

increase in cytosolic calcium (could lead to increase in cAMP)
eg. in the airways

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6
Q

what is type 2 adenyl cyclase?

A

-cytosolic enzyme (AC10)

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7
Q

how is AC10 activated?

A

by increases in HCO3- (bicarbonate) and Ca2+

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8
Q

what are the OFF mechanisms?

A
  1. inhibit cAMP production
  2. breakdown cAMP (by phosphodiesterase)
  3. remove cAMP from cell
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9
Q

how is cAMP production inhibited?

A

-G alpha i reduces AC activity
-opposes stimulation by G alpha s
-lowers cAMP levels

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10
Q

how is cAMP broken down?

A

-phosphodiesterases
-11 isoforms but 8 breakdown cAMP
-expression is tissue specific
-degrade enzyme by breaking the phosphodiester bond

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11
Q

how are phosphodiesters important in ‘shaping’ the local cAMP signal?

A

it affects the:
duration
amplitude
spatial localisation

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12
Q

what inhibits PDEs?

A

caffeine

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13
Q

how are PDE inhibitors used?

A

clinically

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14
Q

what are the 3 PDE inhibitors that raise cAMP inside cells?

A

cilastazol
milrinone
roflumilast

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15
Q

what does cilastazol do?

A

-PDE3 inhibitor
-used for peripheral vascular disease
-cAMP causes vasodilation = improved blood flow

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16
Q

what does milrinone do?

A

-PDE3 inhibitor
-used for failing hearts
-cAMP increases heart rate and inotropy

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17
Q

what does roflumilast do?

A

-PDE4 selective inhibitor
-used of COPD (chronic obstructive pulmonary disease)
-cAMP relaxes airway smooth muscle = reduced obstruction

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18
Q

how is cAMP removed from cells?

A

-by ABC transporters that actively pump cAMP out the cell

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19
Q

what is the summary of the cAMP pathway?

A
  1. stimulatory agonists working through alpha s - impact plasma membrane form of adenyl cyclase to make cAMP which activates protein kinase A
  2. inhibitory agonists (alpha i) that block release of cAMP from adenyl cyclase
20
Q

what is the cAMP pathway?

A
  1. cAMP acts through cAMP-dependent protein kinase A (PKA)
  2. this catalyses transfer of terminal phosphate of ATP to serene or threonine residues on selected proteins
  3. this phosphorylation can influence either the localisation or activity of the substrate
21
Q

how is cAMP released from stimulatory agonsists and where does it go?

A
  1. by GPCRs where alpha subunit is bound to adenylyl cyclase on plasma Membrane surface
  2. soluble adenylyl cyclase activates cAMP and goes to the ABCC4 transporter
22
Q

what inhibits stimulatory agonists?

A

cholera toxin

23
Q

what does EPAC do?

A

-binds to cAMP
-stimulates phospholipase C isoform (PLCe) and Rap1
-this releases DAG and IP3

24
Q

what are the problems with the linear pathway?

A
  1. different agonists increase cAMP levels but produce DIFFERENT responses in SAME cell
  2. some physiological agonists produce cAMP dependent responses but don’t change global cAMP levels
25
Q

what do these findings suggest?

A
  • changes in cAMP must be highly localised (compartmentalised) to spatially distinct areas inside cells
    -changes in cAMP likely to be agonist-specific
26
Q

how can cAMP signalling be compartmentalised?

A

1.have GPCRs localised to different regions of the cell
2. restrict diffusion of cAMP from pm to cytosol
3. target PKA to distinct sites and substrates in cells

27
Q

what are some examples of cAMP being compartmentalised?

A

-CFTR activity in epithelial cells
-isolated heart cell response to different cAMP agonists

28
Q

how do PDEs restrict diffusion?

A
29
Q

what is patch clamp?

A

an experimental method to study how ions flowing through channels can change membrane potential.

30
Q

what are the 4 types of patch clamp configuration?

A

outside-out, cell attached, whole cell, inside-out

31
Q

what is the cell attached patch experiment?

A

-adenosine added to bath in exp 1 and pipette in exp 2
- in exp 1 = not much happens-channel cant be activated, forskolin added to bath soon and activates all AC - big increase in cAMP in cell
- in exp 2 = makes cAMP and activates some CFTR - antagonist added and shows less activity

31
Q

what is the cell attached configuration?

A

tight contact created between pipette and plasma membrane

32
Q

what does inhibiting PDE4 do?

A

-eliminates compartmentalised cAMP signalling in airway cells

33
Q

how do MRP transporters alter cAMP levels?

A

-involved in regulating cAMP deponent processes in epithelial cells
eg. CFTR and intestinal Cl- secretion

34
Q

what occurs in intestinal Cl- secretion with MRP4?

A

-monolayer of intestinal cells put into Ussing chamber to measure short circuit current
-cAMP = membrane permeable analogue
-glybenclamide = blocker of CFTR

35
Q

how can cAMP signalling be compartmentalised?

A

-target PKA (effector) to distinct sites in cells
-happens through A kinase anchoring proteins (AKAPs)

36
Q

what is the function of cAMP?

A

-passes info down signalling pathway activating cAMP dependent protein kinase (PKA)
-cAMP binding to PKA induces conformational change = release and activation of catalytic subunits

37
Q

what are AKAPs?

A
  • PKA2 binds to AKAP via PKA docking domain on Rll
    -R submits bind cAMP but just change confirmation and can phosphorylate substrates nearby
    -AKAPs have own targeting domain - brings PKA close to substrates
    -help assemble signalling complexes to form signalling hubs or signalsomes
38
Q

what is the role of AKAPs in activation of CFTR?

A

-from inside out patch
-ATP alone doesn’t activate channels
-PKA plus ATP = channel activation
-if PKA present via AKAP it should be able to:
1. activate CFTR by cAMP alone (+ATP)
2. block activation by cAMP by preventing PKAll binding to AKAP

39
Q

what is HT31P?

A

-control peptide
-doesnt affect PKAll-AKAP interaction

40
Q

what is HT31?

A

-short peptide
-disrupts PKAll-AKAP interaction

41
Q

what is the conclusion of the experiment?

A

-PKA must be anchored very close to CFTR in the excised patch and must be involve an AKAP
-control did nothing

42
Q

what study was done by Ezrin (AKAP) targeting PKA to CFTR?

A

-Ezrin targets PKA to CFTR
-binds via NHERF1

43
Q

what are the properties of NHERF1?

A
  1. has binding domain for CFTR
  2. ERM domain that binds to Ezrin
44
Q

what does activation of CFTR require?

A

AKAP, ezrin (linked to CFTR by scaffold protein), NHERF1

psc2002 (15 decks)

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