3 - G-Proteins and cAMP Regulation Flashcards
1
Q
Who was Martin Rodbell?
A
A scientist who worked on the missing link between adrenal receptors and AC
2
Q
Which scientist believed in a transducer?
A
Rodbell
3
Q
What is a transducer and what does it do?
A
A transducer converts one type of signal (binding of adrenaline to receptor) into another type of signal (stimulation of adenylyl cyclase)
4
Q
What techniques did Rodbell employ?
A
In vitro reconstitution of purified membranes
5
Q
What components were part of Rodbell’s purified membranes?
A
The receptor, adenylyl cyclase, and the unknown transducer
6
Q
What was the pathway that Rodbell was testing?
A
Purified membrane + hormone + ATP -> cAMP
7
Q
What was the result when Rodbell added purchased ATP?
A
Some ATP produced a response, while some didn’t
8
Q
Why did purchased ATP have different responses in Rodbell’s experiments?
A
Some ATP samples were contaminated with GTP, which activated the transducer
9
Q
What was the result when Rodbell added pure ATP?
A
There was no response
10
Q
Why did pure ATP have no effect in Rodbell’s experiments?
A
The transducer needed GTP to function, which wasn’t present
11
Q
What was the result when Rodbell added GTP?
A
There was a response
12
Q
What did Rodbell observe about GTP levels in his experiment, and what does this suggest?
A
He noticed that GTP levels decreased and GDP levels increased. This suggests that a phosphorylation event was happening with the transducer
13
Q
Why was there a response when Rodbell added GTP?
A
The transducer required GTP to function
14
Q
What was the result when Rodbell added non-hydrolyzable GTP?
A
There was a prolonged response
15
Q
Why did non-hydrolyzable GTP prolong the response in Rodbell’s experiments?
A
The GTP could not be dephosphorylated to GDP, so AC could not be turned off
16
Q
What was the conclusion of Rodbell’s experiments?
A
The transducer required GTP to function, and it was the link between the receptor and AC
17
Q
What was the signal transduction model after Rodbell’s experiments?
A
A hormone binds to a receptor, which activated a GTP transducer, which then activates AC to make cAMP
18
Q
Who was Alfred Gilman, and what did he do?
A
Gilman did not believe in Rodbell’s transducer, and sought to disprove it
19
Q
What experiments did Gilman do?
A
Used S49 cyc- mutants to investigate transducer
20
Q
What is an S49 cell?
A
A cancer cell that continuously divides
21
Q
How can S49 cells stop dividing?
A
By adding cAMP
22
Q
If S49 cells are treated with adrenaline, what is the response and why?
A
They will stop dividing, since the adrenaline response produces cAMP, and cAMP causes S49 cells to stop dividing
23
Q
What are “cyc-“ mutants?
A
S49 cells that (suppossedly) lost adenylyl cyclase
24
Q
If there was a mutation in AC in S49 cells, what would be the expected outcome if they were treated with adrenaline?
A
They would keep dividing, since no cAMP could be produced by AC
25
How can it be proven that the mutation in S49 cyc- cells is not in the receptor?
Confirm with radioactive ligand binding
26
What were the 3 selection criteria of the Gilman experiments?
1. Growth in presence of adrenaline
2. Retain receptors that bind to adrenaline
3. Do not respond to forskolin
27
What were the results of the Gilman experiments?
Obtained cells that bound to adrenaline, but produced no adrenaline response, but still responded to forskolin
28
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
29
What was the conclusion of the Gilman experiments?
There must have been a mutation in another part of the pathway (transducer)
30
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
31
What was the assumed control condition of the Gilman experiments?
The cyc- cells lacked AC, and adding adrenaline would not lead to cAMP
32
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
33
What protein did the cyc- cells actually not have?
G proteins
34
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
35
How many proteins did the final, purified sample had (to find the transducer)?
3
36
What does cholera toxin do?
Covalently links NAD to a substrate, and causes an increase in cAMP (somehow activates AC)
37
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
38
What were the bands for the transducer?
