2 - An Adrenaline Rush through the Second Messenger cAMP Flashcards
What response does adrenaline mediate?
Fight or flight response
True or false: The stress system has many different responses
True: many different organs react in different ways
How can a hormone have different effects (3 ways) on different cell types?
- Different receptors for the hormone
- Different signal transduction pathways
- Different proteins for carrying out the response
Liver cells and smooth muscle cells have the same receptor for adrenaline, but have different responses. How is this possible?
Different intercellular proteins changes the response
Skeletal blood vessels and intestinal blood vessels have similar intercellular proteins, but respond different to adrenaline. How is this possible?
Different receptors for adrenaline
What is a high affinity / low affinity ligand?
Ligands that have strong binding / weak binding (respectively) to a particular receptor
What are the three basic logic circuits for signal transduction?
Additive (OR), more than additive (AND) (synergistic), and less than additive (NOT) (antagonistic)
What does an “additive” response correspond to (in boolean)?
OR
What does a “more than additive” response correspond to (in boolean)?
AND
What does a “less than additive” response correspond to (in boolean)?
NOT
What response does an OR boolean correspond to?
Additive
What response does an AND boolean correspond to?
More than additive
What response does a NOT boolean correspond to?
Less than additive
What are the responses of molecules A and B in an additive response?
A -> response
B -> response
A + B -> response
What are the responses of molecules A and B in a synergistic response?
A -> 0
B -> 0
A + B -> response
What are the responses of molecules A and B in an antagonistic response?
A -> response
B -> 0
A + B -> 0
What does the graph look like for an additional response?
Two individual curves add together to get the combined curve
What does the graph look like for a synergistic response?
The individual curves are very low, but the combined curve is very high
What does the graph look like for an antagonistic response
The combined curve is inbetween the two individual curves (one of them being very low)
In a ligand binding curve, what are the axes?
X: Concentration
Y: Binding %
What is the use of a radioligand?
Track binding on a receptor through radioactivity
What is Bmax?
The maximal number of binding sites
Where is Bmax on a saturation curve?
At 100% binding
What is Kd?
The affinity of ligand for the receptor
Where is Kd on a saturation curve?
At 50% binding
What are the two major types of receptors?
Direct receptors, and indirect receptors
What are some features of a direct receptor?
Fast signaling, and a highly correlated binding and response
What are some features of an indirect receptor?
Slow signaling, with binding and response not well correlated
For a direct receptor, what does the curve of % of maximal response look like?
Closely follows the binding curve
For an indirect receptor, what does the curve of % of maximal response look like?
Sharp increase in the beginning (fewer receptors to get a maximal response), does not closely follow binding curve
What is an example of a direct channel?
Acetylcholine ion channel (simple binding opens up ion channel)
How are the binding curves of a direct and indirect receptor different?
Nothing; they look the same
How are the % of maximal response curves of a direct and an indirect receptor different?
The direct receptor follows the binding curve, while the indirect receptor reaches maximum much sooner
Why is a direct receptor faster?
Linear relationship (one receptor leads to one intermediate protein)
Why is an indirect receptor slower?
Has an integrated response, where many intermediate proteins are integrated together for a response)
Why is an indirect receptor more efficient (takes fewer receptors to achieve a maximal response)?
The many intermediate proteins allow for more amplification, and thus a greater signal
Who is Earl Sutherland?
Scientist who studied cAMP and adrenaline response in the liver
Who studied cAMP and adrenaline response in the liver?
Earl Sutherland
What decade was Earl Sutherland’s research in?
1970s
How did Earl Sutherland prove that cAMP was a second messenger?
Knock out of cAMP, and adding adrenaline, produced no response
What was the conclusion of Earl Sutherland’s first experiments?
There must be something between adrenaline and cAMP to produce the fight/flight response
What does adenylyl cyclase do?
Converts ATP into cAMP
What enzyme converts ATP into cAMP?
Adenylyl cyclase
What structural changes are done by adenylyl cyclase to convert ATP into cAMP?
- Create a ring with the phosphate (connected twice to the ribose sugar)
- Release a pyrophosphate (P-P)
What does phosphodiesterase do?
Converts cAMP into AMP
What enzyme converts cAMP into AMP?
Phosphodiesterase
What structural changes are done by phosphodiesterase to convert cAMP into AMP?
Remove the ring by adding water
What enzyme does caffeine inhibit?
Phosphodiesterase
What process does caffeine inhibit in the body?
The breakdown of cAMP into AMP
How does caffeine affect the adrenaline response in the body?
Caffeine inhibits phosphodiesterase, keeping cAMP levels high, and thus prolonging the adrenaline response
What are the effects of caffeine in the body?
