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

Question 1

What are AGS proteins?

Answer 1

Activation of G-protein signalling proteins

Question 2

What are RGS proteins?

Answer 2

Regulators of G-protein signalling proteins

Question 3

What are the 4 different types of GTPases?

Answer 3

Heterotrimeric
Translational
Large
Small

Question 4

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

Answer 4

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

Question 5

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

Answer 5

Receptor recognition
GDP bound G alpha subunit recognition
Effector recognition

Question 6

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

Answer 6

It acts as a GEF

Question 7

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

Answer 7

Galphas GTPase activity

Question 8

What are the different isoforms of G alpha?

Answer 8

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

Question 9

What is the function of Gs?

Answer 9

Stimulates AC

Question 10

What is the function of Gi?

Answer 10

Inhibits AC

Question 11

What is the function of Gq/11?

Answer 11

Activates PLC

Question 12

What is the function of G12/13?

Answer 12

Scaffold for regulators

Question 13

What are the different isoforms of Gbeta?

Answer 13

Gbeta 1-5

Question 14

What are the different isoforms of Ggamma?

Answer 14

Ggamma 1-13

Question 15

How do Gbetagamma subunits move to their effector?

Answer 15

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

Question 16

What is the stoichiometry of GPCR monomer to Gprotein?

Answer 16

1:1

Question 17

What is the stoichiometry of GPCR dimer to Gprotein?

Answer 17

1:1

Question 18

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

Answer 18

The c-terminal of G alpha

Question 19

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

Answer 19

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.

Question 20

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

Answer 20

Calcium levels are much easier to track than changes in cAMP

Question 21

What does Galpha t1/t2 target?

Answer 21

activates cGMP-PDE Retinal

Question 22

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

Answer 22

Activates GIRKs

Inhibits P/Q N-type calcium channels

Question 23

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

Answer 23

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

Question 24

What are the main tools used to investigate GPCRs?

Answer 24

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

Question 25

How can Aluminium flouride be used to study GPCRs?

Answer 25

A general activator of G proteins, mimicking GTP

Question 26

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

Answer 26

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

Question 27

How can cholera toxin be used to study GPCRs?

Answer 27

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

Question 28

How can pertussis toxin be used to study GPCRs?

Answer 28

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

Question 29

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

Answer 29

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

Question 30

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

Answer 30

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

Question 31

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

Answer 31

Koff

Question 32

What is the function of RGS proteins?

Answer 32

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

Question 33

Are RGS proteins specific to one subfamily of G proteins?

Answer 33

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

Question 34

What are RH domains?

Answer 34

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

Question 35

What are GDIs?

Answer 35

Guanine nucleotide dissociation inhibitors

Question 36

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

Answer 36

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.

Question 37

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

Answer 37

RGS9-1

Question 38

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

Answer 38

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

Question 39

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

Answer 39

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

Question 40

What does p115 rho-GEF do?

Answer 40

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

Question 41

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

Answer 41

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