Architectures of signalling pathways LECTURE 2

Major components of signalling processing pathways

1. Chemical signal→ pheromones, hormones, local hormones etc

2. Receptor→ G protein, tyrosine kinase-linked ion channel linked

3. Transducer→ G protein, non-receptor tyrosine kinases

4. Amplifier→ adenylate cyclase, phospholipase C

5. 2nd Messengers→ cAMP,iP3, Ca2+, DAG

6. Effectors→ Protein kinases, Ca2+ binding proteins, troponin C

7. Response elements→Enzymes, sec vesicles, contractile proteins, ion channels, transcription elements

8. Response→ Metabolism, secretion, contraction, excitbility, gene transciption, cell growth

Singalling processing pathways have 5 principles

1. Information transfer

2. Amplification

3. Down regulation

4. Heterogeneity and concept of diversity

5. Dependence on functional properties of membranes

1. Information transfer

two basic mechanisms:

1. Conformational coupling→ highly reversible (conformational change)

   • Chemical signal to receptor

   • Receptor→ G protein (con change)

   • G protein→ amplifier (con change)

   • 2nd messenger to protein kinase (conf change)

2. Covalent modification→highly reversible

   • protein kinase → proteine kinase

   • protein kinase→ response element

2. Amplification

• Hormones are present at low concentrations

  →have ability to amplify the initial signal

  • as much as 10^6

• Can be at a number of steps in the signalling cascade

One signal molecule→ multiple downstream proteins

3. Down regulation (reset)

Once the chemical signal is removed:

• need to return to unstimulated state

• reversal of each step of signal transduction cascade

e.g (with covalent modification seen above:

• phosphatases reverse the effect of kinase activity

• target protein in dephosphoylated state when no signal

If no down reg:

→ no point in signalling

→ cannot respond to new stimulus

4. Heterogeneity and the concept of diversity (catering specific pathways)

• many of the components come in multiple forms

• different signalling pathways can interact with each other

→ OVERALL: can mix and match and interact with each other to construct precise signalling pathways that the cell needs

5. Dependence on functional properties of membranes

• Most pathways are initially on the cell membrane

  → Membrane properties are important for this

  • fluidity

  • laterla mobility

  • asymmetry of membrane proteins

NOT IN STEROID HORMONES ACTION

• do not initiate signal at the cell membrane

Examples of signalling pathways

1. Steroid hormones→receptor is nuclear protein

2. Ion channel-linked receptors

Pathways that involve secondary messengers:

3. cyclic AMP signalling pathway

4. Phosphoinositide signalling pathway

5. Tyrosine kinase-linked receptors

Steroid hormone action e.g testosterone

1. Hydrophobic→ diffuse across the cytoplasmic membrane of target cell

2. bind reversibly to specific steroid hormone receptors in the cytosol

3. Causes displacement of an inhibitory protein from the receptor

4. Exposes the DNA-binding domain

Steroid hormone receptor:

1. DNA binding site→ Inactive when inhib protein complex bound (and stuck in cytosol)

2. Steroid/ligand site→ once bound→ inhib protein complex gone (DNA binding site exposed)→ can now move to nucleus

3. Transciption activating domain

4. hinge region

Receptor example→ forms a dimer

Steroid hormone action→ what happens to the hormone-receptor complex

1. enters the nucleus

2. directly regulates the transcription of specific primary response genes

   • encodes transciption factors

3. These TFs signals to activate secondary response genes

   → Amplifies the response and invokes real response

Because transcription is involved…

• This is a slow process

  → Used in development!\

  e.g used for athlete cheating

How is it down regulated?

1. Lower steroids

2. favours dissociation from receptor

3. No receptor in nucleus

4. No more primary or secondary transciption

Ion channel-linked receptors: What used by

• neurotransmitters

  → e.g acetylcholine

RAPID

Ion channel-linked receptors: E.g with ACh

1. ACh binds to sites on outside of the target cell

2. Induce conformational change

3. Allows Na+ entry

4. Membrane depolarisation

5. muscle contraction

How does this depolarisation happen?

• Both voltage gradient and concentration of Na+ act in the same direction

  → large net influx of Na+

  • up to 30 000 ions per channel per ms

How is pathway downregulated

1. ACh is hydrolyses by acteylcholineesterase

   • found in neuromuscular junction

2. Na+ must be pumped out by Na+-ATPases

The nicotinic ACh receptor

→ When no ACh→ 10% chance open

→ When 2 ACh→ 90% chance open

What is Curare?

• Antagonist of ACh

  • block the ACh receptor in the muscle cells

Antagonist

( blocks and no activation )

What is nicotine

• Stimulates the receptor

• in the absence of ACh

  → agonist

( binds and forms some sort of response )

Novichok

• Acetylcholineasterase inhibitor

→ No down regulation

continuous stimulation

Why are secondary messengers needed?

• The receptor not always directly involved in doing the response

• (As seen above)

• Help relay the signal in the cytosol

Examples of secondary messengers

What is Cyclic AMP

• synthesised from ATP by adenylate cylase

• secondary messenger

In mammals, the adenylate enzyme is embedded in the plasma membrane, what does this mean the pathway requires?

• a soluble messenger is required

• for it to communicate with cytosolic target enzymes

cAMP signalling pathway steps (Gs is used)→ e.g with adrenaline

1. chemical signal binds to membrane receptor with 7 transmembrane domains

2. induces a conformational change

3. Cytoplasmic domain III of the receptor activates→ G protein (Gs)

4. Adenylate cyclase (membrane bound) synthesises cyclic AMP from ATP (Amplification)

5. cAMP acts through protein kinase→ induce response e.g glycogen breakdown(Amplification)

ALSO

6. cAMP opens Na+ channels in sense organs

7. Membrane depolarisation and information transfer

What are these G-proteins?

