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Pharmacology > cAMP > Flashcards

cAMP Flashcards

1
Q

What was the first second messenger to be isolated?

A

cAMP

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2
Q

What are the basal levels of cyclic nucleotides in the cell?

A

100 nM

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3
Q

What sort of increase in cyclic nucleotide is required to produce a maximum response in most tissues?

A

2- to 3- fold

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4
Q

Which enzyme catalysed the formation of cAMP from ATP?

A

Adenylyl Cyclades

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5
Q

How many genes for adenylyl cyclase (AC) have been identified in mammals?

A

10

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6
Q

Of the 10 mammalian AC genes, how many are bound to the plasma membrane?

A

9

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7
Q

AC10 is a soluble enzyme insensitive to G proteins. What is it regulated by?

A

Bicarbonate

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8
Q

The 10 different isoforms of AC differ in regulation and tissue distribution. In which organ are all 10 isoforms found?

A

The brain

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9
Q

AC1-AC9 adenylyl cyclases are regulated by a number of factors. Give some examples.

A
  1. Activity increased by Gs
  2. Activity decreased by Gi (BUT not for all, eg AC2)
  3. Gby subunits (eg increase AC2 and decrease AC1)
  4. Activity increased by Ca/calmodulin (AC1)
  5. Activity decreased by [Ca]i (AC5)
  6. PKC and PKA phosphorylation of proteins
  7. Activated by FORSKOLIN
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10
Q

G-beta-gamma subunits can regulate AC activity. Give an example of an isoform whose activity it:

  1. Increases
  2. Decreases
A
  1. AC2

2. AC1

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11
Q

Give an example of an AC isoform that is not affected by Gi-linked receptor activity.

A

AC2

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12
Q

Different AC isoforms are regulated in different ways. What does this heterogeneity of properties allow for?

A
  1. Cell-specific responsiveness to different extracellular signals (depending on which AC isoforms are present)
  2. “Cross-talk” with other signalling pathways affecting Ca++ and PKC activity
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13
Q

What terminates cAMP signalling?

A

Breakdown to 5’AMP by phosphodiesterase enzymes

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14
Q

What does the vast majority of cAMP signalling result from?

A

It binding to and activating PKA

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15
Q

Whilst the vast majority of cAMP signalling results from it binding to and activating PKA, which channels can it bind?

A

Cyclic Nucleotide Gated Channels

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16
Q

Which G-proteins does cAMP regulate? How?

A

Monomeric G-proteins such as Rap1 by activating Epac (Exchange protein activated by cAMP) 1 and 2 which are GEFs for Rap1

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17
Q

How does cAMP regulate the monomeric G protein Rap1?

A

By activating Epac1/2 (Exchange protein activated by cAMP) which are GEFs for Rap1

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18
Q

What is Epac1/2?

A

Exchange Protein Activated by cAMP

It is a GEF for Rap1

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19
Q

Epac 1 and Epac 2 are GEFs for which protein?

A

Monomeric G protein Rap1

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20
Q

What is PKA?

A

Protein kinase A - ie cAMP-dependent kinase

It is a serine-threonine kinase

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21
Q

What type of kinase is PKA?

A

Serine-threonine kinase

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22
Q

What consensus recognition sequence does PKA phosphorylate?

A

K/R - K/R - X - S/T - I/V/L

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23
Q

Give three examples of the signalling functions of cAMP

A
  1. Regulation of gene transcription
  2. Control of excitation-contraction coupling in the heart
  3. Regulation of metabolism
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24
Q

How is efficient and specific signal transduction achieved by cAMP?

A

It is often concentrated into MICRODOMAINS

  1. the receptors, AC and PKA are compartmentalised by AKAPs
  2. Local pools of cAMP are generated by PDEs which break down cAMP
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25
Q

What is the purpose of concentrating cAMP mediated signalling into microdomains?

A

Allows efficient and specific signal transduction to occur

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26
Q

How are receptor, AC, and PKA compartmentalised?

A

By A-Kinase Anchoring Proteins (AKAPs)

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27
Q

How do AKAPs enable efficient and specific signal transduction by cAMP?

