The beta2 adrenergic receptor and cAMP Flashcards
What are G protein-coupled receptors (GPCRs ) ?
They are α-helical integral membrane proteins
What are G-proteins ?
G-proteins are heterotrimeric (αβγ) membrane-associated proteins that bind GTP/GDP
What do G-proteins mediate?
Signal transduction from GPCRs to other target protein
What is the structure of the G-protein coupled receptors?
- 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)
What is the structure of the β-adrenergic receptor?
- 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
Binding of hormone to receptor produces ?
Conformational change in the receptor which creates a binding site for the G protein
Binding of the G protein produces ?
Conformational shift in the alpha subunit, causing the nucleotide binding pocket to open, and GDP is displaced by GTP
GTP binding causes ?
The alpha subunit to dissociate from the beta-gamma subunits
The Gβγ subunits are released from the unoccupied receptor and may activate?
Their own downstream signalling
The Gβγ subunits are released from the unoccupied receptor and may activate ?
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
After a finite time, the GTPase activity of the Gαsubunit is ?
Activated and GTP is hydrolysed to GDP and Pi
Gα with GDP bound has a ?
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
What do G proteins act as ?
Molecular switches: When GDP bound they are “off” and when GTP is bound they are “on”
What both play a role in adrenaline signalling to regulate glycogen metabolism?
Signalling by GTP binding and Signalling by Phosphorylation
What is β2AR GPCR ?
This is the receptor, binding in this case adrenaline
What is Gs heterotrimeric G protein ?
This is the transducer - converts the signal across the membrane and activates the amplifier
What enzyme is the amplifier?
The amplifier is the enzyme adenylate cyclase, which makes 3’5’-cAMP
What is 3’-5’cAMP ?
3’-5’cAMP is the second messenger – acts intracellularly to activate downstream effector molecules like PKA
Structure of Adenylate cyclase?
- 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
What can adenylate cyclase catalyse ?
The formation of cAMP
What is the role of 3’5’-cAMP?
To relay the primary message from the hormone into the cell
What does cAMP phosphodiesterase (PDE) catalyse ?
It catalyses hydrolysis of cyclic bond to make AMP (not second messenger)
Explain the The β2-AR signal cascade and glycogen ?
- 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)
Degradative reactions are increased by ? and inhibited by?
Degradative reactions are increased by phosphorylation and biosynthetic reactions inhibited by it
Why is phosphorylation such a common way of regulating enzymes ?
- 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
Receptor stimulation leads to?
A rise in cAMP, which activates PKA
PKA catalytic subunits go into nucleus, where they ?
Phosphorylate and activate CREB
What does activated CREB form a complex with ?
Activated CREB and the co-activator CBP/P300 forms a complex
CREB complex binds to CRE regulatory elements in promoters of multiple genes, stimulating ?
Stimulating transcription
What does Guanine nucleotide exchange factors (GEFs) help stimulate ?
The exchange of GDP for GTP when signalling is activated
What does the GTPase activator proteins (GAPs) strongly stimulate ?
The GTPase activity of the alpha subunit, enhancing the breakdown of GTP to GDP
What are GEFs and GAPs generally known as ? and what does this help to determine ?
- Regulators of G protein signalling (RGS) for heterotrimeric G proteins
- This helps determine how long the G protein will remain active
What are adaptor proteins ?
They are noncatalytic proteins that hold together other protein molecules that function together
What do AKAPs have ? and what do they act as ?
AKAPs (A kinase anchoring proteins) have multiple, distinct protein-binding domains, and acts as scaffolds for signalling proteins
What is AKAP5 targeted to ?
AKAP5 is targeted to rafts in the cytoplasmic side of the plasma membrane by a PIP3 binding domain and palmitoyl groups
What does AKAP5 have binding sites for ?
It has binding sites for for the β2AR, AC, PKA and a phosphatase, bringing them all together in the membrane
What kind of signal is adrenaline meant to be ?
Adrenaline is meant to be a short-acting signal. The hormone will dissociate from the receptor
Hydrolysis of GTP in the α subunit of the G-protein resets?
Resets the original configuration as it then reassociates with the βγ subunit, so AC is switched off and no more cAMP is made
When switching off the signal. The receptor is phosphorylated and inactivated by a protein called?
β-arrestin kinase (βARK). This recruits β-arrestin, resulting in receptor endocytosis: it is later recycled back to the cell surface
Explain the effect of cholera toxin on GTPase cycle ?
- 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
Explain the Amplification of responses ?
- 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)
