W8L1

Question 1

Signal transduction pathways are often portrayed as a linear set of reactions

Answer 1

Pathways however are not always linear:
they can converge, diverge or cross-talk

1. Pleiotropy: one signal can elicit multiple outcomes
   - GPCR activation may trigger multiple signalling cascades
   - e.g. in transcription, ion channel activity, cell proliferation, cell survival
2. Convergence: multiple signals can activate a common outcome
   - RTKs activate PI3 kinase alpha (PI3 kinase phosphorylates the #3 position on phosphatidylinositol in the PM; PI Kinase is dimer with catalytic and irregulatory domains, there are 4 different types of catalytic domains: PI K alpha, beta, gamma, and delta) via p85 (has SH2 recognition domain on it)
   - GPCRs activate PI3 kinase gamma via p101
   - p85 and p101 have different preferences for PI3K subtypes
   - In spite of functional overlap, RTK and GPCR signals may produce different outcomes

Question 2

Signal Integration

Answer 2

Protein Y is activated only when both sites are phosphorylated

Inhibiting one or the other signal may be sufficient to block biological function

Question 3

When Cell Communication Goes Wrong….

Answer 3

Normal blood sugar regulation.
(1) Food enters body
(2) Food broken down, sugar enters
bloodstream
(3) Sugar stimulates cells in pancreas to
release insulin
(4) Insulin travels through blood,
stimulates liver, muscle and fat to take up sugar for later use

When cell communication goes wrong…

Type I Diabetes - No insulin signal; unable to produce insulin signal [insufficient ligand]
Type II Diabetes - No response; do produce insulin, but target cells lose ability to respond to the insulin, leading to hyperglycemia and abnormal insulin secretion.

1. Losing the signal (Type I Diabetes)
   - In type I diabetes: pancreatic cells that produce insulin are lost; insulin signal is also lost: sugar accumulates to toxic levels in blood
   - Type I Diabetes tends to occur earlier in life
   - Without treatment, diabetes can lead to kidney failure, blindness and heart disease in later life.
   - Treatment: inject insulin [more ligand]
2. When Target Ignores the Signal (Type II Diabetes )
   - End result is the same: blood sugar levels become dangerously high
   - Type II Diabetes tends to occur to people later in life
   - One available treatment for Type II Diabetes is glucagon-like peptide 1 receptor agonists, which lower blood glucose levels

Question 4

Receptor families

Answer 4

A family - can be genetically related, can have characteristic domains that are the similar/the same/conserved within the family, repeats of the same domain that indicates a family, convergent evolution (does not need to be closely genetically related)

Receptor structure family:

1. Ligand-gated ion channels
2. 7TM receptors
   - GPCR
3. 1TM receptors
   - kinase-linked receptors
   - enzyme-linked receptors
4. Nuclear receptors (no TM domains)

Oligomerization is important for all classes of receptors

Question 5

Ligand-gated ion channels

Answer 5

• multiple subunits form a pore in the PM
• agonist binding triggers or activates opening
  – agonist is typically a nt
  – agonist binds the subunits within the ion channel that contains an agonist binding site, this causes an opening to allow ions to flow down their concentration gradients
• N-terminus and C-terminus can be on either side, depending on which family of ligand-gated ion channels
• between 4 to 5 TM domain and between 4 to 5 subunits

Question 6

7TM receptors

Answer 6

7TM receptors are GPCRs

• extracellular N-terminus (amino terminus), intracellular C-terminus (carboxy terminus)
• ligand binds to TM core or extracellular domain
• agonist / ligand causes conformational change of receptor, allowing it to activate the G protein and trigger intracellular signalling
• Intracellular domain typically couples to G proteins
• evidence shows that 7TM can form oligomerization dimers for larger aggregates but can also function as monomers in vitro; not known in vivo if preferential signalling is through monomeric receptors or multimeric receptors

