Cell Signalling

Question 1

Overview of the signalling pathway

Answer 1

1. Ligand
2. Receptor
3. Intracellular signalling proteins
4. Effector proteins
5. Functions (metabolic enzyme/gene expression/altered cell shape or movement)

Question 2

Cell Receptors

Answer 2

• Cell signalling is diverse
• Receptors are signal transducers that bind low concentrations of ligand with high affinity
• Bind hydrophilic ligands while small hydrophobic signals can pass through the bilayer directly

Question 3

Signalling Cascade

Answer 3

1. Primary Transduction
2. Relay (scaffold proteins)
3. Transduce and amplify
4. Integrate/spread/anchor/modulate
5. Effector protein activation
6. Gene transcription`

Question 4

Types of Receptors

Answer 4

Cell Surface Receptors

1. Ion channel coupled receptors
2. G protein coupled receptors
3. Enzyme coupled receptors

Nuclear Receptors
- bind to intracellular ligands to alter gene transcription like steroids or vitamins

Question 5

Phosphorylation Dependent Signalling Switch

Answer 5

• Serine/threonine kinases that phosphorylate molecules to create cascades (signal amplification)
  1. Signal in causes ATP hydrolysis so kinase phosphorylation
  2. Loss of P by protein phosphatase when signal leaves causes switch ‘off’

Question 6

GTP Binding Protein

Answer 6

1. Signal causes release of GDP and binding to GTP - switch ‘on’
2. GTP hydrolysis via GTPase activity when signal leaves causes GDP binding and inactivation

Question 7

Two Types of GTP binding proteins

Answer 7

1. G proteins (trimeric gtp binding proteins)

2. monomeric GTPase1

Question 8

G protein coupled receptor Signalling

Answer 8

• Single polypeptide folded into a globular shape and embedded in the cell membrane
• 7 looping segments forming a cylindrical structure
• GPCR interacts with g proteins.
• G protein has an alpha, beta, gamma subunit. The alpha unit binds to either GTP or GDP depending on active state
• Ligand binding leads to a conformational change activating a trimeric GTP binding protein coupling the receptor to a membrane channel

Question 9

GPCR Process

Answer 9

1. Ligand binding to GPCR causes a conformational change in a subunit that triggers GPCR and G protein interaction. GTP replaces GDP in the alpha unit.
   - GCPR acts as a GEF to make the alpha unit release its GDP
2. The G protein dissociates into the GTP bound alpha subunit and a beta/gamma dimer which diffuses laterally to interact with other membrane proteins
3. Activated complex regulates membrane target protein activity
4. When the GTP is hydrolyzed to GDP (alpha unit is a GTPase), the subunits become inactive and the GPCR associates with the g protein (molecular switch)
   - G proteins can affect ion channels in the membrane to alter ion permeability (eg. acetylcholine affecting heart rate)

Question 10

Regulating GTP binding proteins

Answer 10

GAP (gtp-ase activating proteins) = switch off
GEF (guanine exchange factors) = switch on
1. GEF exchanges GDP for GTP and activates monomeric GTPase
2. GAP hydrolyzes GTP so GDP binds again, leading to inactivated monomeric GTPase

Question 11

GTPase RAS

Answer 11

• GTPase switch
• Family of monomeric GTPases
• switches between 2 conformational states depending on whether GTP or GDP binds
• has covalently attached lipid groups anchoring protein to the cytoplasmic face of the membrane
• this change allows other proteins to distinguish between active/inactive RAS
• active RAS binds and activates downstream pathways.
  RAS Gef = uptake of GTP and activation
  RAS Gap = GTP hydrolysis and inactivation
• the indirect coupling of a self-phosphorylating RTK receptor to a RAS gef drives Ras into the active state.
  The short lived RAS signals must be converted to long ones to promote longer lasting effects, and this is done through the MAP kinase module.

Question 12

Cell Signalling

Answer 12

Receptors are often transmembrane proteins and a signal can affect different cells in different ways. This is because of differences in intracellular signalling proteins, effector proteins, activated genes, etc.
Induces cell response according to predetermined state.
Intracellular signalling proteins relay signals inside the cell and alter effector proteins that alter cell behaviour. They act like switches - go from an inactive to active state when signal is received.

Question 13

How to signals stay strong?

Answer 13

1. high affinity and specificity of interactions between signalling molecules and receptors
2. downstream target proteins can ignore background signals
3. robust signalling systems in the face of signal variability

Question 14

Signalling Complexes

Answer 14

Signalling molecules are often localised by scaffold proteins into scaffolding complexes that lead to higher local concentrations so are faster and more efficient.
Induced proximity = a signal triggers signalling complex assembly commonly used to relay signals along a pathway.
Induced proximity = a signal triggers signalling complex assembly/commonly used to relay signals along a pathway.
1. preformed signalling complex on scaffold protein
2. assembly of signalling complex on activated complex
3. assembly of signalling complex on phosphoinositide docking sides

Question 15

Adaptation

Answer 15

Enables cells to respond to changes in signal molecule concentrations over a wide range, ie. a strong signal response will modify the cell’s signalling machinery to make it less responsive to the same signal (negative feedback).
This can occur by receptor inactivation or pathway inhibitor production.

Question 16

GPCR desensitization

Answer 16

1. receptor sequestration (moved to cell interior)
2. receptor down regulation (destroyed in lysosomes)
3. receptor inactivation (altered to prevent interaction)
   Can be phosphorylated, and the receptors bind to the arrestin family. This prevents the receptor from interacting with G proteins.

Question 17

MAP Kinase

Answer 17

Active Ras phosphorylates Raf protein which phosphorylates MAP kkk, which phosphorylates MAP KK, which phosphorylates MAP K, which phosphorylates and inactivates Ras to shut off module.

Question 18

RAS and Cancer

Answer 18

Ras is the most frequent mutation found in cancers.
Specific mutations at codons 12,13,61 in the Ras genes are associated with tumorogensis.
Those mutations favor constitutive activation of Ras, meaning that the gene is always “turned on,” and there is overproduction of the protein. The mutation also increases GTP binding, leading to overactivity.

Question 19

RTK (receptor tyrosine kinases)

Answer 19

Ligand binding to domain activates tyrosine kinase domain on cytosolic side of membrane.
This leads to phosphorylation of tyrosine side chains on the cytosolic part of the receptor, creating phosphotyrosine docking sites for signal relay proteins.
Ligand binding causes the receptors to dimerize, bringing the two cytoplasmic kinase domains together and thereby promoting their activation - they phosphorylate each other.