A

They compartmentalise receptors, AC and PKA

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28
Q

How does Forskolin affect AC activity?

A

Activates it

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29
Q

How does PKA exist in its unstimulated state?

A

As an inactive TETRAMER consisting of:
2 x regulatory subunits
2 x catalytic subunits

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30
Q

How many isoforms are there of each of PKA’s subunits?

A

4 isoforms of the regulatory subunit (R1alpha, R1beta, RIIalpha, RIIbeta)

3 isoforms of the catalytic subunit (Calpha, Cbeta, Cgamma)

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31
Q

How many classes of PKA exist based on their composition of regulatory subunits?

A

2: PKAI and PKAII

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32
Q

What is the difference between the two classes of PKAs, PKAI and PKAII?

A

PKAI
Contains RI subunits. Low affinity for AKAPs. Mainly found free in cytoplasm. Has high affinity for cAMP- response to low [cAMP]

PKAII
Contains RII subunits. High affinity for AKAPs. Usually docked to these scaffolding proteins- localised to specific cellular targets

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33
Q

PKAI is mainly found in the cytoplasm whilst PKAII is mainly found docked to AKAP scaffolding proteins. Why is this?

A

PKAI has low affinity for AKAPs whilst PKAII has high affinity for them.

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34
Q

How do PKI and PKII differ in their subunit composition?

A

PKI contains RI regulatory subunits whilst PKII contains RII regulatory subunits

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35
Q

How many molecules of cAMP bind to each R subunit of PKA?

A

2

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36
Q

What happens when cAMP binds to PKA?

A

2 molecules of cAMP bind to each PKA R subunit, causing a conformational change in the regulatory subunits so that the catalytic subunits are released.

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37
Q

Why does phosphorylation of a protein lead to its conformational change?

A

The phosphate group carried a negative charge

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38
Q

Why does phosphorylation affect the function of a protein?

A

Because the negatively charged phosphate group changes the confirmation of the protein

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39
Q

What does cAMP stand for?

A

Cyclic 3’,5’ adenosine monophosphate

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40
Q

Give some examples of cellular targets for PKA

A

1) neurotransmitter biosynthesis
2) GPCRs
3) ion channels
4) cytoskeletal proteins
5) transcription factors
6) protein phosphatase inhibitors

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41
Q

PKA can target transcription factors. Give an example of such a transcription factor

A

CREB protein (cAMP-response element binding protein)

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42
Q

What does CREB stand for?

A

cAMP response element binding protein

43
Q

PKA can target protein phosphatase inhibitors. Give an example.

A

DARPP-32 (dopamine and cAMP-regulated phosphoprotein)

44
Q

PKA can target proteins involved in neurotransmitter biosynthesis. Give an example

A

Tyrosine hydroxylase (rate-limiting enzyme in dopamine, noradrenaline and adrenaline synthesis)

45
Q

Give two examples of GPCRs targeted by PKA

A

B-adrenoceptors

M1 muscarinic receptors

46
Q

DARPP-32 is an important target for PKA in the brain. What is it?

A

A protein phosphatase inhibitor

47
Q

How do protein kinases and phosphatases integrate inputs into neurones?

A

Cross talk between them

48
Q

Medium spiny neurones in the striatum receive what type of signals?

A

Excitatory glutaminergic inputs

Inhibitory dopaminergic signals

49
Q

How does PKA stimulate the inhibition of protein phosphatase 1 (PP1)?

A

Dopamine stimulates PKA which phosphorylates DARPP-32 at T34 allowing it to inhibit PP1

50
Q

On which residue does PKA phosphorylate DARPP-32?

A

T34

51
Q

PKA can stimulate DARPP-32 to inhibit PP1. How can this action be countered?

A

NMDA receptor mediated increases in intracellular calcium which activates calneurin. Calneurin (PP2B) dephosphorylates DARPP-32 which stops it from inhibiting PP-1.

52
Q

PKA phosphorylates and activates DARPP-32, which inhibits PP1. Which protein prevents DARPP-32 from doing this? How?

