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Pharmacology > Receptor-G Protein Interactions > Flashcards

Receptor-G Protein Interactions Flashcards

1
Q

What type of receptor represents a major therapeutic target, giving rise to the largest single fraction of the prescription drug market?

A

GPCRs

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2
Q

What are GEFs?

A

Guanine nucleotide exchange factors

…proteins responsible for the activation of small GTPases

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3
Q

How might GPCRs be known as GEFs for Ga-GDP/Gby complexes?

A

Upon agonist activation of GPCRs, they undergo a conformational change which subsequently catalyses the exchange of GDP for GTP on the Ga subunit.

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4
Q

What is the rate-limiting step of the Ga guanine nucleotide cycle?

A

GDP release

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5
Q

How does the exchange of GTP for GDP cause dissociation of the Gby dimer from Ga?

A

Binding of GTP induces a conformational change in three flexible ‘switch regions’ of the Ga subunit, leading to dissociation of the Gby dimer, and adoption of the conformation capable of interacting with effectors

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6
Q

Based on their sequence homology and differential regulation of effectors, G-proteins are grouped into which four classes?

A

G-alpha-s
G-alpha-i/o
G-alpha-q
G-alpha-12/13

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7
Q

How does deactivation of G-protein signalling occur?

A

The intrinsic hydrolysis of GTP to GDP by the G-alpha subunit, which occurs at a rate that varies among the G-protein subfamilies

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8
Q

Deactivation of G-protein signalling is brought about by the intrinsic hydrolysis of GTP to GDP by the G-alpha subunit. How can these hydrolysis rates be enhanced?

A

Hydrolysis rates can be dramatically enhanced by members of a superfamily of ‘Regulators of G-protein Signalling’ (RGS) proteins that serve as GTP-ase Accelerating Proteins (GAPs)

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9
Q

What are RGS proteins, and what is their function in the guanine nucleotide cycle?

A

‘Regulators of G-protein Signalling’ proteins

…they act as GAPs to dramatically enhance hydrolysis of GTP to GDP, and thus deactivation of G-protein signalling

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10
Q

What are GAPs? Which proteins act as GAPs for heterotrimeric G proteins?

A

GTPase Accelerating Proteins - they increase the rate of GTP hydrolysis

…RGS proteins (Regulators of G-protein Signalling) and phospholipase C-beta (PLC-beta) isoforms

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11
Q

What are GTPases?

A

A superfamily of HYDROLASE enzymes that can bind and hydrolyse guanosine triphosphate (GTP)

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12
Q

What are the 4 main subfamilies of the superfamily of GTPases?

A

1) small GTPases (e.g. Ras, Rac, Rho)
2) large GTPases (e.g. Dynamin)
3) translational GTPases (e.g. EF-Tu)
4) heterotrimeric GTPases (G proteins)

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13
Q

Give 3 examples of ‘small GTPases’

A

Ras
Rac
Rho

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14
Q

Give an example of a ‘large GTPase’

A

Dynamin

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15
Q

Give an example of a ‘translational GTPase’

A

EF-Tu

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16
Q

Give an example of a ‘heterotrimeric GTPase’

A

G protein

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17
Q

What are heterotrimeric G-proteins composed of?

A

Three distinct subunits:

1) alpha (40kDa)
2) beta (35kDa)
3) gamma (15 kDa)

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18
Q

In heterotrimeric G-proteins, what is the alpha subunit responsible for?

A

1) receptor recognition
2) effector recognition
3) GTP/GDP binding / GTPase ACTIVITY
4) interaction with regulatory proteins (e.g. RGS proteins)

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19
Q

in heterotrimeric G proteins, what is the beta-gamma subunit responsible for?

A

1) receptor recognition
2) GDP-bound G-alpha-subunit recognition
3) effector recognition

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20
Q

In which form is a G-protein heterotrimeric and inactive?

A

In its GDP-bound form

21
Q

GPCR acts as WHAT to promote GTP-for-GDP exchange?

A

a GEF (guanosine nucleotide exchange factor)

22
Q

What are GEFs responsible for?

A

The activation of GTPases

23
Q

Both Ga and Gby subunits can regulate _____?

A

Effectors, e.g. ion channels

24
Q

Which GEFs are responsible for the activation of heterotrimeric G-protein GTPases?

A

G protein-coupled receptors

25
Q

What is GDP binding stabilised by?

A

Proteins known as GDP dissociation inhibitors (GDIs)

26
Q

How do GAPs act to enhance GTP hydrolysis?

A

They act ALLOSTERICALLY by stabilising the catalytic active site on G-alpha in a conformation that is favourable for hydrolysis.

27
Q

What terminates G-protein effector regulation?

