The beta2 adrenergic receptor and cAMP

Question 1

What are G protein-coupled receptors (GPCRs ) ?

Answer 1

They are α-helical integral membrane proteins

Question 2

What are G-proteins ?

Answer 2

G-proteins are heterotrimeric (αβγ) membrane-associated proteins that bind GTP/GDP

Question 3

What do G-proteins mediate?

Answer 3

Signal transduction from GPCRs to other target protein

Question 4

What is the structure of the G-protein coupled receptors?

Answer 4

• Have 7 transmembrane alpha-helical domains (H1-H7) with N-terminus and ligand-binding domain on extracellular side and C-terminus on cytosolic side
• Four extracellular segments (E1-E4) and four cytosolic segments (C1-C4)

Question 5

What is the structure of the β-adrenergic receptor?

Answer 5

• Folds on itself so it forms a barrel-like structure in the membrane (alpha helices shown as cylinders)
• Adrenaline, the ligand, binds in the middle
• This induces a conformational change transmitted to the cysolic side of the receptor
• The G-protein binds to the cytoplasmic side of the protein

Question 6

Binding of hormone to receptor produces ?

Answer 6

Conformational change in the receptor which creates a binding site for the G protein

Question 7

Binding of the G protein produces ?

Answer 7

Conformational shift in the alpha subunit, causing the nucleotide binding pocket to open, and GDP is displaced by GTP

Question 8

GTP binding causes ?

Answer 8

The alpha subunit to dissociate from the beta-gamma subunits

Question 9

The Gβγ subunits are released from the unoccupied receptor and may activate?

Answer 9

Their own downstream signalling

Question 10

The Gβγ subunits are released from the unoccupied receptor and may activate ?

Answer 10

Their own downstream signalling

• The Gα subunit now has a high affinity for the target enzyme and binds to that
• This activates the target enzyme, which then synthesises second messenger
• Second messenger molecules can now act on downstream effector targets

Question 11

After a finite time, the GTPase activity of the Gαsubunit is ?

Answer 11

Activated and GTP is hydrolysed to GDP and Pi

Question 12

Gα with GDP bound has a ?

Answer 12

Low affinity for the enzyme but high affinity for Gβγ therefore it dissociates from the enzyme and reassociates with the Gβγ subunits

• The enzyme is switched off and this regenerates the original conformation
• So there is an intrinsic self-timing mechanism to switch the signal back off

Question 13

What do G proteins act as ?

Answer 13

Molecular switches: When GDP bound they are “off” and when GTP is bound they are “on”

Question 14

What both play a role in adrenaline signalling to regulate glycogen metabolism?

Answer 14

Signalling by GTP binding and Signalling by Phosphorylation

Question 15

What is β2AR GPCR ?

Answer 15

This is the receptor, binding in this case adrenaline

Question 16

What is Gs heterotrimeric G protein ?

Answer 16

This is the transducer - converts the signal across the membrane and activates the amplifier

Question 17

What enzyme is the amplifier?

Answer 17

The amplifier is the enzyme adenylate cyclase, which makes 3’5’-cAMP

Question 18

What is 3’-5’cAMP ?

Answer 18

3’-5’cAMP is the second messenger – acts intracellularly to activate downstream effector molecules like PKA

Question 19

Structure of Adenylate cyclase?

Answer 19

• Adenylate cyclase is a membrane protein with two large intracellular catalytic domains
• The catalytic domains are separate until the Gα subunit binds and brings them together

Question 20

What can adenylate cyclase catalyse ?

Answer 20

The formation of cAMP

Question 21

What is the role of 3’5’-cAMP?

Answer 21

To relay the primary message from the hormone into the cell

Question 22

What does cAMP phosphodiesterase (PDE) catalyse ?

Answer 22

It catalyses hydrolysis of cyclic bond to make AMP (not second messenger)

Question 23

Explain the The β2-AR signal cascade and glycogen ?

Answer 23

• Adrenaline hormone (first message) binds the β2-AR
• This recruits the GαsG-protein with GDP bound
• A conformational change results in the exchange of GDP for GTP
• The G-protein subunits dissociate and the Gαs subunit binds & activates adenylate cyclase
• AC synthesises 3’5’cAMP from ATP
• cAMP acts as a second messenger to activate PKA
• PKA is the effector molecule that phosphorylates downstream targets, e.g. glycogen phosphorylase, phosphorylase kinase (switches on)

Question 24

Degradative reactions are increased by ? and inhibited by?