• Common transducer

• anchored to the plasma membrane

  • ‘G’ due to ability to bind to GTP

G protein’s interactions with GTP/GDP

When bound to GDP:

• inactive

When bound to GTP

• active

Types of G proteins

Heterotrimeric

• Gs and Gq

Monomeric GTPases

• e.g Ras (used in tyrosine kinase pathway)

Why is it important to understand G proteins

• Half of all known drug molecules

  → work by targeting G-protein pathways!

How down regulated?

1. No adrenaline, no conf III change, no G protein activation no cAMP made

2. Need to break down cAMP

   • phosphodiesterase→ AMP

3. Need to rephosphorylate the Protein-P

   • Use phosphatase

4. Na+ needs to be pumped back out

Phosphoionsitide pathway: What are the key enzymes

Phospholipase C beta:

Cleaves phospholipid in membrane from fatty acid tail

• hydrolyses phosphatidyl inositl 4,5 bisphosphate (PIP₂)

→ to form

• inositol 1,4,5-trisphosphate (IP₃) (free to move)

• + diacyl glycerol (DAG) (stays in membrane)

also

PLC:

• plasma-membrane localised enzyme

Phosphoinositide signalling pathway (Gq is used): Smooth muscles→ Uses ACh→ has multiple receptors!

1. Chemical signal binds to membrane receptor with 7 transmembrane domain

2. induces conformational change

3. Cytoplasmic domain III of the receptor activates a G protein (Gq)

4. G protein activates phospholipase C (PLC)

5. PLC hydrolyses PIP₂ → DAG + IP₃

   • acts as 2’ messengers:

6. DAG→ stimulate effector protein kinase C (PKC)

   • → Response: metabolism, secretion, tasciption, proliferation

7. IP₃→diffuses into cytosol→ acts via an IP₃ receptor (IP₃-R) on ion channels

8. Releases Ca2+ sotred in endoplasmic reticulum

9. Internal Ca2+ signal→ further Ca2+ release (amplification!)

10. Ca2+ can act through a protein kinase

or

10. specific binding protein (calmodulin or troponin C)

    • → Induce contraction in muscle cells

      or

    • → act directly on ion channels→ influence excitability

Tyrosine kinase linked receptors: role

• important in cell proliferation

• system transfers information from cell surface→ cell nucleus

Protein phosphorylation cascade→ (no secondary messengers) →with growth factors

1. PDGF induces dimerisation of 2 PDGF receptors

2. 2 tyrosine kinase domains phosphoylates each other on specific tyrosine residues

3. Phosphylated tyrosine residues act as docking sites

4. Bind different amplifiers to form multi-molecular complex → spans several signalling pathways

5. One amplifier: PLC→ forms DAG and IP₃ (does the stuff from above…)

Phosphylated receptor also…

6. Attracts no. of proteins that regulate GTP-binding protein ras

What does the trans-phosphylation of the tyrosine kinase do

• Receptor has tyrosine kinase linked to it

• upon signal→ transphosphoylates

  → now makes sites availabel for ptoeins to bind

  • Other proteins now bind:

    • PLC (from before)

    • GAP and SOS (adaptors for ras)

What is ras?

Monomeric G protein

• e.g of molecular switch in a signalling pathway

How are monomeric G proteins maintained in a default ‘off’ state?

• GTPase-activating proteins (GAPs)

  • promote GTPase activity of monomeric G protein

How are monomeric G proteins switched on?

• Guanine nucleotide exchange factors (GEFs)

  • promote GTP binding

    → therefore the ‘on’ state

    ( coz GTP is bound)

How is ras activated

1. GDP bound→ ras inactive

2. How activated: Son of sevenless (SOS) exchanges GDP for GTP

3. turned off with GAP

Ras GTPase activity:

• has low GTPase activity

BUT

• can inactivate itself with the help of GTPase activating protein GAP

Because of the GAP

• Ras activation is shortlived

→ So needs to amplify the signal: phosphylation cascade

• raf1 (kinases) MAPKKK

• phosphylated MEK→ MEK-P (MAPKK)

• phosphorylates MAPK→ MAPK-P (mitogen activator protein kinase)

→ Inolved in cell proliferation and development response

• Transciptional changes!

Speed of this pathway?-

• Rapid activation of ras

• sustain phosphylation

• Causes slow (sustained) transciptional changes

Oncogenic ras?

• has point mutation

• causes loss of GTPase activity

  → permanently activated

(conformation change)→ if not happened→ massive downstream effects

30% of cancer due to ras mutation

How does (now actiavated) ras contribute to this pathway?

1. Activated rasGTP initiates protein phosphylation cascade

2. → causes phosphylation of mitogen actiavted protein kinase (MAP kinase)→ MAPK-P

3. MAPK-P translocates into the nucleus

4. phosphorylates transcription factors (TF)

5. Activated transcription factors (TF-P)→ initiate transcription of genes responsible for controlling cell proliferation

   • G phase cylins

Used for cell proliferation:

Used for development:

These protein adaptors have SH2 domains

• Recognise specific receptors

• only bind when tyrosine phosphylated

Overall different types of signalling organisation

1. intracellular receptors coupled with transcriptional control

2. plasma membane bound receptors which are ligans inotropic receptors

3. G-protein coupled receptors→ increase concentration of cytosolic second messengers

4. enzyme-linked receptors

Self assessment questions:

1. List five pathways used in intracellular signalling.

2. How are hormonal signals relayed to the nucleus?

3. How can ion channels act as receptors?

4. What is a second messenger?

• Amolecular that relays a signal from a receptor to to target molecules, aplifying and propogating the signal to coordinate a response

5. Name two types of G protein.