Signal Transduction

Question 1

Describe paracrine signalling and give an example.

Answer 1

Paracrine signalling (neurotransmitters) - signalling molecules only affect target cells in close proximity to releasing cells.

Contact-dependant - signal doesn’t travel at all, based on the contact between the signalling cell and target cell.

This is recognized by a receptor exposed on the membrane of the target cell. This is typical of antigen presenting cells.

Question 2

Why do receptors have to be so specific in the way they bind?

Answer 2

Because receptors can bind at high affinity even if there is a low concentration of molecules.

Question 3

What are cell surface receptors?

Answer 3

Receptors that binds ligands which are represented by hydrophilic signalling molecules. They will never be able to enter the cell so needs to bind to an extracellular receptor.

Question 4

What are intracellular receptors?

Answer 4

Receptors that bind ligands which are hydrophobic because they can pass membranes without aid of extracellular receptors.

Question 5

Why is the type of receptor also important in cell transduction?

Answer 5

Because the same type of signal can cause different cells to respond in different ways.

Therefore cell response not only depend on the signals, but also the type of receptor and intracellular mediator.

Question 6

What are the 3 major classes of cell surface receptor proteins?

Answer 6

Ion-channel-coupled receptors
G-protein-coupled receptors
Enzyme-coupled-receptors

Question 7

Describe how ion-channel-coupled receptors work.

Answer 7

Also called transmitter-gated ion channels or ionotropic receptors.

They are involved in rapid signalling, i.e. synaptic signalling which is mediated by a small number of neurotransmitters that can transiently open or close protein ion channel, the ion permeability of PM is changed by binding.

Question 8

Describe how G-protein-coupled receptors work.

Answer 8

Coupled to a GTP binding protein (G protein) this is needed to mediate interaction between receptor and target protein.

An extracellular signal changes the conformation of GPCR, triggering its interaction with a G protein.

The activation of a G protein changes concentration of one or more small intracellular signalling proteins.

Question 9

Describe how enzyme-coupled receptors work.

Answer 9

Also know as catalytic receptors, they are usually single-pass transmembrane receptors.

Has 2 sides - an extracellular face and internal face. The binding of a ligand to the extracellular face causes enzymatic activity on the internal face.

Question 10

What are intracellular signalling molecules?

Answer 10

Intracellular signalling molecules can either be small molecules or other proteins. When they are small molecules, they are often called second messengers.

They are small and hydrophobic, therefore easily diffuses in the cytoplasm.

Other lipid based second messengers which can diffuse in the channels of the PM. Pass signal on by binding to and altering behaviour of selected signalling or effector proteins.

Question 11

What are characteristics of GTP binding proteins?

Answer 11

• Switch between ‘on’ state when GTP is bound and ‘off’ state when GDP is bound.
• Can shut themselves off when hydrolysing GTP.
• 2 major types: Large trimeric GTP-binding proteins (G proteins) help relay signals from G-protein-coupled receptors that activate them.
• Small monomeric GTPase (monomeric GTP-binding proteins) help relay signals from many classes of cell-surface receptors.

Question 12

How are GTP binding proteins regulated?

Answer 12

• GTPase-activating proteins (GAPs) - drives proteins into an ‘off’ state by increasing rate of hydrolysis of bound GTP.
• Guanine nucleotide exchange factors (GEFs) - activates GTP-binding protein by promoting release of bound GDP, which allows new GTP to bind.

Question 13

How do cells avoid unwanted cross-talks and interference between signalling systems?

Answer 13

• High affinity and specificity

- Many downstream target proteins have the ability to ignore other signals.

Question 14

Describe the structure of G-protein-coupled receptors.

Answer 14

• 1 polypeptide chain that threads back and forth seven times across the lipid bilayer.
• Cylindrical structure.
• Often deep ligand-binding site at centre.
• All use G-proteins to relay signal into the cell interior.

Question 15

Describe the activation of G proteins.

Answer 15

Extracellular signal arrives.

By binding to the receptor, signal changes conformation of receptor and thus the G-protein, which can recognise and bind to the receptor.

This binding by alpha subunit opens AH domain where GDP was contained.

GDP released.

Beta and Gamma subunit dissociates and everything’s activated.

Question 16

How do G-protein-coupled receptors regulate the concentration of cAMP?

Answer 16

When an extracellular signal arrives and binds to a receptor, this can be translated very quickly resulting in rapid increase in cAMP.

It needs to be synthesised and removed very quickly.

Adenylyl cyclase is the enzyme for synthesis, while cyclic AMP phosphodiesterases is for breakdown.

Most isoforms of adenylyl cyclase are regulated by Ca2+ and G proteins.

Extracellular signals activate GPCRs that are coupled to stimulatory G proteins (Gs). Activated alpha subunit of Gs binds and activates adenylyl cyclase.

