G protein coupled receptors 1

Question 1

Describe the structure of the G protein-coupled receptor

Answer 1

1. Single polypeptide chain with 7 transmembrane regions (serpentine receptors)
2. Cyto loop C3 and C-terminus interact with G proteins

Question 2

Describe the GPCR Superfamily

Answer 2

1. GPCRs are the largest family of cell surface receptors
2. Important sub-families inc:
   
   • yeast mating factor
   • rhodopsin receptors
   • olfactory receptors
3. Over 50% of all medical drugs target G protein coupled receptors or pathways

Question 3

What are some ligands of G protein-coupled receptors

Answer 3

1. Functionally- Hormones, neurotransmitters or local mediators
2. Biochemically- Small peptides, amino acid and fatty acid Derivatives, Proteins

Question 4

Describe signal transduction through ligand binding

Answer 4

1. Ligand binding causes conformational change, including in the cytosolic domains, that allows recruitment of a trimeric G protein

Question 5

Describe structure of trimeric G-protein

Answer 5

1. Larger, trimeric GTP binding proteins
2. Alpha, beta and gamma subunits
3. Alpha and gamma are tethered to inner leaflet of plasma membranes by lipid tails
4. Alpha has ras domain
5. Alpha has alpha helix domain
6. The nucleotide binding pocket is formed from the Ras domain (structurally related to other GTPases) and alpha helical (AH) domain, which clamps it in place.

Question 6

Describe different classes adn ways of binding of G proteins

Answer 6

1. Two classes of G proteins couple to two different intracellular signalling pathways, Gs and Gq
2. Relatively few different G proteins couple different families of GPCRs to two different intracellular signalling pathways.
3. Some G-proteins associate with their cognate receptors before ligand binding, others only after ligand binding.

Question 7

Describe activation of G protein by GPCR

Answer 7

1. Interaction with the activated receptor causes a conformational change in the Gα sub-unit that promotes release of GDP (the receptor acts like GNRP/GEF for monomeric G-protein Ras ).
2. This allows uptake of GTP which causes further conformational change with two consequences:
3. the G protein dissociates from the receptor;
4. either the α sub-unit dissociates from the β/γ complex or the trimeric complex remains intact but domains on Gα are exposed that allow interaction with a target signalling protein.
5. GTP bound state is active,
6. GDP bound state inactive (like Ras).

Question 8

How is adenylyl cyclase stimulated

Answer 8

1. Activated Gsα (GTP-bound) stimulates adenylyl cyclase
2. Resulting in a rapid rise in cytoplasmic [cAMP] which acts as a second messenger
3. Adenylyl cyclase- imbedded in plasma membrane so in correct place to come into contact with alpha subunit
4. Activates adenylyl cyclase which using ATP to form cyclic AMP- rapid rise in concentration
5. Acts as second messenger

Question 9

How is Gsalpha inactivated

Answer 9

1. Inactivation of Gsα involves stimulation of GTPase activity by the target protein
2. The target enzyme acts as a GTPase activating protein (GAP)
3. Interaction with its target enzyme also activates the GTPase activity of the α/GTP subunit.
4. After a short delay the α subunit hydrolyses GTP to GDP, restoring the inactive α subunit conformation, causing its release from the target enzyme.
5. This restores the resting state unless signal is still present.
6. The target enzyme can act like GAP (GTPase activating protein, as for monomeric GTPases), stimulating the GTPase activity of the alpha subunit.

Question 10

Describe the regulation of G protein GTPase activity

Answer 10

1. Ga hydrolyses GTP to become inactive
2. GTPase is stimulated by:
3. Interaction with substrates (Gs)
4. Regulators of G protein Signalling - (RGS) proteins (Gi and Gq)
5. RGS has similar function for G protein as GAP for Ras,
6. ensuring Galpha remains active for only a brief period.
7. > 20 human RGS proteins, mutated in a variety of diseases (e.g. cardiovascular, neurological, cancer).