Question 7

1TM receptors

Answer 7

• extracellular N-terminus (amino terminus), intracellular C-terminus (carboxy terminus)
• ligand binds to extracellular domain
• most have intracellular enzyme function
• its catalytic domain tends to be tyrosine kinase OR if there is no kinase, then the intracellular domain is able to recruit an intracellular kinase
• tends to form dimers in function

Question 8

Nuclear receptors (no TM domains)

Answer 8

• found in cytosol or nucleus
• nuclear receptors produce their effects in the nucleus, even though it does not necessarily need to reside in the nucleus all the time
• Not TM protein
• agonist-bound receptors function as dimers
• tends to function as dimers

Question 9

A Cell’s Response to a Signal Can Be Fast or Slow

Answer 9

How fast cell responds depends on the nature of the signalling.

Fast: < sec to minutes
i.e. altered protein function

Slow: minutes to hours
i.e. involving changes in gene transcription; new protein synthesis

End result of both fast and slow response:
- changes in cytoplasmic machinery (changes in protein content or protein function within the cell)
- change in cell behaviour

Question 10

Time frame of receptor signalling cascades

Answer 10

All receptors have the potential to initiate slow responses, e.g., protein synthesis

The follow list of 4 are their most immediate effects though:

1. Ligand-gated ion channels (ionotropic receptors)
   - time scale: milliseconds
   - examples: nicotinic, ACh receptor
   - important for neurons and neuromuscular junctions, where rapid response is required
2. G-protein-coupled receptors (metabotropic)
   - time scale: seconds
   - examples: muscarinic, ACh receptor
   - GPCRs can couple to ion channels, and increase or decrease ion channel activity
   - takes longer than ligand-gated ion channels because there are more biological steps. sometimes all the steps are close-by each other, but sometimes there are second messengers which require more time
3. Kinase-linked receptors
   - time scale: minutes (or hours)
   - examples: cytokine receptors
4. Nuclear receptors
   - these are transcription factors; their only effect is to alter the rate of gene transcription
   - time scale: hours
   - examples: oestrogen receptor

All 4 families have potential to impact gene transcription and protein synthesis - they can all produce slow effects

Question 11

Nuclear Receptors

Answer 11

Produces function in the nucleus

Intracellular receptors

Some signalling molecules [ligands] diffuse across membranes and bind to receptors in either cytosol or nucleus

Ligands are hydrophobic (lipid soluble)

If receptor is in cytosol, receptors translocate to the nucleus after ligand binding

Many diverse ligands, but have similar mechanism of action – regulate gene transcription

Question 12

Nuclear receptor types

Answer 12

Type I –Steroid Receptors
▪ bind to steroid hormones
▪ cytoplasmic - reside in cytoplasm
▪ upon activation: receptors will dimerize, the chaperon protein will dissociate to expose a nuclear localization sequence on the protein, and it will move into nucleus
▪ in nucleus, it binds to DNA, and then produces its effects
▪ forms homodimers aka homomers

Type II –RXR heterodimers
▪ e.g., thyroid hormone Receptor, retinoic acid Receptor (RAR, RXR), PPARs
▪ predominantly in nucleus
▪ heterodimerize with retinoid X receptor (RXR)

Type III
▪ similar to Type I

Type IV
▪ function as monomers or homodimers

Question 13

Structure of nuclear receptors

Answer 13

3 domains (from N terminus to C terminus):
1. AF1
- transactivation
- contains binding sites for proteins that can regulate the activity of the receptor
2. DBD
- DNA binding domain
- can also contribute to receptor dimerization
- recognizes particular gene sequences, and is where the receptor binds to the chromosome
3. LBD
- ligand binding domain
- transactivation, transrepression, dimerization functions

Steroid receptor homodimers
- i.e. ER, GR
- 2 of the same dimers come together
- binds to palindromic sequences of DNA

RXR heterodimers
- i.e. PPARs/LXRs
- 2 different dimers come together
- binds to direct DNA repeats
- n=1; PPAR/RXR
- n=4; LXR/RXR

Question 14

Nuclear receptors - overview

Answer 14

*Ligands include steroid hormones, thyroid hormones, vitamin D and retinoic acid, as well as certain lipid-lowering and antidiabetic drugs.

