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Flashcards in GPCRs Deck (102):
1

What is the structure of a GPCR

7 transmembrane proteins in a single chain.
3 intra and 3 extra cellular loops
N-terminus extracellular
C-terminus intracellular

2

Name the 5 families of GPCRs

Secretin
Adhesion
Glutamate
Frizzled/Taste2
Rhodopsin

3

What is the special feature of the Adhesion family of GPCRs

Large N-terminus which includes similar regions to adhesion molecules

4

What are the special features of the Glutamate family of GPCRs

Mainly neuronal
Smaller extracellular region
Can be ligand gated ion channels

5

What are the special features of the Frizzled/Taste2 family of GPCRs

Generally involved in controlling development and cell growth

6

What are the special features of the Rhodopsin family of GPCRs

Biggest family of GPCRs.
Include amine receptors, light receptors, olfactory receptors etc.

7

How was the structure/function relationship of GPCRs determined?

They created chimeras. Fused two different receptors and measured response

8

What region of a GPCR determines function

Function is usually controlled by intracellular loops.
Specifically intracellular loop 3 and carboxyl (C)-terminus.

9

How does a GPCR signal?

Receptor activation causes the alpha subunit of the G-protein to exchange a GDP for a GTP.
The GTP bound alpha subunit is able to activate an effector molecule

10

How does a GPCR signal switch off

Each G-protein has an intrinsic GTPase. It hydrolyses the GTP to give GDP (inactive)

11

What are the 4 stages of the G-protein cycle

Basal state
Receptor activation
Effector modulation
GTP hydrolysis

12

What happens in the basal state of the G-protein cycle

Nothing. Alpha bound to GDP

13

What happens in the receptor activation state of the G-protein cycle

Agonist activates receptor
G-protein coupled and GDP exchanged for GTP
G-protein subunits dissociated

14

What happens in the effector modulation state of the G-protein cycle

Effector is bound to the G alpha subunit

15

What happens in the GTP hydrolysis state of the G-protein cycle

GTP bound to alpha subunit dissociates.
Alpha G protein bound to GDP
Subunits re-associate

16

What is the role of the alpha subunit of a G-protein

Bound to GDP/GTP
Activates effector molecule

17

What is the role of the beta subunit of a G-protein

Propeller structure that creates stability

18

What makes up a G-protein

Alpha subunit
Beta subunit
Gamma subunit

19

What is the effector pair of a Gs protein

Adenylate cyclase

20

What is the effector pair of a G (alpha i) protein

Adenylate cyclase

21

What is the effector pair of a G (alpha o) protein

K+ and Ca2+ channels

22

What is the effector pair of a G (alpha t) protein

cGMP phosphodiesterase

23

What is the effector pair of a G (alphah gust) protein

cGMP phosphodiesterase and possibly adenylate cyclase

24

What is the effector pair of a G (q / 11) protein

Phospholipase C - beta

25

What is the effector pair of a G (12 / 13) protein

Rho A
Misc small proteins

26

What are the main downstream signalling molecules associated with GPCRs

Adenylyl cyclase
cAMP
Protein Kinase A (PKA)
Phosphodiesterase
RGS (regulator of G-protein signalling)

27

Name a common downstream signalling pathway

Adenylyl (adenylate) cyclase activation -> increased cAMP levels -> Increased PKA levels -> lots of intracellular pathways

28

How does cAMP activate protein kinase A

PKA is made up of four subunits - two catalytic subunits, and two regulatory subunits.
cAMP binds to and mediates the dissociation of the regulatory subunits, enabling PKA to function.

29

What is Forskolin and why is it used?

It is a drug that increases the effect of GPCRs coupled to a Gs protein. Usually used in labs to show effects of adenylyl cyclase as it bypasses the need for GPCR activation

30

Give an example of a downstream event triggered by PKA activation

Calcium-handling proteins activated, increases force of contraction in the heart

31

Which G-proteins change intracellular levels of cAMP

Gs - increases
Gi - decreases

32

Define phospholipase

An enzyme that hydrolyses phospholipids (e.g. cAMP) into fatty acids and other lipophilic substances

33

What are the three groups of Protein Kinase C

Calcium-dependent, DAG-activated (cPKC; conventional)
Calcium-independent, DAG-activated (nPKC; novel)
Calcium-independent, DAG-non-responsive (aPKC; atypical)

34

What does adenylyl cyclase do

Synthesises cAMP from ATP.

35

How is adenylate cyclase regulated by GPCRs

Gs alpha protein stimulates AC effector. (e.g. beta-adrenoceptor)
Gi alpha protein inhibits AC effector (e.g. alpha2-adrenoceptor; and CB1 cannabinoid receptor)

36

What is the structure of adenylate cyclase (AC)

Two transmembrane domains; C terminus is bound to the N terminus of the other

37

How many isoforms of Adenylate Cyclase (AC) have been identified

9

38

How does adenylate cyclase act as a catalyst

The two cytoplasmic C-domains in the second transmembrane domain interact.

