Block D Lecture 2 - Molecular Signalling

Question 1

What are the 2 different mechanisms in which a GPCR can be desensitised?

Answer 1

Homologous (agonist-specific) and heterologous

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Question 2

What occurs in homologous desensitisation?

Answer 2

1. The activated receptor is phosphorylated by a specific kinase (GPCR kinase / GRK). This promotes binding of arrestin.
2. The phosphorylated receptor (P-R) then binds to arrestin, causing it to lose its ability to associate with a G protein.
3. This also causes the receptor to undergo endocytosis, as arrestin promotes internalisation, removing the receptor from the membrane

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Question 3

What occurs in heterologous desensitisation?

Answer 3

It occurs as a result of phosphorylation of one type of receptor as a result of activation of kinase by another receptor. E.g PKA or PKC

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Question 4

What do GPCR kinases (GRKs) phosphorylate in GPCRs?

Answer 4

They phosphorylate specific residues on the C-terminal tail of the GPCR

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Question 5

How does arrestin prevent a GPCR from associating with its G protein?

Answer 5

It outcompetes the G-protein for the site of the receptor, causing the receptor to lose G-protein signalling

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Question 6

When can desensitisation become an issue?

Answer 6

When an agonist is used a lot

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Question 7

How does salmeterol take advantage of the desensitisation system to stay active longer?

Answer 7

1. It causes less phosphorylation of the β2-adrenoceptor by β adrenoceptor kinase (β-ARK)
2. This leads to less internalisation of the receptor
3. Even though its a partial agonist, the above results in it being able to produce the same amount of cAMP/PKA dependent phosphorylation of the receptor
4. These effects lead to reduced desensitisation, which results in salmeterol being able to produce effects for a longer period of time then for example, salbutamol

(Slide 8)

Question 8

How can cAMP / cGMP second messengers be turned off?

Answer 8

By the action of phosphodiesterases which terminate the signal

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Question 9

How can Inositol 1,4,5-trisphosphate second messenger be turned off?

Answer 9

By a series of phosphatases progressively removing phosphate to eventually generate inositol

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Question 10

What is roflumilast?

Answer 10

A selective phosphodiesterase (PDE) type IV inhibitor, which acts insides the cells of the airways. It increases cAMP concentration and causes relaxation of the airways and dampens down inflammation

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Question 11

How does Viagra act?

Answer 11

It inhibits phosphodiesterase (PDE) type V inside smooth muscle cells of the penis, increasing cGMP concentration which causes relaxation and increases blood flow. It is used for erectile disfunction

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Question 12

What are 3 examples of mutations in G proteins and GPCRs which can lead to disease?

Answer 12

Loss-of-function mutations

Gain-of-function mutations

Polymorphisms

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Question 13

What do loss-of-function mutations in G proteins and GPCRs result in?

Answer 13

It blocks signalling in response to the corresponding agonist

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Question 14

What do gain-of-function mutations in G proteins and GPCRs result in?

Answer 14

It leads to constitutive, agonist-independent activation of signalling

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Question 15

What do polymorphisms in G proteins and GPCRs result in?

Answer 15

Can give rise to multiple issues, such as G-protein coupling, receptor trafficking or changes in internalisation

(Slide 13)

Question 16

What do mutations in the vasopressin V2 receptor result in?

Answer 16

It being unable to get to the plasma membrane from the ribosome, as it doesn’t fold properly, or it can block interaction with Gs G protein, again due to bad folding / movement.

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Question 17

What is Vaptan and how does it work?

Answer 17

It is a vasopressin V2 receptor antagonist / invest agonist which binds to a misfolded V2 mutant and helps it get to the plasma membrane. It is used in the treatment of nephrogenic diabetes insipidus (NDI)

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Question 18

What is nephrogenic diabetes insipidus?

Answer 18

A rare kidney disorder that causes the body to produce too much urine and lose too much water

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Question 19

What do kinase-linked receptors mediate?

Answer 19

The actions of a wide variety of protein mediators, including growth factors, cytokines and hormones

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Question 20

How are kinase-linked receptor effects usually exerted?

Answer 20

Gene expression (gene transcription level)

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Question 21

What do receptor tyrosine kinases (RTKs) and serine/threonine kinases do?

Answer 21

RTKs - phosphorylate tyrosine residues

Serine / threonine kinases - Phosphorylate serine and / or threonine residues

(Slide 22)

Question 22

What are cytokine receptors?

Answer 22

As they lack intrinsic enzyme activity, they associate with and activate, a cytosolic tyrosine kinase

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Question 23

What are the steps of the growth factor receptor pathways (MAPK)?

Answer 23

1. A growth factor like EGF binds to bind to their specific receptor tyrosine kinases (RTKs) on the cell surface.
2. Binding induces dimerization
3. The (RTKs) undergo auto-phosphorylation via tyrosine residues on their intracellular domains
4. Phosphorylated RTKs recruit adaptor proteins like Grb2 (Growth Factor Receptor-Bound Protein 2)
5. Grb2 associates with SOS (Son of Sevenless), a guanine nucleotide exchange factor (GEF)
6. SOS activates Ras, a small GTPase, by exchanging GDP for GTP
7. GTP-bound Ras activates Raf, a serine/threonine kinase and the first kinase in the MAPK cascade
8. Raf phosphorylates and activates MEK, MEK does the same to ERK (MAPK).
9. Activated ERK translocates to the nucleus, where it phosphorylates various transcription factors

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Question 24

How is the level of RTK regulation usually controlled?

Answer 24

By the antagonizing effect of tyrosine phosphatases

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Question 25

What are 2 ways which constitutive activation of RTKs may occur?

Answer 25

Activation by mutation - a mutation at the receptor that makes them always active

Activation by amplification - overexpression of the receptors and / or the ligands

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Question 26

What 2 classes of drugs are used on growth factor signalling pathways?

Answer 26

Enzyme inhibitors and antibodies

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Question 27

What is the mechanism of action of enzyme inhibitors used in growth factor signalling, and what can they be used to treat?

Answer 27

They bind to the ATP-binding site of the target kinase, preventing phosphorylation of the downstream kinases. Can be used to treat cancer and angiogenesis

(Slide 28)

Question 28

What are the mechanisms of action of antibodies used in growth factor signalling and what can they be used to treat?

Answer 28

They can prevent a ligand from binding to its receptor, bind directly to growth factors (preventing them activating the receptor) or induce internalization and degradation of growth factor receptor. Can be used to treat cancer, prevent angiogenesis or modulate the immune system

(Slide 28)

Question 29

How can growth factor receptors build up resistance to drugs used to treat cancer?

Answer 29

Answers Include:

Can have mutations allowing them to work in a ligand-dependent matter

Can acquire a mutation to make them resistance to a once effective drug

Can become resistant after cells undergo oncogenic shift

(Slide 32)