3 - G-Proteins and cAMP Regulation Flashcards

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1
Q

Who was Martin Rodbell?

A

A scientist who worked on the missing link between adrenal receptors and AC

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2
Q

Which scientist believed in a transducer?

A

Rodbell

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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)

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4
Q

What techniques did Rodbell employ?

A

In vitro reconstitution of purified membranes

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5
Q

What components were part of Rodbell’s purified membranes?

A

The receptor, adenylyl cyclase, and the unknown transducer

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6
Q

What was the pathway that Rodbell was testing?

A

Purified membrane + hormone + ATP -> cAMP

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7
Q

What was the result when Rodbell added purchased ATP?

A

Some ATP produced a response, while some didn’t

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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

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9
Q

What was the result when Rodbell added pure ATP?

A

There was no response

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10
Q

Why did pure ATP have no effect in Rodbell’s experiments?

A

The transducer needed GTP to function, which wasn’t present

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11
Q

What was the result when Rodbell added GTP?

A

There was a response

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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

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13
Q

Why was there a response when Rodbell added GTP?

A

The transducer required GTP to function

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14
Q

What was the result when Rodbell added non-hydrolyzable GTP?

A

There was a prolonged response

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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

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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

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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

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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

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19
Q

What experiments did Gilman do?

A

Used S49 cyc- mutants to investigate transducer

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20
Q

What is an S49 cell?

A

A cancer cell that continuously divides

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21
Q

How can S49 cells stop dividing?

A

By adding cAMP

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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

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23
Q

What are “cyc-“ mutants?

A

S49 cells that (suppossedly) lost adenylyl cyclase

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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

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25
Q

How can it be proven that the mutation in S49 cyc- cells is not in the receptor?

A

Confirm with radioactive ligand binding

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26
Q

What were the 3 selection criteria of the Gilman experiments?

A
  1. Growth in presence of adrenaline
  2. Retain receptors that bind to adrenaline
  3. Do not respond to forskolin
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27
Q

What were the results of the Gilman experiments?

A

Obtained cells that bound to adrenaline, but produced no adrenaline response, but still responded to forskolin

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28
Q

What was the puzzling consequence of the results of the Gilman experiments?

A

The cells had functional receptors and functional AC, but still couldn’t make cAMP

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29
Q

What was the conclusion of the Gilman experiments?

A

There must have been a mutation in another part of the pathway (transducer)

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30
Q

What was the assumed condition of the Gilman experiments?

A

The cyc- cells lacked AC, and adding AC would lead to cAMP in the presence of adrenaline

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31
Q

What was the assumed control condition of the Gilman experiments?

A

The cyc- cells lacked AC, and adding adrenaline would not lead to cAMP

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32
Q

What was the actual control condition of the Gilman experiments?

A

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

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33
Q

What protein did the cyc- cells actually not have?

A

G proteins

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34
Q

How were the transducer proteins verified and purified?

A

Proteins from the membrane were added back into the system to see if they would change the response

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35
Q

How many proteins did the final, purified sample had (to find the transducer)?

A

3

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36
Q

What does cholera toxin do?

A

Covalently links NAD to a substrate, and causes an increase in cAMP (somehow activates AC)

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37
Q

How was cholera toxin used to verify the transducer?

A

Cholera toxin was used to bind radioactive NAD to the transducer proteins that interacted with AC

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38
Q

What were the bands for the transducer?

A

The alpha subunits of G proteins

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39
Q

In wild type cells with cholera toxin and radioactive NAD, what would be the expected blot and why?

A

Two bands corresponding to the G protein transducer, since cholera would bind NAD to these proteins

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40
Q

In wild type cells without cholera toxin and with radioactive NAD, what would be the expected blot and why?

A

No bands, because cholera cannot bind NAD to the proteins

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41
Q

In cyc- cells without cholera toxin and with radioactive NAD, what would be the expected blot and why?

A

No bands, because cholera cannot bind NAD to the proteins

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42
Q

In cyc- cells with cholera toxin and radioactive NAD, what would be the expected blot and why?

A

No bands, because cyc- cells were missing the G protein transducer that cholera would bind NAD to

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43
Q

What was the purpose of the blots?

A

Validate G proteins as transducer of adrenaline response

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44
Q

What is a G-protein coupled receptor (GPCR)?

A

Plasma membrane receptor that works with the help of a G-protein

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45
Q

What is the basic function of a G-protein?

