Cell Signalling IV

Question 1

What are enzyme-coupled receptors and what are they often associated with?

Answer 1

Enzyme-coupled receptors are transmembrane proteins with their ligand-binding domains on the outer surface of plasma membrane.

The cytosolic side is often associated with a kinase enzyme most common class of these proteins act through receptor tyrosine kinases

Question 2

How do receptor tyrosine kinases carry out their functions?

Answer 2

Binding of ligand to receptor activates tyrosine kinase domain on cytosolic part of receptor creating phosphotyrosine docking sites for intracellular molecules.

Question 3

What is a good example of a molecule that signals through receptor tyrosine kinases?

Answer 3

Insulin

Question 4

Do enzyme coupled receptors have 7 transmembrane domains like GPCRs?

Answer 4

No they typically only have a single transmembrane domain with the exception of insulin receptor which contains 3 subunits held together by disulfide bonds

Question 5

How are receptor tyrosine kinases activated and what are they like prior to activation?

Answer 5

RTKs exist as monomers where kinase domain is inactive

Binding of ligand brings 2 monomers together to form a dimer and so the 2 kinase domains phosphorylate each other and activate other domains.

Tyrosine phosphorylation also leads to phosphorylation of other tyrosine residues and generate docking sites for intracellular signalling proteins

Question 6

How does IRS1 (docking protein) transmit signal from intracellular domain of RTK?

Answer 6

Phosphotyrosine binding (PTB) domain binds to RTK and binds to other proteins such as Grb2 adaptor protein which then binds to sos and scaffold proteins (via sh3 domains) and to RTK (via SH2 domain)

Question 7

How do intracellular signals bind to each other and remain near the plasma membrane?

Answer 7

They bind to phosphoinositide docking sites which are in the plasma membrane

Question 8

How do cytosolic proteins bind to RTKs?

Answer 8

via SH3 or SH2 domains

Question 9

What does phospholipase C-gamma do?

Answer 9

Very similar function to phospholipase C-beta. They dock onto the phosphorylated RTK. They act as enzymes which have SH2 and SH3 domains

Question 10

What domain is required to bind to phosphorylated tyrosines?

Answer 10

SH2 domains.

Question 11

What are the common proteins that bind to RTKs at phosphorylated sites?

Answer 11

PI3-kinase

GTPase-activating protein (GAP)

phospholipase C-gamma (PLCgamma)

Question 12

What protein mediates signalling by most RTKs?

Answer 12

the GTPase superfamily Ras

Question 13

What does Ras do?

Answer 13

Functions as a molecular switch which is active with GTP bound and inactive when bound to GDP.

Question 14

What does Grb2 do?

Answer 14

It recognizes a specific phosphotyrosine, binds via SH2 domain, recruits Sos which stimulates inactive Ras

Question 15

What are the 2 types of regulatory proteins that control activity of monomeric GTP-binding proteins?

Answer 15

Guanine nucleotide exchange factors (GEFs)

GTPase-activating proteins (GAPS)

Question 16

What do the regulatory proteins that control activity of monomeric GTP-binding proteins do?

Answer 16

GEFs promote exchange of GDP for GTP

GAPS stimulate hydrolysis of GTP to GDP to switch off GTP-binding proteins

Question 17

How do RTKs activate Ras?

Answer 17

Ras-GEF (aka sos) is activated by Grb2. It does this by binding to the SH3 domains. Ras-GEF activates Ras by exchanging GDP for GTP which activates downstream signals.

Question 18

What does Ras do?

Answer 18

Ras and Rho relay signals from cell-surface receptors.

Question 19

How do GTP and GDP influence the action of Ras and other GTP-binding proteins?

Answer 19

Like other GTP-binding proteins Ras functions as a molecular switch active with GTP and inactive with GDP.

Question 20

How is Grb2 activated?

Answer 20

It recognizes a specific phosphotyrosine which it binds to via SH2 domain

Question 21

What does Grb2 do to Ras?

Answer 21

It recruits Ras-GEF (AKA Sos) which stimulates inactive Ras to become active Ras.

Question 22

How are active GTPases inactivated?

Answer 22

GAPS or GTPase activating proteins stimulate hydrolysis of GTP to GDP to switch off GTP-binding proteins.

Question 23

What do members of the Ras superfamily have in common?

Answer 23

The Ras and Rho family function from receptor tyrosine kinases

Question 24

How does Ras relay a signal?

Answer 24

Active Ras activates Raf (MAP kinase kinase kinase)

Raf then phosphorylates Mek (MAP kinase kinase)

Mek then phosphorylates Erk (MAP kinase)

Erk then phosphorylates Protein X and Y which change protein activity and transcription factors which affect gene expression.

Question 25

What does MAP stand for?

Answer 25

Mitogen-Activated Protein

Question 26

How does PI3 Kinase get activated?

Answer 26

It binds to a phosphorylated tyrosine via an SH2 group

Question 27

What happens when PI3K is activated?

Answer 27

Inositol phospholipid is activated by PI3K which provides it with a third phosphate group.

Phosphorylated inositol phospholipid binds to protein kinase 1 which then phosphorylates Akt

Protein kinase 2 then phosphorylates Akt which relays the signal.

Question 28

How do cytokine receptors alter gene transcription?

Answer 28

They use enzyme-coupled receptors which also associate with cytoplasmic tyrosine kinases.

Question 29

How do enzyme-coupled receptors alter gene transcription?

Answer 29

In the abscence of a bound ligand the JAK kinases are poorly active.

When ligand/cytokine binds a conformational change takes place to being JAK kinases close together and induces cross phosphorylation

Cross phosphorylation induces conformational change that activates kinases to phosphorylate other tyrosine residues in cytosolic domain of the receptor forming docking sites for STAT transcription factors.

STAT translocates to nucleus and induces gene transcription.

Question 30

How are STAT and JAK associated with changes in gene expression?

Answer 30

Phosphotyrosine residues on the receptor recruit STATs

Bound STAT is phosphorylated by JAK

Phosphorylated STATs dimerize

Dimerized STAT moves into the nucleus and binds DNA to activate target gene transcription

Question 31

How do RTKs and GPCRs activate signalling?

Answer 31

RTKs and GPCRs activate overlapping signaling pathways