Tyrosine-kinase associated receptors

Question 1

What is a tyrosine kinase associated receptor?

Answer 1

An enzyme coupled receptor where the kinase domain is non-covalently attached to the receptor.

Question 2

What is the name of the signalling pathway downstream of tyr kinase associated receptors?

Answer 2

The JAK-Stat pathway

Question 3

Give examples of tyr kinase associated receptors.

Answer 3

Cytokine receptors, TCRs, and TGF-β receptors.

Question 4

Give examples of signal proteins that activate the JAK-Stat pathway.

Answer 4

Interferons, erythropoeitin and growth hormone.

Question 5

Why is growth hormone considered a hormone despite acting via the JAK-Stat pathway?

Answer 5

Produced in the pituitary gland and distributed throughout the body. Stimulates the JAK-Stat pathway via the production of IGF1 which goes on to actviate a tyr kinase associated receptor.

Question 6

What is growth hormone used to treat?

Answer 6

Stunted growth and short stature.

Question 7

What is the outcome of the JAK-Stat pathway when activated by interferons? When are interferons used as a drug treatment?

Answer 7

Activation of macrophages and increased resistance to viral infection. Used in antiviral therapy, e.g. in hepatitis treatment.

Question 8

What is the outcome of the JAK-Stat pathway when activated by erythropoeitin?

Answer 8

Differentiation of erythorocyte progenitors to give erythrocytes (RBCs).

Question 9

Why is erythropoeitin taken by athletes?

Answer 9

To increase RBC production, in order to give them a higher oxygen capacity.

Question 10

What are cytokines?

Answer 10

Small proteins (15-25kDa) secreted by immune cells that are used to modulate different immune responses.

Question 11

What are chemokines?

Answer 11

A subset of cytokines that act as chemoattractants - result in the migration of immune cells to a site of infection.

Question 12

What are hormones?

Answer 12

Small organic molecules, peptides or proteins that are produced by endocrine glands and distributed throughout the body in the bloodstream. Have a wide variety of effects on target cells.

Question 13

What are growth factors?

Answer 13

Cytokines or hormones that stimulate cell growth, proliferation and differentiation.

Question 14

What is JAK?

Answer 14

Janus kinase - with two kinase domains, one of which is catalytically active. The second kinase domain negatively regulates the activity of the first kinase domain. Non-covalently associated to the receptor..

Question 15

What is Stat?

Answer 15

Signal transducer activated by JAK.

Question 16

Give an overview of the JAK-Stat pathway.

Answer 16

1. Cytokine (or hormone) binding results in dimerisation of the tyr kinase associated receptor.
2. Dimerisation allows cross phosphorylation of the JAK kinases bound to each receptor monomer.
3. Cross phosphorylation is recognised by Stat proteins, via their SH2 domains.
4. Phosphorylation of Stat proteins by JAKs.
5. Dimerisation of phosphorylated Stat proteins.
6. Stat dimer moves into the nucleus and activates transcription of the target gene - with a cytokine responsive element.

Question 17

Why is the JAK-Stat pathway a faster way to activate gene expression than the Ras-MAPK pathway?

Answer 17

Only has one intermediate - Stat - which goes straight into the nucleus to activate gene transcription. In comparison, the Ras-MAPK pathway has more intermediates and it takes longer for the information to reach the nucleus.

Question 18

What is the main disadvantage of the JAK-Stat pathway in comparison to the Ras-MAPK pathway?

Answer 18

The Ras-MAPK pathway is more complex (has more intermediates) and can therefore have more complex regulation. There are less levels of regulation in the JAK-Stat pathway.

Question 19

What did early studies using the growth hormone conclude about tyr kinase associated receptor dimerisation?

Answer 19

Suggested ligand induced dimerisation

Question 20

Describe the binding sites in a tyr kinase associated receptor.

Answer 20

The receptor is not symmetric and forms a 4 helix bundle - cannot have identical interfaces. One binding site is stronger than the other, this is where the ligand binds. The second binding site is weaker and is how one receptor binds to another receptor.

