Lecture 3 - Introduction to signalling and cell surface receptors

Question 1

Paracrine, juxtacrine, and autocrine: what do they involve?

Answer 1

Paracrine - diffusible signal from an adjacent cell

Juxtacrine - signal molecule directly attached to an adjacent cell

Autocrine - signal arises from the responding cell itself (can be positive or negative feedback)

Question 2

What is the general signalling mechanism?

Answer 2

Extracellular stimulus interacts with receptor

Receptor activates intracellular pathways, which may include the nucleus, cytoplasm, proteins, etc

Intracellular pathways cause altered protein synthesis and/or cytoplasmic machinery

Altered PS/CM causes altered cell activity

Question 3

Signalling pathway components

Answer 3

• Extracellular stimulus - 100s in higher eukaryotes; mostly chemical; may act at very low concentrations (<10-8M)
• Receptor protein - High affinity for ligand, binding activates a cascade of intracellular events
• Signalling machinery - Multiple molecular changes: relay and amplify information
• Effectors - Produce the response
• ‘Off’ switch - Mechanism for restoring basal activity once stimulation ceases

Question 4

Cell surface receptors

Answer 4

• Ligand-gated ion channels (nicotinic acetylcholine/GABAₐ receptor)
• G-protein coupled receptors (muscarinic acetylcholine receptors, adrenoreceptors)
• Enzyme-coupled receptors ()PDGF, insulin, and growth hormone receptors

Question 5

Nicotinic receptor: what is its ligand, what movement does it facilitate, and what does it do?

Answer 5

Ach

Na+ entry into the cell

Induce an action potential in neurones

Question 6

Enzyme-coupled receptor: what is its ligand, what activity does it facilitate, and what types are there?

Answer 6

Ligand binding activates enzyme activity inside the cell

Enzyme activity may be a feature of the receptor molecule itself, or of a distinct protein with which it is closely associated.

Multiple types - receptor tyrosine kinases, receptor threonine/serine kinases, protease-linked receptors.

Question 7

Receptor Tyrosine Kinases (RTKs): what do they do, what are some key features, and what do they do?

Answer 7

Membrane enzymes that phosphorylate proteins on the side chains of tyrosine

• Span the membrane once
• Contain a cytoplasmic tyrosine kinase domain (active site) - receptor and enzyme in one
• Most (EGF, PDGF, etc) dimerise due to aid from mobile monomers
• Dimeric structure is essential for activation

Activation of receptor switches on kinase activity – leading to phosphorylation of the receptor and of target proteins on tyrosine residues

Question 8

Why is RTK dimerisation essential for function?

Answer 8

• Dimerisation brings the active sites of the monomers close together
• The monomers do trans autophosphorylation - each monomer phosphorylates the others’ activation loops
• Tyrosine kinase activation increases significantly - the receptor activates more and more tyrosine residues throughout itself
• Binding proteins are recruited to the phosphotyrosines and may be phosphorylated

Question 9

Dynamic RTK recruitment: what does it do, how do recruited proteins recognise the RTK, what proteins are typically recruited, and what do they do?

Answer 9

Enhances efficiency - brings pathway components together and may bring enzymes close to their substrates

Recruited protein recognises specific phosphotyrosine residues – amino acid sequence context

Multiple effectors recruited - signal bifurcation (transduce the signal further) occurs

Question 10

Signalling adaptor proteins: what are they, what do they do, what examples are there, and what do they do?

Answer 10

Not enzymes - no intrinsic activity

• Bind signalling proteins via various interaction domains
• Help assemble multi-protein complexes at activated receptors

IRS-1: major substrate of the Insulin receptor, recruited via PTB domain - its tyrosine phosphorylation recruits further proteins

SHC: has both PTB and SH2 domains - recruited to activated Insulin Receptor

Grb2: recruited to activated EGF receptor and to Y-phosphorylated SHC via SH2 domain

Question 11

Grb-2: how is it recruited to RTKs, what other domains does it have, and what does it do?

Answer 11

SH2 domain recruits itself to activated RTKs (e.g. EGF receptor)/other Y-phosphorylated proteins

2 SH3 domains - bind proline-rich peptides

Recruit Sos to the membrane, Sos activates ras, a small GTPase

Question 12

Small GTPases: what are they, what are they activated by, what do they do, and what forms of it exist?

Answer 12

GTP binding proteins – they bind the guanine nucleotides GDP and GTP

Guanine nucleotide exchange factors (GEFs) cause the exchange of GDP for GTP – switching the protein on

Intrinsic GTPase activity of the protein hydrolyses GTP to GDP – switches the protein off (happens slowly; speeded up by GTPase Activating Proteins (GAPs))

Ras (3 isoforms, K, H and N Ras) frequently activated by RTKs

Question 13

Ras: where is it found, what is it activated by, and how many SOS molecules activate Ras?