Increases heart rate, dilates blood vessels
How do blood vessels get affected by caffeine in the body, and why?
Blood vessels dilate, because it delivers more oxygen
What does adenosine do in the brain?
Binds to receptors to promote drowsiness
How does caffeine interact with adenosine receptors?
Caffeine blocks the receptors, and prevents the adenosine response
Why does a person remain alert after having caffeine?
Caffeine blocks adenosine receptors, thus preventing drowsiness
Why does adenosine build up during the day?
Byproduct of ATP consumption during the day (energy)
What are the effects of caffeine in the brain?
Prevent drowsiness, blocks dilating blood vessels, elevating mood
How do blood vessels get affected by caffeine in the brain, and why?
Blood vessels do not dilate, because dilating blood vessels occurs as a response to inhibitory sleep signals (sent by adenosine). Since caffeine blocks these responses, the blood vessels do not dilate
What molecule inhibits neurons through adenosine receptors?
Adenosine (inhibits neurons to promote drowsiness)
How does dopamine interact with dopamine receptors?
Binds to the receptors to improve mood
How does adenosine interact with dopamine receptors?
Binds to adenosine receptors associated with dopamine receptors, and inhibits dopamine binding, thus decreasing alertness
How does caffeine interact with dopamine receptors?
Binds to adenosine receptors associated with dopamine receptors, and prevents adenosine from binding. This allows dopamine to bind, thus elevating mood
How does caffeine affect dopamine levels in the brain?
Caffeine increases dopamine levels by slowing down rate of dopamine resorption
What does caffeine, amphetamines, cocaine, and heroin have in common?
All increase dopamine levels by slowing down rate of dopamine resorption (addictive properties)
How does caffeine elevate mood?
Prevents adenosine from blocking dopamine binding, and increases dopamine levels. Dopamine binding elevates mood
What is an agonist?
A molecule that binds to a receptor to produce a response
What is an antagonist?
A molecule that binds to a receptor to block access to agonists
For adenosine receptors, is adenosine an agonist or an antagonist?
Agonist
For adenosine receptors, is caffeine an agonist or an antagonist?
Antagonist
What was Earl Sutherland’s first model?
Adenylyl cyclase was the receptor (hormone binds to adenylyl cyclase)
What is now known to be false about Earl Sutherland’s first model?
Adenylyl cyclase is not the receptor, it just converts ATP into cAMP
What was Earl Sutherland’s second model?
Adenylyl cyclase is part of a receptor complex (adenylyl cyclase is associated with some receptor)
How were Earl Sutherland’s models tested (general steps)?
- Purify adenylyl cyclase and receptor
- Show they don’t work individually
- Reconstitute components together to produce a response
What does NEM (N-ethylmaleimide) do?
Blocks adenylyl cyclase (prevents ATP -> cAMP)
What compound inhibits adenylyl cyclase?
NEM
What does forskolin do?
Activates adenylyl cyclase (without hormone)
What compounds activates adenylyl cyclase (without a hormone)?
Forskolin
How were Earl Sutherland’s models tested (lab techniques)?
- Pharmacological manipulation of cAMP
2. Live cell reconstitution
What was the purpose of Earl Sutherland’s second experiments?
Show that the adrenaline response needs the adrenaline receptor as a separate entity from adenylyl cyclase
What protein(s) do turkey red blood cells (TRBCs) have (in the Sutherland experiments)?
Adrenaline receptors, and adenylyl cyclase
What protein(s) do adrenal cortical cells (ACCs) have (in the Sutherland experiment)?
Adenylyl cyclase
How come TRBCs and ACCs could not be fused together directly?
TRBCs have everything you need, so there would be no new information about the system
What does TRBC stand for?
Turkey red blood cells
What does ACC stand for?
Adrenal cortical cells
What are the steps Sutherland took to combine the cells and create the system?
- Kill adenylyl cyclase in TRBCs
- Confirm adrenaline still binds to adrenaline receptors
- Confirms ACCs still respond to forskolin
- Do reconstruction, and test whether it responds
What is the purpose of Sutherland killing adenylyl cyclase in TRBCs?
Prevent adrenaline response in TRBCs by removing adenylyl cyclase
How did Sutherland kill adenylyl cyclase in TRBCs?
Through NEM
What is the purpose of Sutherland confirming adrenaline still binds to adrenaline receptors?
Show that the adrenaline receptors can still bind to adrenaline
How did Sutherland confirm adrenaline still binds to adrenaline receptors?
Radioactive ligand binding curves
What is the purpose of Sutherland confirming that ACCs still respond to forskolin?
Show that adenylyl cyclase is still active
How did Sutherland confirm that adenylyl cyclase was still working in ACCs?
Through forskolin
What is a heterokayron?