The alpha subunits of G proteins
39
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
40
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
41
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
42
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
43
What was the purpose of the blots?
Validate G proteins as transducer of adrenaline response
44
What is a G-protein coupled receptor (GPCR)?
Plasma membrane receptor that works with the help of a G-protein
45
What is the basic function of a G-protein?
Act as an on-off switch
46
When is a G-protein active?
When GTP is bound
47
When is a G-protein inactive?
When GDP is bound
48
If GTP is bound to a G-protein, what state is it in?
Active
49
If GDP is bound to a G-protein, what state is it in?
Inactive
50
When a signal is inputted, how does a G-protein change?
It loses GDP and binds to GTP to become activated
51
When is the signal outputted though a G-protein?
When it is activated
52
How does a G-protein stop being activated?
Hydrolysis of GTP to GDP
53
How does a G-protein go from activated to inactivated?
Hydrolysis of GTP to GDP
54
How does a G-protein go from inactivated to activated?
Exchange of GDP for GTP
55
What is meant by "G-protein has GTPase activity"?
The G-protein itself can hydrolyze GTP into GDP
56
What happens to a GPCR when a ligand binds to it?
It becomes activated
57
What happens to a G-protein when a GPCR is activated?
It stimulated GDP/GTP exchange of the alpha subunit of the G-protein
58
What subunit is activated by a GPCR?
Alpha subunit
59
What happens to the G-protein once it is activated?
It dissociates into an alpha subunit and a beta/gamma subunit
60
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
61
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
62
What are the three broad subclasses of trimeric GPCR linked effector proteins?
Adenylyl cyclase, phospholipase C, and ion channels
63
What is meant by a "timer system"?
aG is a GTPase, so it can cleave GTP by itself to inactivate itself
64
How does the alpha subunit reunite with the beta/gamma complex?
Inactivate aG has a high affinity for beta/gammaG
65
What is the typical pathway for a GPCR?
Effector proteins -> 2nd messengers -> target proteins
66
What does Gs mean?
Stimulatory G-protein
67
What does Gs do?
Stimulates an effector protein
68
What are the 7 steps in adrenaline signaling (full model)?
1. Adrenaline binds to receptor
2. GDP is replaced by GTP in aGs, activating it
3. aGs moves to AC, and activates it
4. AC converts ATP into cAMP
5. cAMP activates PKA
6. PKA phosphorlyated proteins to response to adrenaline
7. cAMP is degraded by PDE to AMP, reversing activation of PKA
69
What times the time frame of activation of a GPCR mediated response depend on?
Inherent GTPase activity of the G-protein
70
What does GEF stand for?
Guanine nucleotide-exchange factor
71
What do GEFs do?
Promotes replacement of bound GDP molecule for new molecule of GTP (thus activating G-protein)
72
What is another name for G-proteins?
GTPases
73
True or false: GEFs activate the inherent GTPase activity
False: it just activates the aGs subunit, not changing the rate of the GTPase reaction
74
What does GAP stand for?
GTPase accelerating/activating proteins
75
What do GAPs do?
Stimulate the rate of GTP hydrolysis to GDP (thus inactivating G-protein)
76
True or false: GAPs activate the inherent GTPase activity
True: GAPs increase the natural rate of GTP hydrolysis
77
If a G-protein is bound with GDP, which assistant proteins will act on it?
GEFs
78
If a G-protein is bound with GTP, which assistant proteins will act on it?
GAPs
79
What catalyzes PDE?
PKA
80
What is the structure of PKA when it is inactive?
Two regulatory subunits binded to two catalytic subunits
81
How does PKA become activated?
cAMP binds to the regulatory subunits, releasing the catalytic subunits, which are now active
82
How does cAMP stimulate its own degradation?
cAMP activates PKA, which activates PDE, which breaks down cAMP into AMP
83
What G-protein activates AC?
aGs
84
True or false: Adrenaline signaling can lead to changes in gene expression
True: the signals can interact with CREB, and change gene expression
85
What does Gi mean?
