GPCRs 1

Question 1

What are GPCRs

Answer 1

1. Are the largest and most diverse group of membrane receptors in eukaryotes.
2. Have 7 transmembrane loops
3. They are membrane receptors that are coupled to intracellular effector systems primarily via a G protein

Question 2

What are examples of GPCRs

Answer 2

1. muscarinic AChRs,
2. adrenoceptors,
3. dopamine receptors,
4. 5-HT (serotonin) receptors,

Question 3

Describe the structure of GPCRs

Answer 3

1. Single polypeptide chain with an extracellular N-terminal domain of varying length, and an intracellular C-terminal domain.
2. Contain G-protein which comprises of three subunits (α, β, γ), the α subunit possessing GTPase activity.
3. The G protein interacts with a binding pocket on the intracellular surface of the receptor.

Question 4

Describe the mechanism of GPCRs

Answer 4

1. When an agonist binds there is a conformational change in the receptor - the α subunit binds GTP, dissociates and is then free to activate an effector (e.g. a membrane enzyme).
2. In some cases, the βγ subunit is the activator species.
3. Activation of the effector is terminated when the bound GTP molecule is hydrolysed, which allows the α subunit to recombine with βγ.

Question 5

What are the different types of GPCRs

Answer 5

1. Gs- coupled receptors
2. Gq-coupled receptors
3. Gi/Go coupled receptors

Question 6

What does Gs-coupled receptors do

Answer 6

1. Activation of adenylyl cyclase in the catalysis of ATP–>AMP
2. Causing increase in cAMP levels

Question 7

What does Gq-coupled receptors do

Answer 7

1.Activates phospholipase C
2. increasing production of second messengers inositol trisphosphate and diacylglycerol (see pp. 36–38) thus releasing Ca2++ from intracellular stores and activating protein kinase C (PKC)

Question 8

What do Gi/Go coupled receptors do

Answer 8

1. Inhibits adenylyl cyclase, decreasing cAMP formation
2. Open K+ channels
3. Inhibit Ca++ channels
4. Which it does depends on how close to receptor the secondary messenger system is

Question 9

What happens when a agonist binds to a GPCR

Answer 9

1. When a GPCR is activated by an agonist this induces small changes in residues around the ligand-binding pocket that translate to larger rearrangements of the intracellular regions of the receptor that open a cavity on the intracellular side of the receptor into which the G protein can bind, resulting in a high-affinity interaction of aß? and the receptor.
2. This agonist-induced interaction of aß? with the receptor causes the bound GDP to dissociate and to be replaced with GTP (GDP–GTP exchange)
3. which in turn causes dissociation of the G protein trimer, releasing a–GTP from the ß? subunits these are the ‘active’ forms of the G protein
4. This diffuses in the membrane and can associate with various enzymes and ion channels, causing activation of the target

Question 10

Describe GPCR structure to membrane

Answer 10

1. Tethered to the membrane
2. Subunits stay very close to receptor

Question 11

What is desensitisation

Answer 11

1. Agonist-induced loss of function
2. Same concentration of agonist
   a. Different intervals between application and washing off
   b. Longer time inbetween means no desensitisation
   c. Short intervals results in desensitisation
3. MOST GPCRs undergo this process
4. Tachyphylaxis - Synonym
5. Similar effect as irreversible antagonist- results in partial agonist behaviour
6. Receptors that have been activated are then non functional for a bit – less available receptors

Question 12

How can GPCR desensitisation be studied

Answer 12

1. Any functional response that involves GPCR activation
   a. Functional assays – eg. guinea pig ileum
   b. Cell-based assays – eg. inhibition of calcium release measuring cAMP levels
   c. Electrophysiology – eg. opening of potassium channels
   d. GTPgamma[35]S

Question 13

How deos GTPgamm[35]S work

Answer 13

1. Radioactive GTP that binds to protein
2. Gamma makes it irreversible
3. Sulfur is radioactive
4. Energy of GTP is used up – converts to GDP
5. Returns to receptor and then GTPgammaS binds irreversibly- only binds to receptors which have been activated

Question 14

Describe the GPCR desensitisation mechanism

Answer 14

1. GRK causes receptor desensitization
2. On receptor activation GRK2 and GRK3 are recruited to the plasma membrane by binding to free G protein βγ subunits.
3. GRKs then phosphorylate the receptors in their activated (i.e. agonist-bound) state - when G-protein is not bound
4. The phosphorylated receptor serves as a binding site for arrestins, intracellular proteins that block the interaction between the receptor and the G proteins producing a selective homologous desensitisation.
5. Enhances affinity of arrestin to bind to receptor
6. Arrestin has low affinity to unphosphorylated receptor but high to phosphorylated
7. Can’t signal anything

Answer 15

1. The sequence of GPCRs includes certain residues (serine and threonine), mainly in the C-terminal cytoplasmic tail, which can be phosphorylated by specific GPCR kinases (GRKs) and by kinases such as PKA and PKC.
2. What is phosphorylated in desensitisation

Question 16

What is the purpose of homeostasis

Answer 16

1. Homeostasis – protects cells from excessive activation (GRK2 knockout mouse is embryonically lethal)

Question 17

What is a problem with desensitisation

Answer 17

1. Problematic when using agonists as drugs – not many GPCR agonists–
2. Beta 2 adrenergic receptor target for inhalers
3. Tolerance means response can decline

Question 18

What is drug tolerance

Answer 18

1. When continued use of a drug requires increased dose of drug for equivalent effect
2. ie. Agonist-induced loss of receptor function (desensitization)

Question 19

How could you reduce desensitisation/drug tolerance

Answer 19

1. Inhibit desensitization itself: eg. GRK inhibitor?
2. Allosteric modulators?
3. Functional selectivity?
4. Agents to affect receptor internalization?
5. Inverse agonists?

Question 20

How can allosteric modulators

Answer 20

1. Have no agonist function in themselves
2. Enhance or inhibit agonist-induced signalling- eg. benzodiazepines at GABAA receptors
3. Makes it easier to target subtypes of receptor
4. E.g. 5HT – 12 GPCR all with similar binding points
5. Still allows endogenous agonist to bind
6. Could circumvent agonist-induced receptor desensitization
7. Most high-throughput screening misses them!

Question 21

What are orthosteric binding sites

Answer 21

1 The orthosteric sites are the sites for binding of the substrates or competitive inhibitors of enzymes and agonists or competitive antagonists of receptors. - normal agonist binds
2. Allosteric sites are away from these sites but their binding to the protein can change its conformation.

Question 22

What different effects can allosteric modulators binding have

Answer 22

1. Positive affinity- Becomes more potent
2. Negative affinity
3. Positive efficacy- Max response goes up
4. Negative efficacy

Answer 23

1. Positive allosteric modulator - may be able to overcome desensitisation
2. Shown to work in GABAB receptor
3. GS39783 is modulator