Answer 13

Attached to the membrane by a lipid anchor

Activated by Sos (son of sevenless), recruitment of SOS to the membrane (via Grb2) allows it to access Ras; Ras activated: GDP-GTP exchange

1 SOS may activate multiple Ras molecules - amplification

Question 14

MAPK cascade: what is it, what does it consist of, what does it cause to occur, and is it an example of amplification?

Answer 14

Powerful and ancient signalling pathway: thought to be present in all eukaryotes

Consists of 3 different protein kinases that are activated sequentially:
* When activated, K1 phosphorylates and activates K2
* K2 then phosphorylates K3 in the cascade, again causing its activation
* K3 phosphorylates multiple target proteins

Sequential phosphorylation of kinases in this way = ‘kinase cascade’

Can provide significant signal amplification

Question 15

‘The MAP kinase cascade’

Answer 15

General format:

Raf activates MEK on its serine/threonine residues, MEK activates ERK1/ERK2 on its tyrosine/threonine residues (dual specificity - normally only either tyrosine or ser/thr activated but MEK can do both)

ERK1/ERK2 will then go on to

Question 16

What are MAPK, MAP2K, and MAP3K and what examples of them are there?

Answer 16

MAPK - Mitogen-activated kinase (ERK1/2)

MAP2K - Mitogen-activated kinase kinase (MEK)

MAP3K - Mitogen-activated kinase kinase kinase (ERK1/2)

Question 17

Role of the Membrane in MAPK activation

Answer 17

Ras is associated with the membrane due to its lipid anchor, Ras GTP recruits Raf to the membrane

Membrane targeting is an essential part of activation

Question 18

MAPK module protein scaffolding

Answer 18

To prevent inappropriate activation of the wrong module, since there are several modules used for different pathways that have some overlapping activators, some kinases are immobilised on protein scaffolds

This increases efficiency by co-localising the correct proteins, but reduces amplification - some freely-diffusible steps still needed in the pathway

Question 19

PI3K: what is it, what does it do, and what are the reactions involving it?

Answer 19

Phosphoinositide 3-kinase

Phosphorylate headgroup of inositol lipids at position 3 of the inositol ring

Phosphatidylinotisol (4,5) bisphosphate (PI(4,5)P₂) + PI3K -> Phosphatidylinotisol (3,4,5) trisphosphate (PI(3,4,5)P₃)

TL;DR: PIP₂ + PI3K -> PIP₃

Question 20

PI3K class IA: what are they, what do they do, where do they function, what classes are there, what are the classes activated by, and what subunits do they have?

Answer 20

Phosphoinositide 3-kinase class IA

Phosphorylate headgroup of PI(4,5)P₂, producing PIP₃

Recruited to the membrane where the substrate is located

• Class IA (activated by RTKs/Ras-GTP)
• Class IB (activated by heterotrimeric G proteins/Ras-GTP)

Heterodimers: 1 catalytic and 1 regulatory subunit

Question 21

Class IA PIP2(?): what are its subunits, how big are its subunits, what do these subunits do, and what is its secondary messenger?

Answer 21

Class IA: 110kDa catalytic subunit (p110 a, b or d) smaller regulatory subunit (p85 / p55).

The regulatory subunit has an SH2 domain that mediates interaction with phosphotyrosines on activated RTK or signalling adaptor protein.

Product (PIP₃) is a second messenger

Question 22

Secondary messengers: what are they and what key features are there of them?

Answer 22

Small, intracellular, non-protein molecules that rapidly increase during stimulation and allow for signal amplification

• Typically low concentration in unstimulated cells – rapidly destroyed or removed
• May diffuse rapidly.
• Levels drop quickly once stimulation ceases.
• Interact with specific binding proteins – recruitment and/or allosteric activation
• Often promote protein phosphorylation (direct or indirect activation of protein kinases)

Question 23

PIP3: where is it found, where is it generated, what do downstream binding proteins contain, how is it removed, and in what ways are they removed?