A

Act as an on-off switch

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46
Q

When is a G-protein active?

A

When GTP is bound

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47
Q

When is a G-protein inactive?

A

When GDP is bound

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48
Q

If GTP is bound to a G-protein, what state is it in?

A

Active

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49
Q

If GDP is bound to a G-protein, what state is it in?

A

Inactive

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50
Q

When a signal is inputted, how does a G-protein change?

A

It loses GDP and binds to GTP to become activated

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51
Q

When is the signal outputted though a G-protein?

A

When it is activated

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52
Q

How does a G-protein stop being activated?

A

Hydrolysis of GTP to GDP

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53
Q

How does a G-protein go from activated to inactivated?

A

Hydrolysis of GTP to GDP

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54
Q

How does a G-protein go from inactivated to activated?

A

Exchange of GDP for GTP

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55
Q

What is meant by “G-protein has GTPase activity”?

A

The G-protein itself can hydrolyze GTP into GDP

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56
Q

What happens to a GPCR when a ligand binds to it?

A

It becomes activated

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57
Q

What happens to a G-protein when a GPCR is activated?

A

It stimulated GDP/GTP exchange of the alpha subunit of the G-protein

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58
Q

What subunit is activated by a GPCR?

A

Alpha subunit

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59
Q

What happens to the G-protein once it is activated?

A

It dissociates into an alpha subunit and a beta/gamma subunit

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60
Q

True or false: Once dissociated, the alpha subunit of a G-protein is the only thing that can cause an effect

A

False: depending on the system, the beta/gamma complex can also cause an effect

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61
Q

True or false: The alpha and beta/gamma subunits of a G-protein could interact in the same pathway

A

True: both have the ability to cause an effect, depending on the system

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62
Q

What are the three broad subclasses of trimeric GPCR linked effector proteins?

A

Adenylyl cyclase, phospholipase C, and ion channels

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63
Q

What is meant by a “timer system”?

A

aG is a GTPase, so it can cleave GTP by itself to inactivate itself

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64
Q

How does the alpha subunit reunite with the beta/gamma complex?

A

Inactivate aG has a high affinity for beta/gammaG

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65
Q

What is the typical pathway for a GPCR?

A

Effector proteins -> 2nd messengers -> target proteins

66
Q

What does Gs mean?

A

Stimulatory G-protein

67
Q

What does Gs do?

A

Stimulates an effector protein

68
Q

What are the 7 steps in adrenaline signaling (full model)?

A
  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
Q

What times the time frame of activation of a GPCR mediated response depend on?

A

Inherent GTPase activity of the G-protein

70
Q

What does GEF stand for?

A

Guanine nucleotide-exchange factor

71
Q

What do GEFs do?

A

Promotes replacement of bound GDP molecule for new molecule of GTP (thus activating G-protein)

72
Q

What is another name for G-proteins?

A

GTPases

73
Q

True or false: GEFs activate the inherent GTPase activity

A

False: it just activates the aGs subunit, not changing the rate of the GTPase reaction

74
Q

What does GAP stand for?

A

GTPase accelerating/activating proteins

75
Q

What do GAPs do?

A

Stimulate the rate of GTP hydrolysis to GDP (thus inactivating G-protein)

76
Q

True or false: GAPs activate the inherent GTPase activity

A

True: GAPs increase the natural rate of GTP hydrolysis

77
Q

If a G-protein is bound with GDP, which assistant proteins will act on it?

A

GEFs

78
Q

If a G-protein is bound with GTP, which assistant proteins will act on it?

A

GAPs

79
Q

What catalyzes PDE?

A

PKA

80
Q

What is the structure of PKA when it is inactive?

A

Two regulatory subunits binded to two catalytic subunits

81
Q

How does PKA become activated?

A

cAMP binds to the regulatory subunits, releasing the catalytic subunits, which are now active

82
Q

How does cAMP stimulate its own degradation?

A

cAMP activates PKA, which activates PDE, which breaks down cAMP into AMP

83
Q

What G-protein activates AC?

A

aGs

84
Q

True or false: Adrenaline signaling can lead to changes in gene expression

A

True: the signals can interact with CREB, and change gene expression

85
Q

What does Gi mean?

A

Inhibitory G-protein

86
Q

What does Gi do?

A

Inhibits an effector protein

87
Q

How does aGs interact with AC?