A

G-alpha GTPase activity

28
Q

What happens to G-alpha once its GTP has been replaced with a GDP?

A

It re-associates with free G-beta-gamma subunits

29
Q

What type of structure is the beta-gamma interaction domain in heterotrimeric G-proteins?

A

Coiled-coil

30
Q

What modification allows the alpha and gamma subunits of a heterotrimeric G-protein to be tethered to the plasma membrane?

A

They are LIPIDATED

31
Q

What modification allows the alpha and gamma subunits of a heterotrimeric G-protein to be tethered to the plasma membrane?

A

They are LIPIDATED

32
Q

What is the problem with the monocycle:

                 GEF (GPCR)
             Kon --->  G-GDP                        G*GTP    ----> EFFECTOR
A

When the activating stimulus is removed and fractional activity drops, the return to the resting steady state is predicted to be slow because then both Kon and Koff are low.

An example of why this is a problem is represented by the effects of turning a light switch on and off. When the light comes on, we perceive it v. quickly because rhodopsin rapidly activates transducin (Gt), the visual G protein. However, the intrinsic rate of hydrolysis of Gt-bound GTP is slow. If this rate were the only determinant of the deactivation rate, it would take us a while to perceive darkness when the light is switched off.
This highlights the importance of a GAP to increase Koff and thus give physiologically fast deactivation.

33
Q

What are the different Ga, Gb and Gy sub-families?

A

Ga: Gs, Gi/o, Gq/11, G12/13

Gb: Gb1-5

Gy: Gy1-13

34
Q

G proteins are extremely diverse. How many of each type of subunit have been found?

A

17-20 alpha subunits
5 beta subunits
12 gamma subunits

35
Q

Theoretically, approximately how many different Galpha-beta-gamma combinations are there?

A

~1000

36
Q

There are four main G-alpha subunit subfamilies. What are they, and what are their main effectors?

A

G-alpha-s: adenylyl cyclase activation
G-alpha-i/o: adenylyl cyclase inhibition, ion channel modulation
G-alpha-q/11: PLC activation
G-alpha-12/13: scaffolds for regulators of monomeric GTPases

37
Q

Which part of the G-alpha subunit is critical for receptor-G protein coupling specificity?

A

Its extreme C-terminal end

38
Q

How can the GPCR specificity of a G protein be changed?

A

Switching the C-terminal 5 amino acids of the G-alpha subunit for the last 5 C-terminal amino acids of a different G protein

39
Q

What are AGS proteins? What do they do?

A

Activators of GPCR Signalling proteins - change the activation state of G-proteins

40
Q

Why is the G protein beta-gamma dimer so stable?

A

Coiled coil interaction domain

41
Q

What is the function of G-alpha(12/13) proteins?

A

Scaffolds for regulators of monomeric GTPases

42
Q

Which is the biggest family of G-alpha protein subtypes?

A

G-alpha(i/o)

43
Q

Which G-alpha protein subfamily are G-alpha(15/16) in?

A

G-alpha(q/11)

44
Q

Name 4 tools that can be used to investigate Receptor-G protein interactions

A
  1. aluminium fluoride
  2. non-hydrolysable analogues of GTP and GDP
  3. bacterial toxins that ADP-ribosylate G proteins
  4. G protein-specific antibodies
45
Q

Why is aluminium fluoride a good tool to investigate receptor-g protein interactions?

A

GDP.AIF4- is a general ACTIVATOR of G proteins: it mimics GTP occupancy of the G-alpha subunit

46
Q

Give an example of a non-hydrolysable analogue of GTP. How can it be used as a tool to investigate Receptor-G protein interactions?

A

GTP-gamma-S. It binds to G-alpha subunits but is resistant to hydrolysis - this causes persistent G-alpha activation

47
Q

How do cholera and pertussis toxins affect G proteins?

A

Cholera toxin covalently modifies G-alpha(s) proteins TURNING OFF the ability to hydrolyse GTP
Pertussis toxin covalently modifies G-alpha(i/o) TURNING OFF the ability to undergo GTP-for-GDP exchange

48
Q

How can G protein-specific antibodies be used as tools to investigate Receptor-G protein interactions?

A

Can be used to block R-G or G-E interactions

Can be used to IMMUNOPRECIPITATE specific G-alpha subunits to assess R-G coupling

49
Q

What are the two experimental steps in defining a G protein sub-population activated by a particular GPCR?

A
  1. [35S]-GTPyS binding step (add agonist to prep containing GPCR in the presence of [35S]-GTPyS
  2. Stop assay, solubilise membrane, recover G-alpha-[35S]-GTPyS complexed by IP and use a scintillation counter to detect radioactivity

Pharmacology (13 decks)

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