Answer 24

Degradative reactions are increased by phosphorylation and biosynthetic reactions inhibited by it

Question 25

Why is phosphorylation such a common way of regulating enzymes ?

Answer 25

• A phosphoryl group adds two negative charges to a modified protein, changing electrostatic interactions
• A phosphate group can form three or more hydrogen bonds with a tetrahedral geometry, making them highly directional and specific
• The process is readily reversible, allowing for molecular switching
• The free energy of phosphorylation is large: phosphorylation can change the conformational equilibrium between different functional states by the order of
• The kinetics can be adjusted to meet the timing needs of a physiological process
• The unique geometry can be recognised by other proteins, facilitating protein-protein interactions
• ATP is the cellular energy currency: you can link the energy status of the cell to the regulation of metabolism
• A number of amino acid residues can be phosphorylated allowing a range of responses

Question 26

Receptor stimulation leads to?

Answer 26

A rise in cAMP, which activates PKA

Question 27

PKA catalytic subunits go into nucleus, where they ?

Answer 27

Phosphorylate and activate CREB

Question 28

What does activated CREB form a complex with ?

Answer 28

Activated CREB and the co-activator CBP/P300 forms a complex

Question 29

CREB complex binds to CRE regulatory elements in promoters of multiple genes, stimulating ?

Answer 29

Stimulating transcription

Question 30

What does Guanine nucleotide exchange factors (GEFs) help stimulate ?

Answer 30

The exchange of GDP for GTP when signalling is activated

Question 31

What does the GTPase activator proteins (GAPs) strongly stimulate ?

Answer 31

The GTPase activity of the alpha subunit, enhancing the breakdown of GTP to GDP

Question 32

What are GEFs and GAPs generally known as ? and what does this help to determine ?

Answer 32

• Regulators of G protein signalling (RGS) for heterotrimeric G proteins
• This helps determine how long the G protein will remain active

Question 33

What are adaptor proteins ?

Answer 33

They are noncatalytic proteins that hold together other protein molecules that function together

Question 34

What do AKAPs have ? and what do they act as ?

Answer 34

AKAPs (A kinase anchoring proteins) have multiple, distinct protein-binding domains, and acts as scaffolds for signalling proteins

Question 35

What is AKAP5 targeted to ?

Answer 35

AKAP5 is targeted to rafts in the cytoplasmic side of the plasma membrane by a PIP3 binding domain and palmitoyl groups

Question 36

What does AKAP5 have binding sites for ?

Answer 36

It has binding sites for for the β2AR, AC, PKA and a phosphatase, bringing them all together in the membrane

Question 37

What kind of signal is adrenaline meant to be ?

Answer 37

Adrenaline is meant to be a short-acting signal. The hormone will dissociate from the receptor

Question 38

Hydrolysis of GTP in the α subunit of the G-protein resets?

Answer 38

Resets the original configuration as it then reassociates with the βγ subunit, so AC is switched off and no more cAMP is made

Question 39

When switching off the signal. The receptor is phosphorylated and inactivated by a protein called?

Answer 39

β-arrestin kinase (βARK). This recruits β-arrestin, resulting in receptor endocytosis: it is later recycled back to the cell surface

Question 40

Explain the effect of cholera toxin on GTPase cycle ?

Answer 40

• Cholera toxin transfers the ADP-ribose group from NAD+ onto the Gαs subunit (ADP-ribosylation)
• This blocks the GTPase activity of Gαs and prevents hydrolysis of the GTP to GDP, keeping the alpha subunit in its active form
• Prevents adenylate cyclase being switched off, resulting in persistent production of cAMP

Question 41

Explain the Amplification of responses ?

Answer 41

• The activation of a few GPCRs leads to the activation of few adenylyl cyclase enzymes
• Every active adenylyl cyclase enzyme makes several cAMP molecules, thus activating several PKA enzymes
• These activate thousands of glycogen-degrading enzymes in the liver tissue
• At the end, tens of thousands of glucose-1-phosphate molecules are available for glycolysis (or conversion to glucose: liver)