A

Stimulate it, thus produces higher cAMP levels

88
Q

How does aGi interact with AC?

A

Inhibits it, thus producing lower cAMP levels

89
Q

What are some examples of stimulatory hormones?

A

Epinephrine, glucagon, ACTH

90
Q

What are some examples of inhibitory hormones?

A

PGE1, adenosine

91
Q

What is the general way for desensitization occur for GPCRs?

A

Blocking activate GPCRs from turning on additional G-proteins

92
Q

What does GRK stand for?

A

G-protein coupled receptor kinase

93
Q

What do GRKs do?

A

Phosphorylate a GPCR to help stop the signal

94
Q

How does phosphorylating a GPCR lead to desensitization?

A

Arrestin binds to phosphorylated GPCR, preventing G-protein from being activated

95
Q

What do arrestins do?

A

Bind to phosphorylated GPCRs to prevent G-proteins from being activated

96
Q

What does RGS stand for?

A

Regulators of G-protein signaling

97
Q

What do RGSs do?

A

Hydrolyze GTP into GDP (same thing as GAPs)

98
Q

What is the physiological consequence of retinitus pigmentosa?

A

A mutation in rhodopsin GRK (disease)

99
Q

What happens to a GPCR when arrestin is bound to it?

A

Cause an endocytotic event

100
Q

Once a GPCR is in an endosome, what are the possible outcomes?

A

Degradation, or recycling (reinsertion of a clean GPCR)

101
Q

How is desensitization seen in terms of a response?

A

A lower response compared to the initial

102
Q

After a long period of time, what fraction of the initial response is the new response?

A

Full (no more desensitization)

103
Q

What is homologous desensitization?

A

Both receptors are sensitized separately (sensitizing A doesn’t affect B)

104
Q

If two receptors experience homologous desensitization, and ligand A is added, what is the response if another ligand A is added?

A

Desensitization (lower response)

105
Q

If two receptors experience homologous desensitization, and ligand A is added, what is the response if ligand B is added?

A

High initial response (no desensitization)

106
Q

What is heterologous desensitization?

A

Both receptors are sensitized together (sensitizing A also affects B)

107
Q

If two receptors experience heterologous desensitization, and ligand A is added, what is the response if another ligand A is added?

A

Desensitization (lower response)

108
Q

If two receptors experience heterologous desensitization, and ligand A is added, what is the response if ligand B is added?

A

Lower response (desensitization of B also occurs)

109
Q

If two receptors experience homologous desensitization, what can you say about the regulatory molecule?

A

It is only a downstream output of one receptor

110
Q

If two receptors experience heterologous desensitization, what can you say about the regulatory molecule?

A

It is a common downstream output of both receptors

111
Q

What stimulates GRK to phosphorylate a GPCR?

A

If the GPCR is activated (ligand bound)

112
Q

What does clathrin do?

A

Helps with endocytosis

113
Q

True or false: Arrestins can lead to signaling events

A

True: arrestin coated endosomes can be signaling inputs themselves

114
Q

Who discovered arrestins?

A

Robert Lefkowitz

115
Q

What is an example of a response to an arrestin-endosome signal?

A

Inhibit transcription of receptors (time to not be responsive)

116
Q

True or false: G-proteins can directly regulate ion channels

A

True: these G-proteins can open ion channels (such as with acetylcholine)

117
Q

In acetylcholine signaling, which subunit activates the potassium channel?

A

Beta/gamma subunit

118
Q

In acetylcholine signaling, how is the signal turned off?

A

aGs is inactivated by GTPase activity, and it has a high affinity for beta/gamma complex, thus inactivating it in its trimeric form

119
Q

True or false: 2nd messengers such as cAMP allow for amplification

A

True: one signaling ligand can lead to 10^8 response

120
Q

What three proteins can interact with ion channels (in G-protein signaling)?

A
  1. Effector proteins
  2. aG
  3. beta/gamma subunit
121
Q

True or false: several disorders are caused by defects in receptors or G-proteins

A

True: there are many areas which can lead to disease if mutated

122
Q

What does cholera toxin do to G-proteins?

A

Constantly activates aGs by inhibiting GTPase activity, thus always stimulating AC to produce cAMP

123
Q

What are the symptoms of cholera?

A

Diarrhea, dehydration, and death

124
Q

How does cholera interact with aGs?

A

Cholera covalently binds NAD to aGs, which prevents GTPase activity

125
Q

Why does cholera toxin lead to dehydration?

