GPCR

Question 1

Which receptors are the largest of all receptor superfamilies?

Answer 1

GPCR

Question 2

All GPCR have a highly conserved structure, how many TMSD do they have?

Answer 2

7

Question 3

What is the typical structure of GPCR?

Answer 3

1. 7 TMSD
2. N terminus contributes to ligand binding
3. C terminus contributes to regulation
4. TMSD connected by intracellular and extracellular loops
5. TMSD made up of alpha helices
6. Alpha helix 3 always centred towards the structure as it contributes to agonist binding and transduction of information

Question 4

How does usual binding to a GPCR take place (though there is huge diversity?)

Answer 4

Binding of an activating ligand within the pocket is detected by TM3 which undergoes a conformational change resulting in TM5/6 moving in such a way that a G protein binding pocket is revealed.

Question 5

Give an example of unusual activation of a GPCR.

Answer 5

PAR receptors are GPCR activated by proteinases. Eg. Thrombin is an enzyme. When it finds its receptor, it cleaves its amino acids terminus, the remaining amino acid terminus folds into its own ligand binding site to activate it receptor.

Question 6

Why is knowledge of structure of GPCR important?

Answer 6

For rational design of small molecule drugs targetting GPCR.

Question 7

We understand that upon agonists binding to GPCR TM3 pulls TM5/6 in such a way that a cleft opens for what?

Answer 7

For the alpha subunits of heterotrimeric GTP binding proteins.

Question 8

What happens to a trimeric G protein before and after activation?

Answer 8

The G proteins are naturally anchored in the plamsa membrane through lipid modifications. At rest, the alpha subunit has a high affinity for GDP.
Interaction of the apha subunit with an agonist bound receptor induces a conformation change such that GDP dissociates and GTP associates, and the beta gamma subunits can dissociate from the alpha subunit and a free to interact with effector molecules.

Question 9

What is the role of the alpha subunit of a trimeric G protein?

Answer 9

The subunit is actually an enzyme that can hydrolyse GTP. It acts like a guanine nucleotide exchange factor. The rate of this reaction is regulated by RGS (regulators of G protein signalling.)

Question 10

A G protein is always named after …..

Answer 10

Its alpha subunit.

Question 11

There are multiple isoforms of each type of G protein. What differs with each?

Answer 11

The downstream effectors they can interact with.

Question 12

They are many GPCR to G proteins so what has to happen?

Answer 12

G proteins are shared.

Question 13

What do RGS proteins do?

Answer 13

Stimulate GTPase activity of alpha subunits. In some cases the effector itself is an RGS. Hydrolysis of GTP to GDP stops the reaction ie negative feedback.

Question 14

What do acivated trimeric proteins do?

Answer 14

Regulate activities of enzymes that control second messengers.

Question 15

What are second messengers?

Answer 15

Small molecules that carry signals inside of cells.

Question 16

What is local concentration of second messengers determined by?

Answer 16

Rate of production
Rate of diffusion from site of production
Rate of removal.

Question 17

Give examples of second messengers.

Answer 17

cAMP, cGMP, calcium ions, NO

Question 18

What regulates production of cAMP?

Answer 18

Adenylate cyclase (activated by alpha subunit)

Question 19

What removes cAMP?

Answer 19

Phosphodiesterase

Question 20

Adenylate cyclase is located where?

Answer 20

It is a membrane anchored enzyme that is stimulated by G-alpha-S pathway and inhibited by the G-alhpa-I pathway.

Question 21

What are the steps of the Gs pathway?

Answer 21

1. Ligand binds to receptor turning it on
2. Receptor activates trimeric GTPase
3. GTPase activity activates adenylate cyclase
4. cAMP activates Protein Kinase A
5. PKA and Calcium activates Calcium Calmodulin Phosphorylase Kinase
6. Calcium Calmodulin Phosphorylase Kinase activates phosphorylase
7. Phosphorylase converts Glycogen to Glucose

Question 22

How is GPCR signalling switched off?

Answer 22

1. Agonist dissociates from the receptor.
2. GTPase activity of alpha subunit
3. cAMP breakdown by phosphodiesterase
4. Dephosphorylation of enzymes
5. Under long lasting stimulation beta arrestin

Question 23

What effectors do the G-alpha S pathway stimulate?

Answer 23

Stimulates adenylyl cycalde to produce cAMP, and receptor kinases

Question 24

What effectors do the G-alpha I pathway stimulate?

Answer 24

Inhibits adenylyl cyclases, opens potassium channels and closes calcium channels

Question 25

What effectors do the G-alpha Q pathway stimulate?

Answer 25

Activates phoshpolipase C to produce IP3 and Calcium

Question 26

Some trimeric G proteins activate second messengers derived from membrane proteins by actions of enzymes such as what? And what effect does this have?

Answer 26

Phospholipase C

Produces IP3 and calcium release

Question 27

Signalling via lipid derived second messengers is shared across many receptor families allowing opportunities for what?

Answer 27

Cross Tallk

Question 28

Membrane lipids may be targeted by receptor regulated kinases to generate two kinds of second messenger. What are these?

Answer 28

A water soluble second messenger that can diffuse through the cytoplasm eg IP3
A hydrophobic molecule that remains in the membrane eg DAG

Question 29

What are common phospholipids?

Answer 29

Sphingomyelin

Phosphatidylcholine

Question 30

Specificity in signalling comes from selective expression and cellular localisation of signalling molecules. What does this mean?

Answer 30

There are numerous isoforms of PLC.

Question 31

What does IP3 activate?

Answer 31

Calcium

Question 32

What does DAG activate?

Answer 32

Protein Kinase Cs and calcium

Question 33

What structures tend to be conserved throughout PLC?

Answer 33

X and Y domains are the catalytic domain
Plextrin homology domain
C2 domain for calcium binding
EF domain for calcium binding

Question 34

How does DAG activate PKC

Answer 34

DAG binding causes dissociation of an intramolecular pseudosubstrate domain from its own active site.

Question 35

What may DAG also be associated with?

Answer 35

Recruitment of Munc13 to the membrane and stimulation of vesicle docking.

Question 36

What is Lowe Syndrome caused by?

Answer 36

Mutations in OCRL coding for enzymes regulating PIP2 levels causes renal dysfunction, eye dysfunction and major retardation. This shows how highly regulated these must be.

Question 37

What are some processes regulated by calcium signalling?

Answer 37

Hormone secretion
Fertilisation
Muscle Contraction
Cytokinesis

Question 38

Calcium influx into the cytosol is regulated by voltage gated and ligand gated channels in the ER. What is responsible for store refilling?

Answer 38

Store operated channels made up of ORAI and gated by STIM are responsible for store refilling.

Question 39

Information transfer is encoded by the amplitude,duration and frequency of store released calcium signalling and translated by calcium binding proteins for what purpose?

Answer 39

To bring about changes in cell functions.

Question 40

What can Calcium imaging be used to study?

Answer 40

Signalling in a wide variety of cells, in response to many types of stimuli and to gain insight into disease mechanisms and develop new drugs.

Question 41

What can defective Rhodpsin channels cause?

Answer 41

Night blindnessm retinitis pigmentosa

Question 42

What is the action of Cholera?

Answer 42

Cholera toxin ADP ribosylation of R201 inhibits GTP hydrolysis in intestinal epithelium.