10th and 12th Oct - Receptor-G-Protein interactions Flashcards
What are AGS proteins?
Activation of G-protein signalling proteins
What are RGS proteins?
Regulators of G-protein signalling proteins
What are the 4 different types of GTPases?
Heterotrimeric
Translational
Large
Small
What are the functions of the alpha subunit of a heterotrimeric G-protein?
Receptor recognition
Effector recognition
GTP/GDP binding and GTPase activity
RGS interaction
What are the Beta-gamma subunits useful for in a heterotrimeric g-protein?
Receptor recognition
GDP bound G alpha subunit recognition
Effector recognition
What function does the GPCR have in the GTP-GDP cycle of a heterotrimeric G-protein?
It acts as a GEF
What terminates the regulation of the effector by a hetertrimeric g protein?
Galphas GTPase activity
What are the different isoforms of G alpha?
Gs, Gi/o, Gq/11, G12/13
What is the function of Gs?
Stimulates AC
What is the function of Gi?
Inhibits AC
What is the function of Gq/11?
Activates PLC
What is the function of G12/13?
Scaffold for regulators
What are the different isoforms of Gbeta?
Gbeta 1-5
What are the different isoforms of Ggamma?
Ggamma 1-13
How do Gbetagamma subunits move to their effector?
They are attached to the membrane via an isoprene structure, allowing them to diffuse laterally like a monorail
What is the stoichiometry of GPCR monomer to Gprotein?
1:1
What is the stoichiometry of GPCR dimer to Gprotein?
1:1
Which region of the Gprotein is critical for receptor-Gprotein coupling specificity?
The c-terminal of G alpha
How can you alter which g-protein binds to a GPCR?
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.
Why would you want to make a Gs/i GPCR couple to Gq instead?
Calcium levels are much easier to track than changes in cAMP
What does Galpha t1/t2 target?
activates cGMP-PDE Retinal
What does the beta gamma subunit of Gi/o act on?
Activates GIRKs
Inhibits P/Q N-type calcium channels
Why are lipidations of the heterotrimeric G-proteins so important?
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
What are the main tools used to investigate GPCRs?
Aluminium flouride
Non-hydrolysable analogues of GTp and GDP
Bacterial toxins that ADP-ribosylate G-proteins
G-protein Antibodies
How can Aluminium flouride be used to study GPCRs?
A general activator of G proteins, mimicking GTP
How can non-hydrolyzable analogues of GTP and GDP be used to study GPCRs?
They can alter the lifetime of activity leading to persistent G alpha activation
How can cholera toxin be used to study GPCRs?
Cholera covalently modifies Galpha s proteins preventing them from being able to hydrolyse GTP
How can pertussis toxin be used to study GPCRs?
Pertussis covalently modifies Galpha i/o proteins preventing them from being able to undergo GTP for GDP exchange therfore impossible to switch off signaling
How can G-protein antibodies be used to study GPCRs?
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
How do you identify which g protein subpoulations are activated by a GPCR?
- [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 - 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
What kinetic factor primarily determines the lifetime of Galpha-GTP?
Koff
What is the function of RGS proteins?
To accelerate the intrinsic GTPase activity of g alpha subunits causing a more rapid switching off of G-protein signalling
Are RGS proteins specific to one subfamily of G proteins?
No they can work on one subfamily of G proteins or several
What are RH domains?
RGS like homology domains which interact specifically with G alpha but do not appear to accelerate GTPase activity
What are GDIs?
Guanine nucleotide dissociation inhibitors
Outline an experiment that shows that RGS4 is present in the heart
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.
Which RGS protein is necessary for normal photoreceptor responses to light exposure?
RGS9-1
How was it identified that RGS9-1 is necessary for normal photoreceptor responses to light exposure?
By using RGS9-1 KO mice which exhibited marked delays in recovery from photobleaching
Do RGS proteins show different responses to GPCRs within a subfamily?
Yes, the GPCR itself plays a role in determining the specificity of the Galpha-RGS protein interaction.
What does p115 rho-GEF do?
It is a multi-functional protein possessing an RH domain. It allows specific GPCRS to activate the monomeric GTPase Rho
How does p115 rho-GEF cause motility/morphological chance in endothelial cells?
Thrombin activates PAR receptors –> G alpha –> beta gamma release –> p115 rho/gef activation –> RhoA activation –> motility/morphological changes