*Receptors are intracellular proteins, so ligands must first enter cells.

*Receptors consist of a conserved DNA-binding domain attached to ligand-binding and transcriptional control domains.

*DNA-binding domain recognises specific base sequences, thus promoting or repressing particular genes.

*Pattern of gene activation depends on both cell type and nature of ligand, so effects are highly diverse.

*Effects are produced as a result of altered protein synthesis and, therefore, are slow in onset.

Question 15

Enzyme-linked receptors

Answer 15

3 main groups:
- Receptor tyrosine kinases
- Cytokine receptors lacking enzymatic activity so they have to recruit kinase
- Natriuretic peptide receptors with guanylyl cyclase activity so they form cyclic GMPs

The receptors function as dimers. All have an extracellular N- terminal binding site, intracellular carboxy terminus, and a single transmembrane domain

Question 16

Receptor tyrosine kinases (RTKs)

Answer 16

• agonist binding to extracellular domain of RTK causes two receptor molecules to associate into a dimer
• for some RTKs, a single agonist molecule binds to both receptors
  – sometimes, the 2 halves of the dimer will form a single binding site and bind to a larger agonist molecule
• dimer formation brings kinase domains of each cytosolic receptor tail into contact
• this activates kinases to phosphorylate adjacent tail on several tyrosines = trans-activation

Question 17

Transduction mechanisms of kinase-linked receptors: The growth factor (Ras/Raf/MAP kinase) pathway

Answer 17

A lot of receptor tyrosine kinases are growth factor receptors, named after the tissue that they were discovered in

1. Growth factor binds to extracellular receptor domain
2. Conformation change dimerization
3. Tyrosine autophosphorylation
4. Phosphorylation of Grb2
   - transphosphorylation
5. Activation of Ras GDP/GTP exchange
   - leads to signalling cascade (kinase cascade)
6. Change in gene transcription in nucleus

Question 18

Receptor tyrosine kinase heterodimerization allows functional flexibility

Answer 18

There are 4 different monomeric receptor species that are encoded by the genome. These can form either homodimers or heterodimers. They differ with activity and agonist preferences
- subtype 1 can form a 1-1 homodimer OR a heterodimer with subtype 2 (1-2 heterodimer)
- subtype 2 can stand on its own. subtype 2 has no activity of its own (does not signal on its on) but can heterodimer bind with other 3 subtypes
- there is 3-3 homodimer, 2-3 heterodimer, 4-4 homodimer, and 2-4 heterodimer

• Binding of epidermal growth factor (EGF) and related peptides to ErbB1, ErbB3 or ErbB4 leads to homodimer or ErbB2 heterodimer formation
• ErbB2 does not bind EGF peptides and is inactive by itself
• Heterodimers have greater activity
• ErbB3 homodimers are inactive but ErbB2/ErbB3 heterodimers are the most active species

Question 19

Transduction mechanisms of kinase-linked receptors. The cytokine (Jak/Stat) pathway.

Answer 19

Transduction mechanisms of kinase-linked receptors. The cytokine (Jak/Stat) pathway. Here, the receptor lacks kinase activity and must recruit one (JAK) for downstream substrate phosphorylation to occur.

The cytokine binds, leading to conformational change and binding of intracellular Jak (Janus kinase) to intracellular domain.
- Then, there is phosphorylation of receptor + Jak
- Then, there is recruitment of other proteins: Jak/Stat pathway

Jak/Stat pathway
- after phosphorylation of receptor + Jak, causing binding and phosphorylation of SH2-domain STAT protein
- leads to dimerization of Stat
- then enters the nucleus to lead to gene transcription (or gene transcription changes) in the nucleus

Question 20

A different kind of 1TM receptor: Natriuretic peptide receptor topology and ligand preferences