39

Where is the AC1 receptor located

Brain retina, adrenal medulla

40

Where is the AC3 receptor located

Olfactory neurones, brain, retina, heart, lung

41

Where is the AC8 receptor located

Brain

42

How do the AC1, AC3 and AC8 receptors work

Activated by calcium/calmodulin
Inhibited by G[alphai] subunits (AC1 and AC3)
Inhibited by G[betagamma] subunits (AC1)

43

Where is the AC2 receptor located

Brain, olfactory tissue, lung

44

Where is the AC4 receptor located

Widespread

45

Where is the AC7 receptor located

Widespread

46

How do the AC2, AC4 and AC7 receptors work

Activated by Protein Kinase C (PKC)
Unaffected by calcium/calmodulin/G[alphai] (unlike AC1,3,8)
Stimulated by G[betagamma] subunits

47

Where are the AC5 and AC6 receptors located

Heart, Brain, Kidney

48

How do the AC5 and AC6 receptors work

Inhibited by free calcium; unaffected by Calcium/calmodulin
Inhibited by protein kinase A (PKA) phosphorylation
unaffected by G[betagamme] subunits

49

Where is the AC9 receptor located

Testis/widespread

50

How does the AC9 receptor work

Inhibited by:
Calcium/calcineurin
G[alphai] subunits
Protein Kinase C (PKC) phosphorylation
Unaffected by G[betagamma] subunits

51

What is the specific activity of Protein Kinase A

Acts as a serine/threonine kinase. Transfers terminal phosphate on ATP onto target. Involves esterification between phosphate group and serine/threonine.

52

What is the consensus sequence recognised by Protein Kinase A

NH2 ---- Arg-Arg-X-Ser/Thr-X

53

What does CREB stand for

cAMP response element binding protein

54

How does Protein Kinase A activation affect the heart? how?

Phosphorylates several calcium handling proteins.
Increases force of contractions

55

What is CREB

A transcription factor that regulates gene expression

56

How does protein kinase A affect CREB

Indirectly
Modifies other proteins that are able to promote/inhibit transcription

57

What genes are targeted by Protein Kinase A activity

Phosphoenolpyruvate carboxykinase (PEPCK) (rate limiting in glyconeogenesis)
AC8, c-fos, glutamate receptor, tyrosine hydroxylase (important in learning and memory)

58

What does PIP2 stand for

phosphatidylinositol-4,5-bisphosphate

59

How is PIP2 affected by GPCR receptor activated

GPCR q effector is phospholipase C.
Phospholipase C cleaves PIP2 (yielding DAG and IP3)

60

What does DAG stand for

Diacyl Glycerol

61

What does IP3 stand for

Inositol-1,4,5-triphosphate

62

How are DAG and IP3 formed

Through PIP2 cleavage by phospholipase C

63

What does DAG do

Activates protein kinase C which then moves from the cytosol to the plasma membrane. PKC then phosphorylates other proteins.

64

What does IP3 do

Stimulates calcium release from the ER by activating calcium-release channels in the ER membrane.

65

Give some examples of protein kinase C inhibitors

Non-selective protein kinase inhibitors (H7, staurosporine etc)
Selective inhibitors (calphostin C etc)

66

What modifications can happen to IP3

Phosphorylated (to IP4/5/6)
Sequentially dephosphorylated - eventually yields inositol (can be used to synthesise phosphatidylinositol PI)

67

What signalling role does IP4 have

Can stimulate Calcium entry (activation of plasma membrane calcium channels maybe?)

68

What does GAP stand for

GTPase Activating Proteins

69

What does a GAP do, how

Promotes GTP hydrolysis
Inserts an amino acid which helps to stabilise the transition state.

70

What does RGS stand for

Regulator of G-protein signalling

71

What do RGS proteins do

Negative regulators of G protein signalling. Stimulate GTP hydrolysis.
Are a type of GAP
Accelerate the rate of GTP hydrolysis

72

What does GEF stand for

Guanine Nucleotide Exchange Factors

73

What do GEFs do

Promote GDP/GTP exchange

74

What can act as a GEF

An activated GPCR
AGS (activator of G-protein signalling)

75

How many families of RGSs are there

2
Modulators and integrators

76

How does RGS4 work

GPCR modulator
Reversibly associates with membranes via N terminus amphipathic alpha-helix.
Targets specific GPCRs
Can bind to PIP3, which blocks the RGS. (stimulated by G[beta/gamma] subunits)(inhibited by calcium/calmodulin)

77

What is the molecular basis of calcium oscillations (RGS4/PIP3)

GPCR activates; intracellular Ca inc
RGS4 forms complex which shuts off Ca mobilisation (Ca dec)
PIP3 binds to RGS4; inhibits; allows Ca mobilisation (Ca inc)
Ca/Cam complex forms. Inhibits PIP3. RGS4 continues. (Ca dec).
Lowered Ca; CaM deactivated. PIP3 able to rebind to RGS4.
PROCESS CONTINUES

78

What does PDE stand for

phosphodiesterase

79

What are phosphodiesterases

Diverse family of enzymes.
Degrade phosphodiester bond in cyclic nucleotices (cAMP, cGMP etc).

