3 - G-Proteins and cAMP Regulation Flashcards
Who was Martin Rodbell?
A scientist who worked on the missing link between adrenal receptors and AC
Which scientist believed in a transducer?
Rodbell
What is a transducer and what does it do?
A transducer converts one type of signal (binding of adrenaline to receptor) into another type of signal (stimulation of adenylyl cyclase)
What techniques did Rodbell employ?
In vitro reconstitution of purified membranes
What components were part of Rodbell’s purified membranes?
The receptor, adenylyl cyclase, and the unknown transducer
What was the pathway that Rodbell was testing?
Purified membrane + hormone + ATP -> cAMP
What was the result when Rodbell added purchased ATP?
Some ATP produced a response, while some didn’t
Why did purchased ATP have different responses in Rodbell’s experiments?
Some ATP samples were contaminated with GTP, which activated the transducer
What was the result when Rodbell added pure ATP?
There was no response
Why did pure ATP have no effect in Rodbell’s experiments?
The transducer needed GTP to function, which wasn’t present
What was the result when Rodbell added GTP?
There was a response
What did Rodbell observe about GTP levels in his experiment, and what does this suggest?
He noticed that GTP levels decreased and GDP levels increased. This suggests that a phosphorylation event was happening with the transducer
Why was there a response when Rodbell added GTP?
The transducer required GTP to function
What was the result when Rodbell added non-hydrolyzable GTP?
There was a prolonged response
Why did non-hydrolyzable GTP prolong the response in Rodbell’s experiments?
The GTP could not be dephosphorylated to GDP, so AC could not be turned off
What was the conclusion of Rodbell’s experiments?
The transducer required GTP to function, and it was the link between the receptor and AC
What was the signal transduction model after Rodbell’s experiments?
A hormone binds to a receptor, which activated a GTP transducer, which then activates AC to make cAMP
Who was Alfred Gilman, and what did he do?
Gilman did not believe in Rodbell’s transducer, and sought to disprove it
What experiments did Gilman do?
Used S49 cyc- mutants to investigate transducer
What is an S49 cell?
A cancer cell that continuously divides
How can S49 cells stop dividing?
By adding cAMP
If S49 cells are treated with adrenaline, what is the response and why?
They will stop dividing, since the adrenaline response produces cAMP, and cAMP causes S49 cells to stop dividing
What are “cyc-“ mutants?
S49 cells that (suppossedly) lost adenylyl cyclase
If there was a mutation in AC in S49 cells, what would be the expected outcome if they were treated with adrenaline?
They would keep dividing, since no cAMP could be produced by AC
How can it be proven that the mutation in S49 cyc- cells is not in the receptor?
Confirm with radioactive ligand binding
What were the 3 selection criteria of the Gilman experiments?
- Growth in presence of adrenaline
- Retain receptors that bind to adrenaline
- Do not respond to forskolin
What were the results of the Gilman experiments?
Obtained cells that bound to adrenaline, but produced no adrenaline response, but still responded to forskolin
What was the puzzling consequence of the results of the Gilman experiments?
The cells had functional receptors and functional AC, but still couldn’t make cAMP
What was the conclusion of the Gilman experiments?
There must have been a mutation in another part of the pathway (transducer)
What was the assumed condition of the Gilman experiments?
The cyc- cells lacked AC, and adding AC would lead to cAMP in the presence of adrenaline
What was the assumed control condition of the Gilman experiments?
The cyc- cells lacked AC, and adding adrenaline would not lead to cAMP
What was the actual control condition of the Gilman experiments?
The cyc- cells lacked an unsuspected G protein transducer, which when added back would allow for the adrenaline response. The receptor and AC were perfectly fine
What protein did the cyc- cells actually not have?
G proteins
How were the transducer proteins verified and purified?
Proteins from the membrane were added back into the system to see if they would change the response
How many proteins did the final, purified sample had (to find the transducer)?
3
What does cholera toxin do?
Covalently links NAD to a substrate, and causes an increase in cAMP (somehow activates AC)
How was cholera toxin used to verify the transducer?
Cholera toxin was used to bind radioactive NAD to the transducer proteins that interacted with AC
What were the bands for the transducer?
The alpha subunits of G proteins
In wild type cells with cholera toxin and radioactive NAD, what would be the expected blot and why?
Two bands corresponding to the G protein transducer, since cholera would bind NAD to these proteins
In wild type cells without cholera toxin and with radioactive NAD, what would be the expected blot and why?
No bands, because cholera cannot bind NAD to the proteins
In cyc- cells without cholera toxin and with radioactive NAD, what would be the expected blot and why?
No bands, because cholera cannot bind NAD to the proteins
In cyc- cells with cholera toxin and radioactive NAD, what would be the expected blot and why?
No bands, because cyc- cells were missing the G protein transducer that cholera would bind NAD to
What was the purpose of the blots?
Validate G proteins as transducer of adrenaline response
What is a G-protein coupled receptor (GPCR)?
Plasma membrane receptor that works with the help of a G-protein
What is the basic function of a G-protein?
Act as an on-off switch
When is a G-protein active?
When GTP is bound
When is a G-protein inactive?
When GDP is bound
If GTP is bound to a G-protein, what state is it in?
Active
If GDP is bound to a G-protein, what state is it in?
Inactive
When a signal is inputted, how does a G-protein change?
It loses GDP and binds to GTP to become activated
When is the signal outputted though a G-protein?
When it is activated
How does a G-protein stop being activated?
Hydrolysis of GTP to GDP
How does a G-protein go from activated to inactivated?
Hydrolysis of GTP to GDP
How does a G-protein go from inactivated to activated?
Exchange of GDP for GTP
What is meant by “G-protein has GTPase activity”?
The G-protein itself can hydrolyze GTP into GDP
What happens to a GPCR when a ligand binds to it?
It becomes activated
What happens to a G-protein when a GPCR is activated?
It stimulated GDP/GTP exchange of the alpha subunit of the G-protein
What subunit is activated by a GPCR?
Alpha subunit
What happens to the G-protein once it is activated?
It dissociates into an alpha subunit and a beta/gamma subunit
True or false: Once dissociated, the alpha subunit of a G-protein is the only thing that can cause an effect
False: depending on the system, the beta/gamma complex can also cause an effect
True or false: The alpha and beta/gamma subunits of a G-protein could interact in the same pathway
True: both have the ability to cause an effect, depending on the system
What are the three broad subclasses of trimeric GPCR linked effector proteins?
Adenylyl cyclase, phospholipase C, and ion channels
What is meant by a “timer system”?
aG is a GTPase, so it can cleave GTP by itself to inactivate itself
How does the alpha subunit reunite with the beta/gamma complex?
Inactivate aG has a high affinity for beta/gammaG