A

Secretion is controlled by cAMP in intenstinal cells, and cholera leads to high cAMP levels

126
Q

What are the symptoms of whooping cough?

A

Severe coughing

127
Q

What bacterium is responsible for whooping cough?

A

Bordetella pertussis

128
Q

What does pertussis toxin do to G-proteins?

A

Inhibits aGi by preventing exchange of GDP to GTP, thus preventing inhibition of AC, leading to production of cAMP

129
Q

Where does cholera toxin act?

A

Intestinal cells

130
Q

Where does pertussis toxin act?

A

Lung cells

131
Q

How does pertussis toxin inhibit aGi?

A

Pertussis covalently binds NAD to aGi, which prevents exchange of GDP for GTP

132
Q

Why does cholera toxin stimulate AC?

A

Keeps aGs signal on by preventing GTPase activity, thus stimulating AC

133
Q

Why does pertussis toxin stimulate AC?

A

Prevents aGi signal by preventing GDP to GTP exchange, thus preventing AC from being inhibited

134
Q

How does aG and beta/gammaG move through the plasma membrane (usually)?

A

Diffusion along the plasma membrane

135
Q

Why does cholera cAMP levels not activate PKA and PDE to break down cAMP?

A

Very high cAMP levels, and localization

136
Q

True or false: evidence suggests that GAPs are regulated by cAMP levels

A

False: there is no such evidence

137
Q

True or false: evidence suggests that GEFs are regulated by GTP levels

A

False: while they need GTP to make the exchange, there is no evidence to suggest they are activated by GTP levels

138
Q

What happens if the receptors aren’t reset?

A

First you get high, then you get addicted

139
Q

Why does a high / addiction occur if receptors aren’t reset?

A

Drugs bind to the receptors more tightly, thus messing up the signaling cascade by not resetting properly

140
Q

How does the body respond to receptors not being reset (high / addiction)?

A

Gene expression is altered to create more receptors to reach physiological response

141
Q

True or false: there are multiple levels of desensitization

A

True: there is desensitization at the receptor level, and at the gene expression level for example

142
Q

What does a full agonist do to the activity?

A

Gets the maximal response

143
Q

What does a partial agonist do to the activity?

A

Gets a partial response

144
Q

What does an antagonist do to the activity?

A

Gets no response

145
Q

What does an inverse agonist do to the activity?

A

Gets the opposite response (reverse signal)

146
Q

What is meant by biased signaling (in terms of GPCR and arrestin)?

A

A ligand can bind to bias the signaling between a G-protein and arrestin

147
Q

How does a molecule bias signaling (in terms of GPCR)?

A

Changes the conformation of GPCR slightly to bias the signaling

148
Q

What is the leading theory about how GPCRs can bias signaling?

A

A “phospho-barcode” on the GPCR allows for biased signaling (how it changes its conformation)

149
Q

What do class A GPCRs do?

A

Recycle GPCRs close to the membrane (by activating ERK)

150
Q

What do class B GPCRs do?

A

Endosomes lead to ERK activation and degradation over a longer distance

151
Q

What do class C GPCRs do?

A

“Kiss-and-run” technique to create an endosome that leads to ERK activation

152
Q

What does “kiss-and-run” refer to?

A

Arrestin briefly binding to a GPCR before moving to a clathrin coated pit (endosome) without the GPCR

153
Q

What are the three general mechanisms of biased signaling?

A

Biased ligand, biased receptor, and biased system (all pre-biased)

154
Q

Why is using biased signaling important?

A

Can develop drugs that bias for one signaling pathway over another

155
Q

Why would a signaling pathway want to be biased for for clinical uses?

A

One signal may have more beneficial effects that the other signaling pathway

156
Q

Would desensitization occur with an antagonist?

A

No, because no response is generated

157
Q

How do vaccines for cholera and pertussis work?

A

They have cell surface receptors of the bacteria so the immune system can recognize it and fight against it

158
Q

How is the beta/gamma subunit of a G-protein inactivated?

A

aG has a high affinity for beta/gamma G when inactivated, thus restoring trimeric form

159
Q

How is cholera treated to prevent the symptoms?

A

IV hydration

160
Q

What would happen to cAMP if cholera and pertussis were added together?

A

cAMP would increase, since cholera stimulates AC, and pertussis prevents inhibition of AC, which both lead to increasing cAMP levels