L18 - Receptor Tyrosine Kinases

Question 1

RTK overview

Answer 1

16 families each with many individual receptors and their ligand partners
Ligands are sometimes specific for one receptor and vice-versa
High affinity or low affinity ligands

Question 2

What are two examples of RTK ligands?

Answer 2

Insulin-like Growth Factor (IGF)

Epidermal Growth Factor (EGF)

Question 3

What does EGF stimulate?

Answer 3

Stimulates proliferation of many cell types

Question 4

What does IGF stimulate?

Answer 4

Stimulates cell growth and survival

Question 5

What are the different types of extracellular domains of RTK?

Answer 5

Immunoglobilin like domain
Cysteine rich domains
Fibronectin types III like domain

Question 6

The intracellular domains of RTKs have?

Answer 6

Kinase activity
They have
- Enzyme-linked receptor
- Tyrosine kinase domain

Question 7

What is canonical RTK activation?

Answer 7

Ligand can dimerise and facilitates receptor dimerisation
- May also reorient existing receptor oligomers
Once positioned correctly, kinase domains phosphorylate each other

Question 8

What is the role of kinase domains phosphorylating each other?

Answer 8

Increases kinase activity
Stabilises receptor in the active state - ligand independent
Kinase phosphorylates other tyrosines in the receptor – creates docking sites

Question 9

Tools for analysis of RTK signalling exploit?

Answer 9

Dimerisation

Question 10

How can you use genetic engineering to create a dominant negative? - Analysis of RTK signalling

Answer 10

Using GE can generate DNA that encodes for a normal receptor that is mutated in the kinase domain
DNA is expressed in an organism at high levels and poisons endogenous receptor
Dominant negative - antimorphic

Question 11

How can you use genetic engineering to create a constitutively active? - Analysis of RTK signalling

Answer 11

Using GE can generate DNA that encodes for a receptor that lacks a ligand binding domain and instead has a homodimerisation domain
DNA is expressed in an organism at normal levels and is ligand independent
Constitutively active

Question 12

HSPGs protein core can either be?

Answer 12

Transmembrane
Tethered
Secreted

Question 13

Where do the long chains of sugar attach to in HSPGs?

Answer 13

• Long chain of sugars attach to protein core – heparin

Question 14

How can long chains of sugar in HSPGs be modified?

Answer 14

Can be modified by sulphation

Modification could result in a code that creates binding sites for specific proteins (e.g. FGF2)

Question 15

What does FGF and its receptor form a complex with?

Answer 15

Form a complex with HSPGs

FGF first forms oligomers on HSPGs

Question 16

What is the structure of the complex formed between FGF and HSPGs?

Answer 16

Proteoglycans form the protein backbone
Sugar chains hang off the extracellular space and interact with FGF ligands
- Recognise the modifications
Interaction helps form a complex with the receptor tyrosine kinases to allow activation

Question 17

How does the phosphorylated receptor transduce the signal?

Answer 17

1. Activated receptor recruits other intracellular signaling proteins to the membrane
2. These proteins bind to phosphorylated tyrosine - docking sites
   - Proteins have binding sites for phosphotyrosine and amino acid side chain
3. Activated signalling proteins relay signal downstream

Question 18

How do proteins recognise the docking sites on the RTL?

Answer 18

SH2 domain in Src recognizes this short phosphopeptide
It specifically recognises
- Phosphotyrosine – glutamic acid – glutamic acid – isoleucine

Question 19

What is an example of how phosphorylated receptor transduces the signal?

Answer 19

GTPase-activating protein activates RAS/MAP kinase
PI 3-kinase activates inositol lipid pathway
PLC-gamma activates inositol lipid pathway
- All 3 have SH2 and SH3 domains
RAS and PI3K are the two main branches in the RTK signalling pathway

Question 20

How does Ras function?

Answer 20

Ras is a superfamily of monomeric GTPases
Ras acts as a GTP switch
- When bound to GTP – active
- When bound to GDP – inactive
- GTP is stable for a certain amount of time before it hydrolyses to GDP

Question 21

Activation of Ras method

Answer 21

1. Binding of GRB2 and Sos couples receptor to inactive Ras
2. Sos promotes dissociation of GDP from Ras
3. GTP binds and Sos dissociates from active Ras

Question 22

The mitogen activated protein kinase module

Answer 22

Ras activates a signalling cascade starting with Map kinase kinase kinase
Final Map kinase is involved in changing protein activity and gene expression

Question 23

What are the effect of the mitogen activated protein kinase module?

Answer 23

Leads to amplification of signal
A rapid and transient response
- Phosphorylated tyrosines are not very stable

Question 24

What are the 4 methods to study signalling?

Answer 24

Visualisation or detection of interactions/signalling
Chemical inhibitors
Misexpression/overexpression
Genetic methods

Question 25

Visualisation or detection of interactions/signalling

Answer 25

Biochemical methods – columns, in vitro | In vivo - Fret

Question 26

Chemical inhibitors

Answer 26

To disrupt signalling by manipulating a particular component - agonist vs antagonist

Question 27

Misexpression/overexpression

Answer 27

Wild-type, constitutively active or dominant negative molecules

Question 28

Genetic methods

Answer 28

Forward/reverse genetics, transgenics, mutation analysis

Question 29

Fluorescence microscopy camera method

Answer 29

1. Light travels through excitation filter 2. Light is then reflected by a dichroic mirror onto a specimen - Dichroic mirrors only reflect shorter wavelengths 3. Emitted fluorescence light is reflected from specimen and travels through dichroic mirror 4. Barrier filter only allows light of one wavelength into the eyepiece - emitted light is recorded

Question 30

What type of camera is used in fluorescence microscopy?

Answer 30

Colour cameras are not sensitive - use grey scale cameras

Question 31

What are | fluorescent protein fusions used for?

Answer 31

Can excite protein and see where it is in the cell | Use excitation and emission wavelengths

Question 32

What is FRET?

Answer 32

Förster resonance energy transfer

Question 33

What is the FRET method?

Answer 33

Different fluorescent protein (GFP and BFP) is attached to different proteins

Question 34

FRET - if individual proteins

Answer 34

Violet excitation of BFP = blue light emission | Blue light excitation of GFP = green light emission

Question 35

FRET - if no protein interaction

Answer 35

Violet excitation | No excitation of GFP = blue light detected

Question 36

FRET - if protein interaction

Answer 36

Violet excitation of compound | Fluorescence resonance energy transfer = green light detected

Question 37

What are the 3 uses of FRET?

Answer 37

Used to study when two proteins come together you stop getting a certain emission Used to study conformational changes of a protein - When it goes through a change it may bring to sub-units together Used to study when a protein is cleaved - When cleaved GFP and BFP are separated - Different light emitted