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Cell and Molecular > L18 - Receptor Tyrosine Kinases > Flashcards

L18 - Receptor Tyrosine Kinases Flashcards

1
Q

RTK overview

A

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

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2
Q

What are two examples of RTK ligands?

A

Insulin-like Growth Factor (IGF)

Epidermal Growth Factor (EGF)

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3
Q

What does EGF stimulate?

A

Stimulates proliferation of many cell types

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4
Q

What does IGF stimulate?

A

Stimulates cell growth and survival

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5
Q

What are the different types of extracellular domains of RTK?

A

Immunoglobilin like domain
Cysteine rich domains
Fibronectin types III like domain

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6
Q

The intracellular domains of RTKs have?

A

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

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7
Q

What is canonical RTK activation?

A

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

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8
Q

What is the role of kinase domains phosphorylating each other?

A

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

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9
Q

Tools for analysis of RTK signalling exploit?

A

Dimerisation

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10
Q

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

A

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

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11
Q

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

A

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

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12
Q

HSPGs protein core can either be?

A

Transmembrane
Tethered
Secreted

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13
Q

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

A

• Long chain of sugars attach to protein core – heparin

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14
Q

How can long chains of sugar in HSPGs be modified?

A

Can be modified by sulphation

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

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15
Q

What does FGF and its receptor form a complex with?

A

Form a complex with HSPGs

FGF first forms oligomers on HSPGs

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16
Q

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

A

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

17
Q

How does the phosphorylated receptor transduce the signal?

A
  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
18
Q

How do proteins recognise the docking sites on the RTL?

A

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

19
Q

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

A

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

20
Q

How does Ras function?

A

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

21
Q

Activation of Ras method

A
  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
22
Q

The mitogen activated protein kinase module

A

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

23
Q

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

A

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

24
Q

What are the 4 methods to study signalling?

A

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

25
Q

Visualisation or detection of interactions/signalling

A

Biochemical methods – columns, in vitro

In vivo - Fret

26
Q

Chemical inhibitors

A

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

27
Q

Misexpression/overexpression

A

Wild-type, constitutively active or dominant negative molecules

28
Q

Genetic methods

A

Forward/reverse genetics, transgenics, mutation analysis

29
Q

Fluorescence microscopy camera method

A
  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
30
Q

What type of camera is used in fluorescence microscopy?

A

Colour cameras are not sensitive - use grey scale cameras

31
Q

What are

fluorescent protein fusions used for?

A

Can excite protein and see where it is in the cell

Use excitation and emission wavelengths

32
Q

What is FRET?

A

Förster resonance energy transfer

33
Q

What is the FRET method?

A

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

34
Q

FRET - if individual proteins

A

Violet excitation of BFP = blue light emission

Blue light excitation of GFP = green light emission

35
Q

FRET - if no protein interaction

A

Violet excitation

No excitation of GFP = blue light detected

36
Q

FRET - if protein interaction

A

Violet excitation of compound

Fluorescence resonance energy transfer = green light detected

37
Q

What are the 3 uses of FRET?

A

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

Cell and Molecular (24 decks)

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