Inhibitory G-protein
86
What does Gi do?
Inhibits an effector protein
87
How does aGs interact with AC?
Stimulate it, thus produces higher cAMP levels
88
How does aGi interact with AC?
Inhibits it, thus producing lower cAMP levels
89
What are some examples of stimulatory hormones?
Epinephrine, glucagon, ACTH
90
What are some examples of inhibitory hormones?
PGE1, adenosine
91
What is the general way for desensitization occur for GPCRs?
Blocking activate GPCRs from turning on additional G-proteins
92
What does GRK stand for?
G-protein coupled receptor kinase
93
What do GRKs do?
Phosphorylate a GPCR to help stop the signal
94
How does phosphorylating a GPCR lead to desensitization?
Arrestin binds to phosphorylated GPCR, preventing G-protein from being activated
95
What do arrestins do?
Bind to phosphorylated GPCRs to prevent G-proteins from being activated
96
What does RGS stand for?
Regulators of G-protein signaling
97
What do RGSs do?
Hydrolyze GTP into GDP (same thing as GAPs)
98
What is the physiological consequence of retinitus pigmentosa?
A mutation in rhodopsin GRK (disease)
99
What happens to a GPCR when arrestin is bound to it?
Cause an endocytotic event
100
Once a GPCR is in an endosome, what are the possible outcomes?
Degradation, or recycling (reinsertion of a clean GPCR)
101
How is desensitization seen in terms of a response?
A lower response compared to the initial
102
After a long period of time, what fraction of the initial response is the new response?
Full (no more desensitization)
103
What is homologous desensitization?
Both receptors are sensitized separately (sensitizing A doesn't affect B)
104
If two receptors experience homologous desensitization, and ligand A is added, what is the response if another ligand A is added?
Desensitization (lower response)
105
If two receptors experience homologous desensitization, and ligand A is added, what is the response if ligand B is added?
High initial response (no desensitization)
106
What is heterologous desensitization?
Both receptors are sensitized together (sensitizing A also affects B)
107
If two receptors experience heterologous desensitization, and ligand A is added, what is the response if another ligand A is added?
Desensitization (lower response)
108
If two receptors experience heterologous desensitization, and ligand A is added, what is the response if ligand B is added?
Lower response (desensitization of B also occurs)
109
If two receptors experience homologous desensitization, what can you say about the regulatory molecule?
It is only a downstream output of one receptor
110
If two receptors experience heterologous desensitization, what can you say about the regulatory molecule?
It is a common downstream output of both receptors
111
What stimulates GRK to phosphorylate a GPCR?
If the GPCR is activated (ligand bound)
112
What does clathrin do?
Helps with endocytosis
113
True or false: Arrestins can lead to signaling events
True: arrestin coated endosomes can be signaling inputs themselves
114
Who discovered arrestins?
Robert Lefkowitz
115
What is an example of a response to an arrestin-endosome signal?
Inhibit transcription of receptors (time to not be responsive)
116
True or false: G-proteins can directly regulate ion channels
True: these G-proteins can open ion channels (such as with acetylcholine)
117
In acetylcholine signaling, which subunit activates the potassium channel?
Beta/gamma subunit
118
In acetylcholine signaling, how is the signal turned off?
aGs is inactivated by GTPase activity, and it has a high affinity for beta/gamma complex, thus inactivating it in its trimeric form
119
True or false: 2nd messengers such as cAMP allow for amplification
True: one signaling ligand can lead to 10^8 response
120
What three proteins can interact with ion channels (in G-protein signaling)?
1. Effector proteins
2. aG
3. beta/gamma subunit
121
True or false: several disorders are caused by defects in receptors or G-proteins
True: there are many areas which can lead to disease if mutated
122
What does cholera toxin do to G-proteins?
Constantly activates aGs by inhibiting GTPase activity, thus always stimulating AC to produce cAMP
123
What are the symptoms of cholera?
Diarrhea, dehydration, and death
124
How does cholera interact with aGs?