Question 11

What regulates cAMP level

Answer 11

1. GPCR (Gs) signalling regulates cytosolic cAMP level
2. Gas activates adenylyl cyclase (AC)
3. AC synthesises cyclic AMP (second messenger)
4. ATP is being used as the substrate to generate the small second messenger signalling molecule 3’,5’-cyclic AMP (cAMP), releasing a pyrophosphate molecule.
5. cAMP phosphodiesterase hydrolyses cAMP
6. There are several classes of AC which, unusually are structurally unrelated but catalyse the same reaction.
7. ATP is also important in signalling as is the phosphate donor in phosphorylation of many signalling proteins

Question 12

How fast are cell responses to cAMP and how is this demonstrated

Answer 12

1. Cell responses to cAMP can be rapid
2. Typically affecting proteins present in the cytoplasm
3. e.g. Stimulation of glycolysis; Inhibition of glycogen synthesis
4. A dye that fluoresces when bound to cAMP is used to illustrates the rapid rise and dispersal of the cAMP second messenger following stimulation of a neuron with the neurotransmitter serotonin.

Question 13

Describe cell responses to cAMP signalling

Answer 13

1. Response to increased [cAMP] is cell type specific
2. Elevated cAMP elicits a cell type-specific response, sometimes to different signals (e.g. adipose).
3. The same signal can affect multiple cell types, sometimes using different receptors (e.g adrenaline in adipose, heart and muscle).

Question 14

What can increased levels of cAMP activate

Answer 14

1. Increased levels of cAMP activate protein kinase A
2. One consequence of the requirement for 4 cAMP molecules to activate PKA is that it helps to generate a sudden or switch-like response to gradually increasing levels of signal
3. PKA substrates are cell type specific, explaining how the same simple second messenger (cAMP) can elicit cell type specific responses.

Question 15

Describe Protein Kinase A

Answer 15

1. PKA (aka A-kinase) is a serine/threonine kinase.
2. Substrates of PKA are cell type specific.
3. Catalytic subunits can activate cytoplasmic and nuclear proteins (transcription factors).

Question 16

What can happen to inactive PKA and give example

Answer 16

1. Inactive PKA can be membrane tethered with other signalling proteins
2. A-kinase anchoring proteins tether inactive PKA to cellular membranes, sometimes along with other signalling molecules (thus acting as signalling scaffolds);
3. e.g. cAMP phosphodiesterase around cardiomyocyte nuclear membrane – keeps local cAMP level low in resting state and when cAMP increases PKA stimulates the phosphodiesterase, creating a negative feedback loop that ensures signal activity is short-lived

Question 17

What brings about rapid energy mobilisation

Answer 17

1. Rapid response to adrenaline for energy mobilisation
2. The same signal stimulates glycogen breakdown (to glucose) and inhibits glycogen synthesis (from glucose), another mechanism to bring about a switch-like response.
3. Protein kinase a phosphorylates phosphorylase kinase etc

Question 18

What type of cAMP response is involved in gene expression

Answer 18

1. Slower cAMP response involve changes in gene expression
2. PKA activates CREB in the nucleus which binds CRE elements at target genes
3. CREB is a transcription factor
4. CREB = cAMP response element binding protein.
5. PKA activates CREB in the nucleus which binds (with cofactor CBP- CREB binding factor) to CRE (cAMP response element) motif in DNA.

Question 19

How are actions of PKA reversed

Answer 19

1. Actions of PKA are reversed by serine/threonine phosphatases
2. Type I- dephosphorylates many substrates of PKA
3. For a sustained response to elevated cAMP levels some cells activate a phosphatase inhibitory protein, which inhibits protein phosphatase I, and can itself be regulated by PKA.
4. Type IIA- broad specificity, most common
5. Type IIB- (calcineurin) most abundant in brain, Ca2+ activated
6. Type IIC- not so common, unrelated to the others

Question 20

What is PKA activity antagonised by

Answer 20

1. PKA activity is antagonised by type I phosphatases.