Cells To Organisms Class > Lecture 6 > Flashcards

Lecture 6 Flashcards

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1
Q

What are receptor tyrosine kinases (RTKs)?

A

cell-surface receptors with intrinsic tyrosine kinase activity.

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2
Q

What types of signals use RTKs?

A

Many different extracellular signalling molecules.

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3
Q

How are RTKs activated in general?

A

By ligand-induced dimerisation.

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4
Q

What two stages are required for RTK activation?

A

Dimerisation followed by autophosphorylation.

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5
Q

What do RTKs link too?

A

They directly link the cell surface to an intracellular enzyme.

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6
Q

What happens during RTK autophosphorylation?

A

The receptor phosphorylates its own tyrosine residues.

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7
Q

What effect does autophosphorylation have on RTK activity?

A

It increases kinase activity.

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8
Q

What sites does autophosphorylation create for signalling?

A

It creates binding sites for adaptor proteins.

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9
Q

When can adaptor proteins bind an RTK?

A

Only after tyrosine residues are phosphorylated.

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10
Q

What types of proteins bind activated RTKs?

A

Adaptor proteins, enzymes, and proteins that modify lipids.

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11
Q

what is dimerisation

A

small similiar molecules bond snd form a larger molecule

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12
Q

Is RTK dimerisation alone sufficient for signalling?

A

No, ligand binding is also required.

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13
Q

Why was the insulin receptor used to test RTK dimerisation sufficiency?

A

It exists permanently as a dimer via disulphide bonds.

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14
Q

What did hybrid RTK–insulin receptor experiments show?

A

Dimerisation alone does not activate signalling.

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15
Q

What conclusion was drawn from hybrid receptor experiments?

A

Ligand occupancy is required for RTK signalling.

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16
Q

What experimental technique demonstrated EGFR dimerisation?

A

Single-molecule FRET imaging.

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17
Q

What does FRET stand for?

A

Fluorescence Resonance Energy Transfer.

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18
Q

Why is FRET useful for detecting dimers?

A

It only occurs when fluorophores are very close together.

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19
Q

What fluorescent ligands were used to study EGFR dimerisation?

A

EGF-Atto532 (green) and EGF-Cy5 (red).

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20
Q

What happens when EGFR forms a dimer with green and red ligands?

A

FRET occurs between the dyes.

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21
Q

What happens to donor fluorescence during FRET?

A

Green fluorescence decreases.

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22
Q

What happens to acceptor fluorescence during FRET?

A

Red fluorescence increases.

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23
Q

What did FRET imaging show about EGFR dimers localisation?

A

EGFR dimers are found at the membrane.

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24
Q

What important protein interacts with RTKs downstream?

A

The small GTPase Ras.