A

Calcineurin. It dephosphorylates DARPP-32, which inactivates it

53
Q

Calcineurin relieves inhibition of PP1 by dephosphosphorylating and inactivating DARPP-32. How is calcineurin activated?

A

By NMDA receptor mediated (and other) increases in intracellular calcium.

54
Q

PKA phosphorylates DARPP-32 on T34. What happens if DARPP-32 is phosphorylated on T75?

A

DARPP-32 would inhibit PKA

55
Q

The ability of DARPP-32 to modulate which type of signalling has been implicated in the actions of caffeine and several drugs of abuse (e.g. cocaine)?

A

dopaminergic

56
Q

cAMP can regulate transcription of genes containing CREs. What are CREs?

A

cAMP response elements

57
Q

Which family of nuclear receptors mediates cAMP regulation of gene transcription?

A

CREB (cAMP-response element binding) proteins.

58
Q

What motif do CREBs contain that allows them to bind DNA?

A

Basic domain/ leucine zipper motif

59
Q

Which genes does the transcription factor CREB modulate?

A

Those containing cAMP response elements (CREs) in their promoters

60
Q

What element must genes possess in their promoters in order to be modulated by the CREB transcription factor?

A

CRE (cAMP recognition element)

61
Q

How does an increase in [cAMP] affect CREB?

A

Increased [cAMP] results in activation of PKA. The catalytic subunit enters the nucleus and phosphorylates CREB at serine 133. Phosphorylated CREB can then interact with CREs on gene promoters and along with co-activator proteins, activates gene transcription.

62
Q

Which residue on CREB is phosphorylated by PKA?

A

Serine 133

63
Q

How is CREB activated?

A

Rise in [cAMP] activates PKA. The PKA catalytic subunit enters the nucleus and phosphorylates CREB on Serine 133

64
Q

What is the result of Serine133 phosphorylation on CREB?

A

It interacts with CREs on gene promoters and (along with other co-activators) activates gene transcription

65
Q

Give some examples of genes whose transcription is regulated by CREB

A

1) c-fos
2) tyrosine hydroxylase
3) neuropeptides
4) genes involved in the mammalian circadian clock

66
Q

cAMP can activate cyclic nucleotide gated channels (CNGs). What type of channels are these?

A

non-selective cation channels

67
Q

What are CNGs?

A

cyclic nucleotide gated channels

68
Q

CNGs are found in a variety of cell types. How are they opened?

A

In response to binding of (typically 4) cyclic nucleotides

69
Q

What do CNGs contribute to?

A

Control of membrane potential and intracellular calcium levels

70
Q

To which cyclic nucleotides can olfactory CNG bind?

A

cAMP and cGMP

71
Q

To which ion channels are CNGs structurally similar?

A

voltage gated K+ channels

72
Q

Which proteins are GEFs for the Ras-like monomeric G protein Rap1?

A

Epac1 and Epac2 (i.e. Exchange protein activated by cAMP 1 and 2)

73
Q

What does Epac stand for? What is their function in cAMP signalling?

A

Exchange protein activated by cAMP. They act as GEFs for the small monomeric Ras-like G proteins such as Rap1, Rac and H-Ras

74
Q

What is Rap1?

A

A small monomeric Ras-like G protein

75
Q

cAMP directly regulates which GEFs?

A

Epac1 and 2

76
Q

Which G proteins does cAMP regulate via the GEFs Epac1 and Epac 2?

A

Rap1, Rac and H-Ras

77
Q

What pathways do the small G proteins Rap1, Rac and H-Ras mediate?

A

Secretion
Cell adhesion
Exocytosis
Gene transcription

78
Q

What are Epacs?

A

exchange proteins activated by cAMP - GEFs

79
Q

Once activated, the C subunits of PKA do not phosphorylate every protein containing a consensus site. Why not?

A

Compartmentalisation

80
Q

Give 4 main mechanisms by which cAMP signalling can be compartmentalised.

A

1) AKAPs (A-Kinase Anchoring Proteins) bind to the R subunits of PKA to localise it to specific regions
2) Targeting of some receptors, G proteins and ACs are targeted to CAVEOLAE
3) Limiting diffusion of cAMP to specific areas using PDEs
4) The multiplicity of AC and PDE isoforms, each differentially regulated

81
Q

How do AKAPs localise PKAs to specific regions?