Fused two cells
What is the result of the Sutherland cell reconstitution?
The system was responsive to adrenaline, thus providing evidence for his second model (associated receptor and adenylyl cyclase)
What are the limitations of the Sutherland studies?
Could not describe the mechanics of how the adrenal receptor worked
What did Lefkowitz and Kobilka study?
How the adrenal receptor worked
Who studied how the adrenal receptor worked?
Leftkowitz and Kobilka
What decade did Leftkowitz and Kobilka study the adrenal receptor?
1980s
What experiments did Leftkowitz and Kobilka perform?
Overexpression of adrenal receptors
When adrenaline was added in Leftkowitz and Kobilka experiments, what was the general pattern between no AR, low AR, medium AR, and high AR?
There was a basal level of expression at no AR, and high / equal levels of expression at the other concentrations
When adrenaline was added in Leftkowitz and Kobilka experiments, what conclusion could be made about the adrenaline receptor?
Indirect receptor (large response at a low amount of receptor)
When adrenaline was not added in Leftkowitz and Kobilka experiments, what was the general pattern between no AR, low AR, medium AR, and high AR?
Increasing response from no -> low -> medium -> high
When adrenaline was not added in Leftkowitz and Kobilka experiments, what conclusion could be made about the adrenaline receptor?
Response level was also driven by receptor density
What is the reason that response level was driven by receptor density?
Receptors dimerize to produce a response
How does adrenaline interact with receptor dimerization?
Adrenaline ligand stabilizes the dimerization, and thus produces the response
What is required to activate adrenal receptors (besides adrenaline)?
Dimerization
What is required to stabilize adrenal receptors?
Adrenaline (ligand)
What happens in strong binding activity?
An extracellular and intercellular molecule both bind to the receptor to get a large response
How many hydrophobic regions are found in the adrenaline receptor?
7
What is the significance of the adrenaline receptor having 7 hydrophobic domains?
Must pass through the membrane 7 times (stitched)
Where are transmembrane proteins made?
ER
What environment does the ER lumen resemble?
Extracellular environment
What environment does the cytosol resemble?
Intracellular environment
What does a hydrophobic start-transfer sequence do?
Causes the protein to move to a translocation channel
What does a translocation channel do?
Allows the protein to be made in the ER lumen (transmembrane proteins)
What does a hydrophobic stop-transfer sequence do?
Causes the protein to move from the translocation channel to the ER membrane (transmembrane)
What does a signal peptidase do?
Cleave the hydrophobic start-transfer sequence if it is found at the beginning of the protein
True or false: A signal peptidase always cleaves the hydrophobic start-transfer sequence
False: If the hydrophobic start-transfer sequence is found in the middle of the protein, it is not cleaved
What does SRP stand for?
Signal recognition particle
What does the SRP do?
Recognize the hydrophobic start-transfer sequence, and directs the protein to the translocation channel on the ER
How is a water soluble protein made in the ER (in terms of the hydrophobic sequences)
The sequences need to be at the beginning and the end; otherwise, there would be transmembrane regions
True or false: transmembrane proteins are made with a defined orientation
True: there is a distinct extracellular and intercellular side, and thus a distinct direction
How are multipass transmembrane proteins made (in terms of the hydrophobic sequences)?
Multiple pairs of these sequences are required, one for each pass
What side of the membrane will the protein sequence between a hydrophobic start sequence and a hydrophobic stop sequence (or the ends of the protein) be in?
Extracellular side
What side of the membrane will the protein sequence between a hydrophobic stop sequence and a hydrophobic start sequence (or the ends of the protein) be in?
Intercellular side
How are signaling events reversible (in terms of energy and delta G)?
There is a low delta G because they are small changes (conformational changes, etc.)
How come there is a basal level of cAMP expression?
Lots of processes use cAMP, which depend on the context
How come adrenal receptors don’t dimerize under physiological conditions without adrenaline?
There is not enough density to drive dimerization
How does chronic caffeine affect adrenal receptors?
Alters the density of adrenal receptors (and other mood receptors), and makes them higher affinity
What determines whether caffeine or adensoine will bind first to the adenosine receptors?
Concentration
Which has the bigger physiological response: caffeine inhibiting phosophodiesterase, or caffeine blocking adenosine receptors?
Caffeine blocking adenosine receptors (20 fold difference)
Why is AMPA (an ion gated channel) either direct or indirect?
Direct, because a binding of the ligand leads to an immediate change in the confirmation to allow calcium to flow into the cell
What is divergent signaling?
Different ligands change the conformation of the receptor differently, and thus goes down different pathways
True or false: when creating a synthetic drug, you must decide whether to have a direct or indirect response
False: the native signaling pathway is already there, and thus it is predetermined whether your pathway is direct or indirect