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25
What adaptor proteins allow RTKs activate Ras?
Through the adaptor protein Grb2.
26
What protein does Grb2 recruit to activate Ras?
SOS (Son of Sevenless).
27
Is Ras a kinase?
No.
28
What is Ras?
A small GTP-binding protein.
29
What does Ras do when activated?
Recruits and activates downstream signalling proteins at the membrane.
30
whats the process of GTP and GDP
GDP gets swapped with GTP which then scticates the protein, GTP is then hydrolysed to form GDP.
31
What are small GTPases?
GTP-binding proteins that act as molecular switches.
32
What molecules do small GTPases bind?
GTP and GDP.
33
When is a small GTPase active?
When bound to GTP.
34
When is a small GTPase inactive?
When bound to GDP.
35
How are small GTPases activated?
By exchanging GDP for GTP.
36
What causes the activity change in small GTPases?
A conformational change.
37
How do small GTPases terminate signalling?
By hydrolysing GTP to GDP.
38
Why don’t small GTPases stay permanently active?
They have intrinsic GTPase activity.
39
What protein enhances GTP hydrolysis?
GAP (GTPase-activating protein).
40
Why are GAPs necessary for the speed of GTPase
Intrinsic GTPase activity is very slow.
41
What protein promotes GDP–GTP exchange?
GEF (guanine nucleotide exchange factor).
42
What proportion of human cancers involve Ras mutations?
Approximately 50%.
43
What effect do Ras mutations have?
They lock Ras in the active form
44
Why are Ras mutations oncogenic?
They cause persistent growth signalling.
45
What domains does wild-type Ras contain?
P-loop, Switch 1, and Switch 2.
46
Which residue in Switch 1 is critical for Ras function?
Threonine 35.
47
Which residue in Switch 2 is critical for Ras function?
Glycine 60.
48
What do these residues coordinate?
The phosphate groups of GTP.
49
What happens when these residues are positioned correctly?
Ras activates downstream effectors.
50
What is the FGF family named after?
Fibroblast growth factor.
51
Do FGFs only stimulate fibroblasts?
No, they have many roles.
52
Give an example of an FGF mutation phenotype.
Dachshund body shape.
53
How many FGF ligands exist in humans?
22
54
What type of molecules are FGFs?
Peptide ligands.
55
What are the major functions of FGFs?
Cell growth, survival, differentiation, and embryonic development.
56
Are FGFs hydrophobic or hydrophilic?
Hydrophilic.
57
Why can’t FGFs cross the plasma membrane?
The membrane is hydrophobic.
58
How is FGF signalling received by cells?
Through cell-surface receptors.
59
Why are FGF receptors important in disease?
Dysregulation leads to cancer and developmental disorders.
60
What type of receptor is the FGF receptor?
A receptor tyrosine kinase.
61
What activity does the intracellular domain of FGFR have?
Tyrosine kinase activity.
62
What additional molecules are required for FGF binding?
Extracellular proteoglycans.
63
What happens after FGFR activation?
Intracellular phosphorylation cascades begin.
64
What major pathway does Ras activate downstream of FGFR?
The MAP kinase cascade.
65
Which kinases form the MAPK cascade? Do
Raf → Mek → Erk.
66
How does the cascade amplify the signal?
Each kinase activates many downstream molecules.
67
How fast is Erk activated after FGF stimulation?
Within minutes or seconds.
68
Where does activated Erk go?
The nucleus.
69
What does Erk do in the nucleus?
Phosphorylates transcription factors.
70
What is the result of Erk-activated transcription factors?
Gene transcription.
71
How many early response genes are activated?
At least 100.
72
How long does gene activation take after FGF stimulation?
30–60 minutes.
73
What is the central dogma of molecular biology?
DNA → RNA → Protein.
74
Why don’t mRNA and protein levels always correlate?
Differences in stability and regulation.
75
What example shows post-transcriptional regulation?
HIF-1α protein degradation in oxygen.
76
What technique separates proteins by size?
SDS-PAGE.
77
What does SDS-PAGE stand for?
Sodium dodecyl sulphate polyacrylamide gel electrophoresis.
78
What role does SDS play?
Denatures proteins and gives uniform negative charge.
79
What role does mercaptoethanol or DTT play?
Breaks disulphide bonds.
80
How are proteins separated in SDS-PAGE?
By molecular weight.
81
Why do smaller proteins migrate further?
They move more easily through the gel.
82
What stain is commonly used to visualise SDS-PAGE gels?
Coomassie Brilliant Blue.
83
What is the limitation of SDS-PAGE alone?
It does not identify specific proteins.
84
Why are Western blots used?
To detect specific proteins.
85
What is the first step of a Western blot?
SDS-PAGE.
86
What happens after electrophoresis in a Western blot?
Proteins are transferred to a membrane.
87
What membranes are used for Western blotting?
Nitrocellulose or PVDF.
88
What binds the protein of interest in a Western blot?
A primary antibody.
89
What binds the primary antibody?
A secondary antibody.
90
Why use secondary antibodies?
Signal amplification and cost efficiency.
91
How is the protein signal detected?
Enzymatic or fluorescent detection.
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