Answer 23

Restricted to the inner surface of the plasma membrane; some mobility in 2 dimensions

Generated at specific locations near sites of receptor activation

Many downstream binding proteins: possess PH or PX domains

Binding proteins recruited to the plasma membrane: a platform for signal organisation

Rapidly removed by dephosphorylation:
* PIP₃ + PTEN -> PI(4,5)P₂
* PIP₃ + SHIP -> PI(3,4)P₂

• PTEN - removes phosphate at position 3
• SHIP - removes phosphate at position 5

Question 24

PI3 kinases

Question 25

Akt

Answer 25

A protein kinase activated downstream of PIP3

S/T kinase

Also known as Protein Kinase B (PKB)

First identified as an oncogene: inappropriate activation of PIP3 / Akt pathway in majority of cancers – reduces apoptosis in cancer cells

Contains a PH domain – binds to PIP3 when it is produced in the membrane

Following recruitment to the membrane, Akt is phosphorylated twice to activate it fully (by PDK1, another PIP3 binding protein and mTORC2).

Stays active until dephosphorylated by phosphoprotein phosphatases

Question 26

Cell signalling: the physiological importance

Answer 26

Essential for survival

Coordination of cells/tissues in multicellular organisms

Stimulation can have powerful effects on cells (and by extension, the whole body), including driving cell division and preventing apoptosis

Need for BALANCE: responses need to be robust, appropriate and optimal; dysfunction of signalling leads to disease

Question 27

Signaling errors/disease

Answer 27

Loss of sensitivity/lack of activation - cells do not respond even if appropriate (e.g. non insulin-dependent (type 2) diabetes), often due to malfunction or loss of receptor or signalling intermediate

Inappropriate activation - Process activated in the absence of stimulus (e.g. cancer), may be due to overexpression, activating mutations or ‘off switch’ malfunction

Question 28

Insulin resistance

Answer 28

Signals from insulin receptor bifurcate – it activates multiple pathways including Ras/MAP kinase and PI 3-K

Insulin resistance: selective loss of IRS signalling.

Multiple S/T phosphorylation of IRS. Glucose regulation lost.

Occurs in obesity, sepsis, type 2 diabetes; risk factor for AD, cardiovascular disease, some cancers, pancreatitis

~25% of adults worldwide have ‘metabolic syndrome’ (includes insulin resistance) (Medscape) – nearly 1.3bn people

Question 29

Over-active RTKs: Gastrointestinal stromal tumours

Answer 29

GIST: Rare tumour (<1% of all GI tumours)

Poor prognosis: insensitive to conventional chemotherapy or radiation

Mutations: increased activity in RTKs:

c-kit (80%)

Platelet derived growth factor receptor A (10%)

Increase in tyrosine phosphorylation and cell division

Imatinib (Gleevec ®):

Answer 30

Imatinib (Gleevec ®):

Specific inhibitor of certain tyrosine kinases (including Kit and PDGFR)

revolutionised GIST treatment

Question 31

PIP3 and disease

Answer 31

Major role in cell survival – elevation in tumour cells makes them resistant to apoptosis:

> 50% of human cancers have elevated PIP3

PIK3CA gene (encodes for a Class 1A PI3K catalytic subunit) is the most frequently mutated gene in solid tumours – over-activation of the kinase / amplification of wild-type

Isoform specific PI 3-K inhibitors now being used for some advanced breast cancers and types of leukaemia

Many potential drugs withdrawn, often due to toxicity.

Question 32

Signal termination mechanisms

Answer 32

Important that cells only produce a response when appropriately stimulated.

Phosphorylated proteins or lipids dephosphorylated by phosphatase enzymes.

Second messengers are destroyed or removed

Receptors can desensitize and may be removed from the membrane by endocytosis (although some can still signal from intracellular compartments)

Balance between signalling and termination ensures that responses are optimized.

Impaired signal termination can cause disease.

Question 33

Small GTPases and disease: Ras and tumours

Answer 33

Small GTPases inactivate themselves by GTP hydrolysis

Mutations reduce the GTPase activity of Ras – loss of ‘off switch’

Frequent in cancer: K-ras is mutated in 30% of human tumours (90% of pancreatic tumours)

GAPs increase GTPase activity of Ras: important for inactivation

Cell-free system – t1/2 1-5h

Inject GTP-ras into oocyte – all GTP hydrolysed in 5min

Ras GAPs recruited by activated RTKs: help to down-regulate signalling; mutations can lead to Ras overactivity

Question 34

Ras-GAPs and disease: Neurofibromatosis type 1

Answer 34

Multiple neurofibromas (benign growths arising from Schwann cells) develop under the skin and in the nervous system

Predisposition to cancers

Affects ~1 in 3000 people

Caused by mutations in the Ras GAP NEUROFIBROMIN (NF1): reduced GAP activity

Over-active Ras (and consequently MAPK & PI 3-kinase) signalling

Question 35

PIP3 removal and disease

Answer 35

Reminder: PIP3 removed by lipid phosphatases

SHIP-1: haematopoetic cells. Reduced activity associated with leukaemia

PTEN

Tumour suppressor: reduced activity predisposes to tumours.