Question 21

What is the main function of dimerisation of tyr kinase associated receptors?

Answer 21

Allows cross phosphorylation of the JAKs bound to each receptor molecule.

Question 22

What do more recent studies of growth hormone receptor dimers?

Answer 22

Suggest that the receptor exists as a constitutive dimer - only parting in the extracellular domain. Ligand binding causes a conformational change in the cytoplasmic region of the receptor - allows pulling apart kinase domains.

Question 23

Describe the growth hormone receptor in the inactive state.

Answer 23

The pseudokinase domains block the kinase domains - unblocked when pulled apart upon ligand binding.

Question 24

Describe the use of a co-receptor by a JAK-associated receptor.

Answer 24

There is the JAK-associated signalling receptor and an interleukin - not involved in signal transduction but without it there is no signal output.

Question 25

What is Lck?

Answer 25

A Src family kinase - tyr kinase that is non-covalently bound to the coreceptor in the TCR complex (CD8/CD4)

Question 26

Describe the activation of the TCR complex.

Answer 26

1. Lck activation by TCR- co-receptor binding antigen-MHC complex on APC. 2. Lck phosphorylates tyrosine residues on the cytosolic region of the T cell receptor. 3. ZAP70 is recruited and uses its SH2 domains to bind the phosphorylated tyrosine residues (ITAM domains) on the T cell receptor. 4. ZAP70 is activated by phosphorylation catalysed by Lck. 5. Activated ZAP70 triggers the response within the T cell.

Question 27

What is caused by ZAP70 deficiency?

Answer 27

Immune deficiency

Question 28

Describe the kinetic segregation model of T cell receptor specificity.

Answer 28

Membrane bound phosphatase (CD45) removes phosphates from the TCR. With both dephosphorylation and phosphorylation occurring the overall phosphorylation level is low - no activation of the T cell. As the T cell and the APC come closer together, due to the MHC-TCR interaction, the large extracellular domains of the phosphatase don't fit into the narrow space between the two cells - pushed aside from the interface between the cells. Leads to an imbalance in phosphorylation - less dephosphorylation occurring, allowing prolonged phosphorylation, ZAP70 recruitment and T cell activation.

Question 29

What are integrins?

Answer 29

Receptors in the ECM - communicate between the actin cytoskeleton and the ECM.

Question 30

Describe the inactive conformation of integrins.

Answer 30

Bent over conformation, ligand binding domain inaccessible.

Question 31

Describe the active conformation of integrins.

Answer 31

Separation of the cytoplasmic domains, extracellular domains become upright.

Question 32

Describe outside-in activation of integrins.

Answer 32

Inducing the separation of the cytoplasmic domains by ligand binding on the outside e.g. typically happens in ECM, fibronectin binds to activate the integrin.

Question 33

Describe inside-out activation of integrins.

Answer 33

Inducing the separation on the inside of the cell to increase adhesion e.g. in blood cell coagulation, platelets become more adhesive because a signalling event inside the platelet switches on an integrin, causing it to bind to fibrin.

Question 34

Describe the signalling downstream of integrins in focal adhesions.

Answer 34

Focal adhesion kinase (FAK) phosphorylates stress fibres and Src kinase. This is a signal for survival that informs a cell that it is attached to the ECM and can continue to grow. If a cell were to separate from the ECM, it would undergo apoptosis – specifically called anoekis.

Question 35

What are focal adhesions?

Answer 35

The point at which stress fibres terminate at plasma membrane - where integrins connect the actin cytoskeleton to the ECM. Approximately 500 proteins are recruited into the focal adhesions - have layered assemblies of proteins.

Question 36

What is the role of focal adhesions in cell migration?

Answer 36

Cell pulls on focal adhesions to migrate, focal adhesions then disassemble behind the cell. Focal adhesions therefore must be able to withstand considerable force.

Question 37

What is super resolution microscopy and what has it been used for?

Answer 37

Modern technique that can distinguish proteins by only 10-20nm - has been used to identify proteins in focal adhesions.