Cholera covalently binds NAD to aGs, which prevents GTPase activity
125
Why does cholera toxin lead to dehydration?
Secretion is controlled by cAMP in intenstinal cells, and cholera leads to high cAMP levels
126
What are the symptoms of whooping cough?
Severe coughing
127
What bacterium is responsible for whooping cough?
Bordetella pertussis
128
What does pertussis toxin do to G-proteins?
Inhibits aGi by preventing exchange of GDP to GTP, thus preventing inhibition of AC, leading to production of cAMP
129
Where does cholera toxin act?
Intestinal cells
130
Where does pertussis toxin act?
Lung cells
131
How does pertussis toxin inhibit aGi?
Pertussis covalently binds NAD to aGi, which prevents exchange of GDP for GTP
132
Why does cholera toxin stimulate AC?
Keeps aGs signal on by preventing GTPase activity, thus stimulating AC
133
Why does pertussis toxin stimulate AC?
Prevents aGi signal by preventing GDP to GTP exchange, thus preventing AC from being inhibited
134
How does aG and beta/gammaG move through the plasma membrane (usually)?
Diffusion along the plasma membrane
135
Why does cholera cAMP levels not activate PKA and PDE to break down cAMP?
Very high cAMP levels, and localization
136
True or false: evidence suggests that GAPs are regulated by cAMP levels
False: there is no such evidence
137
True or false: evidence suggests that GEFs are regulated by GTP levels
False: while they need GTP to make the exchange, there is no evidence to suggest they are activated by GTP levels
138
What happens if the receptors aren't reset?
First you get high, then you get addicted
139
Why does a high / addiction occur if receptors aren't reset?
Drugs bind to the receptors more tightly, thus messing up the signaling cascade by not resetting properly
140
How does the body respond to receptors not being reset (high / addiction)?
Gene expression is altered to create more receptors to reach physiological response
141
True or false: there are multiple levels of desensitization
True: there is desensitization at the receptor level, and at the gene expression level for example
142
What does a full agonist do to the activity?
Gets the maximal response
143
What does a partial agonist do to the activity?
Gets a partial response
144
What does an antagonist do to the activity?
Gets no response
145
What does an inverse agonist do to the activity?
Gets the opposite response (reverse signal)
146
What is meant by biased signaling (in terms of GPCR and arrestin)?
A ligand can bind to bias the signaling between a G-protein and arrestin
147
How does a molecule bias signaling (in terms of GPCR)?
Changes the conformation of GPCR slightly to bias the signaling
148
What is the leading theory about how GPCRs can bias signaling?
A "phospho-barcode" on the GPCR allows for biased signaling (how it changes its conformation)
149
What do class A GPCRs do?
Recycle GPCRs close to the membrane (by activating ERK)
150
What do class B GPCRs do?
Endosomes lead to ERK activation and degradation over a longer distance
151
What do class C GPCRs do?
"Kiss-and-run" technique to create an endosome that leads to ERK activation
152
What does "kiss-and-run" refer to?
Arrestin briefly binding to a GPCR before moving to a clathrin coated pit (endosome) without the GPCR
153
What are the three general mechanisms of biased signaling?
Biased ligand, biased receptor, and biased system (all pre-biased)
154
Why is using biased signaling important?
Can develop drugs that bias for one signaling pathway over another
155
Why would a signaling pathway want to be biased for for clinical uses?
One signal may have more beneficial effects that the other signaling pathway
156
Would desensitization occur with an antagonist?
No, because no response is generated
157
How do vaccines for cholera and pertussis work?
They have cell surface receptors of the bacteria so the immune system can recognize it and fight against it
158
How is the beta/gamma subunit of a G-protein inactivated?
aG has a high affinity for beta/gamma G when inactivated, thus restoring trimeric form
159
How is cholera treated to prevent the symptoms?
IV hydration
160
What would happen to cAMP if cholera and pertussis were added together?
cAMP would increase, since cholera stimulates AC, and pertussis prevents inhibition of AC, which both lead to increasing cAMP levels