Answer 20

3 endogenous ligaments:
- ANP - atrial natriuretic peptide
- BNP - brain natriuretic peptide
- CNP - C-type natriuretic peptide

1. Ligand: ANP, BNP, CNP
   Receptor: NPR-C
   - has no intracellular function domain and is thought to be blind to endogenous agonists
2. Ligand: ANP, BNP
   Receptor: NPR-A (GC-A)
   - ANP and BNP activate NPR-A
3. Ligand: CNP
   Receptor: NPR-B (GC-B)
   - CNP activates NPR-B

Both NPR-A and NPR-B have intracellular kinase homology domain, which binds to dimerization domain, which binds to guanylyl cyclase domain (binds to GTP to form cyclic GMP)
- cyclic GMP is a second messenger (similar to cyclic AMP) and can activate things

Question 21

Enzyme-linked receptors - summary

Answer 21

• Enzyme-linked receptors all share a common architecture, with a large extracellular ligand-binding domain connected via a single α-helix to the intracellular domain.
• Receptors for various hormones (e.g. insulin) and growth factors are tyrosine kinases
• Cytokine receptors activate cytosolic kinases
• Signal transduction generally involves dimerisation of receptors, followed by (auto)phosphorylation of tyrosine residues
• Kinase-linked receptors govern cell growth and differentiation; they also act indirectly by regulating gene transcription.
• A few hormone receptors (e.g. NPR-A) have a similar architecture and are linked to guanylate cyclase.

Question 22

Ligand gated ion channels (ionotropic receptors)

Answer 22

Convert chemical signals into electrical ones

• Rapid signalling in electrical excitable cells – e.g. nerve cells, muscle, synaptic transmission
• ligand binding transiently opens or closes ion channel leading to brief changes in ion permeability across plasma membrane and excitability of target cell
• ligand-gated ion channels, like other types, are selective for which ions they allow across the plasma membrane
• can become refractory if activated too much and undergo conformational desensitization

Question 23

Ligand-gated ion channel structure

Answer 23

LGICs may be made up of both agonist-binding and non- binding subunits. The combination of subunits may vary, which can give rise to multiple receptor subtypes.
- they form pore that remains closed, unless activated by agonist

Many LGICs belong to the Cys-loop superfamily of transmitter-gated ion channels. These are either pentamers (5 subunit) OR tetramers (4 subunit), and each subunit is made up of 4TM-spanning subunits, where both the C- and N-termini are extracellular

▪ 5HT3 receptors
▪ nicotinic acetylcholine receptors
▪ GABAA receptors
▪ glycine receptors

The vast majority of ligand-gated ion channels are activated by endogenous agonists that are also able to activate distinct GPCRs - so nature conserves useful molecules

Ionotropic glutamate receptors:
- are tetramers where each subunit has an extracellular N-terminal domain, three transmembrane, a channel lining re-entrant ‘p-loop’ that pokes into the PM from the cytosol, and an intracellular C-terminal domain

P2X purinergic receptors:
- consist of trimers of 2 TM subunits, each with intracellular C- and N-termini and a large extracellular loop
- there are 6 TM domains within an entire P2X receptor

Question 24

LGIC function and ion selectivity

Answer 24

▪ GABAA and glycine receptors are anion-selective (chloride channels) and decrease excitability

▪ other LGICs are cation selective (Na+, K+ and Ca2+) and tend to be excitatory

▪ some isoforms show selectivity among cations, e.g., AMPA and kainate receptors are relatively impermeable to Ca2+ under normal conditions

▪ Channel opening in response to agonist binding can be sensitive to the number of binding sites occupied.

• Concerted channel opening:
  – there are multiple agonist binding domains within the oligomer, and all of the binding sites need to be occupied by agonists in order for the channel to open
• Subunit-specific channel opening:
  – can have partially opened channel, not all binding sites need to be occupied by agonists
  – conductance depends on how many of the binding sites are occupied
  – having incomplete occupancy results in channel partially open
  – having all binding sites occupied results in channel fully open