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BS3054 > 10th and 12th Oct - Receptor-G-Protein interactions > Flashcards

10th and 12th Oct - Receptor-G-Protein interactions Flashcards

1
Q

What are AGS proteins?

A

Activation of G-protein signalling proteins

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2
Q

What are RGS proteins?

A

Regulators of G-protein signalling proteins

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3
Q

What are the 4 different types of GTPases?

A

Heterotrimeric
Translational
Large
Small

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4
Q

What are the functions of the alpha subunit of a heterotrimeric G-protein?

A

Receptor recognition
Effector recognition
GTP/GDP binding and GTPase activity
RGS interaction

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5
Q

What are the Beta-gamma subunits useful for in a heterotrimeric g-protein?

A

Receptor recognition
GDP bound G alpha subunit recognition
Effector recognition

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6
Q

What function does the GPCR have in the GTP-GDP cycle of a heterotrimeric G-protein?

A

It acts as a GEF

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7
Q

What terminates the regulation of the effector by a hetertrimeric g protein?

A

Galphas GTPase activity

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8
Q

What are the different isoforms of G alpha?

A

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

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9
Q

What is the function of Gs?

A

Stimulates AC

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10
Q

What is the function of Gi?

A

Inhibits AC

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11
Q

What is the function of Gq/11?

A

Activates PLC

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12
Q

What is the function of G12/13?

A

Scaffold for regulators

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13
Q

What are the different isoforms of Gbeta?

A

Gbeta 1-5

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14
Q

What are the different isoforms of Ggamma?

A

Ggamma 1-13

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15
Q

How do Gbetagamma subunits move to their effector?

A

They are attached to the membrane via an isoprene structure, allowing them to diffuse laterally like a monorail

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16
Q

What is the stoichiometry of GPCR monomer to Gprotein?

A

1:1

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17
Q

What is the stoichiometry of GPCR dimer to Gprotein?

A

1:1

18
Q

Which region of the Gprotein is critical for receptor-Gprotein coupling specificity?

A

The c-terminal of G alpha

19
Q

How can you alter which g-protein binds to a GPCR?

A

Alter the last 5aa of the Gproteins alpha subunit C-terminus, to be the same as the endogenous g protein.

I.e. to get Gq to bind to a 5-HT receptor, alter the last 5aa of Gq to match the last 5aa of Gs.

20
Q

Why would you want to make a Gs/i GPCR couple to Gq instead?

A

Calcium levels are much easier to track than changes in cAMP

21
Q

What does Galpha t1/t2 target?

A

activates cGMP-PDE Retinal

22
Q

What does the beta gamma subunit of Gi/o act on?

A

Activates GIRKs

Inhibits P/Q N-type calcium channels

23
Q

Why are lipidations of the heterotrimeric G-proteins so important?

A

They allow the G-proteins to move in 2D rather than 3D, which is important due to the small size of the G-proteins, as moving in 3D would lead to massive diffusion times

24
Q

What are the main tools used to investigate GPCRs?

A

Aluminium flouride
Non-hydrolysable analogues of GTp and GDP
Bacterial toxins that ADP-ribosylate G-proteins
G-protein Antibodies

25
Q

How can Aluminium flouride be used to study GPCRs?

A

A general activator of G proteins, mimicking GTP

26
Q

How can non-hydrolyzable analogues of GTP and GDP be used to study GPCRs?

A

They can alter the lifetime of activity leading to persistent G alpha activation

27
Q

How can cholera toxin be used to study GPCRs?

A

Cholera covalently modifies Galpha s proteins preventing them from being able to hydrolyse GTP

28
Q

How can pertussis toxin be used to study GPCRs?

A

Pertussis covalently modifies Galpha i/o proteins preventing them from being able to undergo GTP for GDP exchange therfore impossible to switch off signaling

29
Q

How can G-protein antibodies be used to study GPCRs?

A

Antibodies raised to the Galpha subtype can be used to block R-G or G-effector interactions they can also be used to immunoprecipitate subunits to assess R-G coupling

30
Q

How do you identify which g protein subpoulations are activated by a GPCR?

A
  1. [35S] GTPgammaS binding step
    - to a membrane preparation containing the GPCR of interest add agonist in the presence of [35S] GTPgammaS. Receptor activation will stimulate [35S] GTPgammaS for GDP exchange on a subpopulation of Galpha proteins
  2. Immunoprecipitation step
    - After stopping the assay and solubilizing the membranes [35S] GTPgammaS complexes are recovered by immunoprecipitation using Galpha protein specific Ab and using a scintillation counter to detect the co-immunopreciupitated [35S] GTPgammaS radioactivity
31
Q

What kinetic factor primarily determines the lifetime of Galpha-GTP?

A

Koff

32
Q

What is the function of RGS proteins?

A

To accelerate the intrinsic GTPase activity of g alpha subunits causing a more rapid switching off of G-protein signalling

33
Q

Are RGS proteins specific to one subfamily of G proteins?

A

No they can work on one subfamily of G proteins or several

34
Q

What are RH domains?

A

RGS like homology domains which interact specifically with G alpha but do not appear to accelerate GTPase activity

35
Q

What are GDIs?

A

Guanine nucleotide dissociation inhibitors

36
Q

Outline an experiment that shows that RGS4 is present in the heart

A

GIRKs are primarily regulated by Gbeta gamma subunits released by Gi/o proteins
In the heart ACh –> M2 receptor activation to regulate GIRKs
In CHO cells GIRKS co-expressed with M2, yet the kinetics are much slower than heart cells. Why?

Co-expressed CHO cells with GIRKS and M2 and RGS4, showed much faster kinetics similar to heart cells.

37
Q

Which RGS protein is necessary for normal photoreceptor responses to light exposure?

A

RGS9-1

38
Q

How was it identified that RGS9-1 is necessary for normal photoreceptor responses to light exposure?

A

By using RGS9-1 KO mice which exhibited marked delays in recovery from photobleaching

39
Q

Do RGS proteins show different responses to GPCRs within a subfamily?

A

Yes, the GPCR itself plays a role in determining the specificity of the Galpha-RGS protein interaction.

40
Q

What does p115 rho-GEF do?

A

It is a multi-functional protein possessing an RH domain. It allows specific GPCRS to activate the monomeric GTPase Rho

41
Q

How does p115 rho-GEF cause motility/morphological chance in endothelial cells?

A

Thrombin activates PAR receptors –> G alpha –> beta gamma release –> p115 rho/gef activation –> RhoA activation –> motility/morphological changes

BS3054 (25 decks)

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