A

They bind to the RII subunit (only Class II PKAs)

82
Q

How can the spatial domains in which cAMP can diffuse be limited to ‘channel’ the signal to specific subcellular regions?

A

Cellular placement of phosphodiesterases (PDEs)

83
Q

How does the existence of multiple isoforms of AC and PDE allow compartmentalisation of cAMP signalling?

A

Each isoform is differentially regulated, so the signal processing will depend on which isoforms are expressed in that particular cell (and/ or subcellular compartment)

84
Q

How is cAMP-mediated signalling terminated?

A

G-alphaS has intrinsic GTPase activity and hydrolyses GTP to GDP and Pi. The presence of GDP on G-alphaS causes it to rebind to the inhibitory Beta-Gamma complex and AC is no longer activated.

PDEs hydrolyse cAMP to AMP. This shifts the equilibrium between free cAMP and cAMP bound to PKA. cAMP dissociates from the regulatory subunit, allowing it to rebind the catalytic subunit, returning the kinase to its inactive form.

Protein phosphatases catalyse hydrolysis of phosphates that were attached to proteins via PKA.

Receptor desensitisation.

85
Q

How are the intracellular levels of cAMP regulated?

A

The balance between the activities of two enzymes: AC and PDE

86
Q

Most ACs are activated downstream from GPCRs such as the B-AR by interactions with the Gas subunit. Which are not?

A

AC10, i.e. the soluble bicarbonate-regulated AC

87
Q

The heterogeneity of properties between different adenylyl cyclase isoforms allows for what? Give an example.

A
  1. cell specific responsiveness to different extracellular signals (depending on which AC isoforms are present)
  2. ‘cross-talk’ with other signalling pathways affecting Ca2+ and PKC activity
88
Q

What is the consensus recognition sequence for PKA?

A

K/R - K/R - X - S/T - I/V/L

89
Q

What is the difference in localisation between PKA1 and PKA2?

A

PKA1 has DIFFUSE CYTOPLASMIC localisation

PKA2 has SPECIFIC LOCAL localisation

90
Q

What does DARPP-32 stand for?

A

Dopamine- and cAMP- Regulated PhosphoProtein, Mw = 32

91
Q

What converts DARPP-32 into a potent inhibitor of PP-1?

A

Phosphorylation at Thr(34) by PKA

92
Q

What is PP-1?

A

A multifunctional serine/threonine protein phosphatase

93
Q

How is DARPP-32 converted into an inhibitor of PKA?

A

Phosphorylation at Thr(75) by Cdk5

94
Q

DARPP-32 plays a key role in integrating a variety of biochemical, electrophysiological, and behavioural responses controlled by which neurotransmitter in particular?

A

Dopamine

95
Q

Which receptors does caffeine act on?

A

A1 and A2 adenosine receptors AND phosphodiesterase

96
Q

How does caffeine act at A1/2 adenosine receptors to exert its actions?

A

Antagonistically

97
Q

How does binding of cAMP activate Epacs?

A

It induced a CONFORMATIONAL CHANGE that releases the AUTO-INHIBITION of the catalytic site, thus allowing Epac to activate the Rap GTPases

98
Q

In its inactive state, how is the GEF activity of Epac inhibited?

A

The N-term REGULATORY region interacts with the catalytic region to inhibit GEF activity

99
Q

Which domain of Epac promotes the exchange of GDP for GTP on Rap GTPases?

A

the CDC25 domain

100
Q

How does Epac signalling differ from other cAMP signalling pathways?

A

It is independent of PKA

101
Q

Which enzymes ‘funnel’ cAMP signals into particular parts of the cell?

A

Phosphodiesterases

102
Q

In the heart, how do 1) Beta-AR and 2) L-type calcium channels keep PKA in localised regions?

A

AKAPs (A-Kinase Anchoring Proteins)

1) AKAP79
2) AKAP18

103
Q

How does strategic localisation of phosphodiesterase affect cAMP levels in the cell?

A

It limits diffusion

Pharmacology (13 decks)

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