80

Why are phosphodiesterases important

Can degrade cyclic nucleotides -
Regulators of signal transduction mediated by these secondary messenger molecules.

81

How many member of the PDE family are there

11 families (PDE1-11)
Over 50 isoforms known

82

What are the key structural features of the PDE family members

Conserved catalytic site (in the C-terminus)
They have regulatory sites/interactions at the N terminus

83

How are the members of the PDE family involved in signalling

Each can be inhibited or stimulated by PKA, PKG, cGMP (and one by calcium/calmodulin)
They each have varying affinity for degrading a specific type of cyclic nucleotide (cAMP, cGMP etc).

84

What is the molecular basis of Antiphasic calcium oscillations

cAMP increases = Calcium decrease (and vice versa)
Activation of store-operated/voltage-gated calcium channels. Simulates Ca/CaM dependent PDE1 activation. Stimulates cAMP hydrolysis for duration of calcium increase.
Anti-phasic because they do the opposite of each other. (PDE1 dependent)

85

What is the molecular basis of Phasic calcium oscillations

cAMP increase = calcium increase
Calcium increase stimulates adenylyl cyclase to increase cAMP production. Calcium decrease causes cAMP hydrolysis by PKA-dependent PDE4 activity.
Phasic because they do the same thing as each other. (PDE4 dependent)

86

How can IP3 signals be terminated

Dephosphorylation to inositol (minus 3 phosphates)

87

What is G protein uncoupling

Occurs when phosphorylated (by specific kinases and G-protein coupled receptor kinases (GRKs))
GRKs interact with agonist activated GPCRs. GRK phosphorylation allows beta-arrestins to bind.
Beta-arrestins uncouple GPCRs from G-proteins

88

What do beta-arrestins do to GPCRs

Uncouple G-proteins, preventing activation
Can target for internalisation in clathrin-coated vesicles.

89

What happens to a GPCR when bound in a clathrin-coated vesicle

Internalised.
Then either degraded (by lysosomes); or dephosphorylated and recycled back to the surface (GRK activity reversed).

90

How does GPCR desensitisation occur

Constant GPCR stimulation leads to phosphorylation by GRK. This binds beta-arrestin which targets the GPCR for internalisation in a clathrin-coated vesicle. From here can be degraded (downregulation) or recycled.

91

What is cholera toxin

Protein toxin that acts intracellularly to enzymatically disrupt GTPase activity of G[Salpha] and G[t].

92

What is the structure of cholera toxin

Made up of A1, A2 and B subunits

93

How does cholera toxin work

B subunits bind to cell surface.
A2 subunit is an 'adaptor piece' which enables the A1 subunit to enter the cell
A1 subunit binds to ARF6 (ADP-ribosylation factor 6). Disrupts GTPase activity.

94

What is pertussis toxin

Protein toxin - responsible for whooping cough

95

What is the structure of pertussis toxin

Made up of A subunits (S1)
and B subunits (S2-5)
S2/S4 and S3/S4 are dimers linked together by S5.

96

How does pertussis toxin work

Released in inactive form. Binds to cell receptor and internalised.
S1 subunit is pathogenic. Activated by many factors (including ATP and reducing agents).

97

What is the mechanism of cholera toxin

Catalyses the transfer of ADP-ribose from NAD+ to an arginine residue on the G[Salpha] GTPase active site.
This prevents GTP hydrolysis.
Therefore the stimulatory G-protein is permanently activated (because no GTPase to switch off)

98

What is the mechanism of pertussis toxin

Catalyses the transfer of ADP-ribose onto a cysteine residue of the inhibitor G[ialpha]. This makes the protein incapable of exchanging GDP for GTP.
Therefore the inhibitory pathway is blocked (and stimulatory continues)

99

What are the cellular effects of cholera toxin

G[Salpha] permanently activated
Massive increase of cAMP. PKA activated
Activates CFTR-mediated chloride efflux in gut epithelia
Rapid loss of fluid in gut ensues.

100

What are the cellular effects of pertussis toxin

G[ialpha] permanently inactivated.
cAMP levels increase. Causes:
Increased insulin secretion (and hypoglycemia)
Cough
Reduced neutrophil/macrophage recruitment (immune)

101

Why are GTP analogues used

Different analogues can cause different effects (such as perma activation or inhibition).
Allows to detect a GPCR, and identify the pathway

102

Give some examples of GTP analogues used

Gpp(NH)p - Guanosine-5'[betagamma-imido] triphosphate; leads to perma activation
GDP/AIF4- (Aluminium fluoride). Activates G proteins. Commonly used.