Opposite signals act through different GPCRs can reduce cAMP levels by activating an inhibitory G protein (Gi), which inhibits adenylyl cyclase.

Question 17

Give examples of toxins that can disrupt the regulation of cAMP.

Answer 17

Cholera toxin alters alpha subunit causing it to no longer hydrolyze bound GTP and stay active. Consequently high cAMP concentration causes severe diarrhea.

Pertussis toxin prevents the alpha subunit of Gi from interacting with receptors and keeping inactive state, therefore unable to regulate target proteins.

Question 18

How does Cyclic-AMP-Dependent Protein Kinases (PKA) mediate the effects of cAMP?

Answer 18

In its inactive state, PKA consists of 2 catalytic subunits and 2 regulatory subunits.

Binding of cAMP to regulatory units changes protein conformation, causing these units to dissociate from complex.

Released catalytic subunits are activated. They can then migrate into the nucleus to phosphorylate transcripion regulatort genes. The catalytic subunits remain in the cytoplasm.

Question 19

Describe the function of NO as a gaseous mediator for blood vessels.

Answer 19

NO in mammals have the function of relaxing smooth muscle in the wall of blood vessels.

Acetylcholine stimumlates NO synthesis by binding to a GPCR of an endothelial cell. This then triggers IP3+ synthesis and Ca2+ release.

This in turn stimulates NO synthase. The NO produced can pass through to membranes of neighbouring smooth muscle cells.

Question 20

What are some other clinical applications for NO?

Answer 20

• Nitroglycerin used for angina (chest pain from inadequate blood flow to heart). Nitroglycerine is converted to NO and relaxes blood vessel muscles to reduce the workload of the heart.
• Viagra - NO binds in a reversible way to iron in the active site of guanylyl cyclase, stimulating synthesis of cGMP.

The normal turnover rate of cGMP is very high. Its regulated by degradation by phosphodiesterase. NO interacts with iron and therefore the enzyme, which inhibits the degradation of phosphodiesterase, thereby increasing the amount of cGMP in the penile blood vessels, keeping it relaxed and consequently the penis erect.

Question 21

What are the differences between G-protein-coupled receptors and enzyme-coupled receptors?

Answer 21

Their cytosolic domain doesn’t associate with a G protein, but instead has either intrisic enzyme activity or associates with an enzyme.

GCPR has 7 transmembrane segments, each enzyme coupled receptor subunit has 1.

Question 22

Describe the structure of protein kinases and how this affects their function.

Answer 22

They have an external part for ligand binding, which when activated by a ligand, leads to the sctication of the tyrosine kinase domain on the cytocylic side.

This means that the tyrosine side chains are phosphorylated, many times they are autophosphorylated. In turn this will create a docking site for other proteins that relay the signal.

Question 23

How does protein kinases work?

Answer 23

Ligand binding causes the receptors to dimerize. This means that the 2 cytoplasmic kinase domains are brought together.

Trans-autophosphorylation (one kinase phosphorylatres the other) generates binding sites for other proteins that are then activated and relays the signal.

Question 24

How does the epidermal growth factor receptor (EGFR) work?

Answer 24

Without ligands, they exist as inactive monomers.

2 ligand molecules are needed to promote dimerizations of external domains.

One kinase domain (activator) pushes the other (receiver) resulting in an activating conformational change in the receiver.

The receiver phosphorylates tyrosines in the C-terminal tails of both proteins, creating docking sites for intracellular molecules.

Question 25

Describe the GTPase and its connection to RTKs.

Answer 25

GTPase Ras mediates most RTKs signalling.

They are active when bound to GTP, inactive when bound to GDP, and is not directly linked to RTK but works together with it.

Question 26

How is Ras linked to RTK?

Answer 26

An activated RTK is bound to an adaptor protein (Grb2) via the protein’s SH2 domain. This domain binds to the phosphotyrosine residue in the activated RTK.

The protein also has SH3 domains, which binds to a protein that act as a guanine exchange factor (GEF) for Ras, called Sos.

Sos changes the GDP bound to the inactivated Ras for GTP, thereby activating Ras.

Question 27

What are the functions of Ras?

Answer 27

Often needed in signalling for cell division and differentiation, therefore some human tumours express hyperactive forms of Ras.

Question 28

Describe Ras and the cascade of substrates it triggers.

Answer 28

Activated Ras phosphorylates Raf, which then uses ATP to phosphorylates Mek, then Mek phosphorylates (ATP) Erk (MAP Kinase).

Erk then enters the nucleus and phosphorylates transcription regulatory factors.

Question 29

What does Ras ultimately activate?

Answer 29

Both activators and repressors are phosphorylated by Erk, therefore genes are not only expressed, but also repressed.

The earliest genes to be activated are immediate early genes, among them are some that stimulates cell proliferation, such as genes encoding for G1 cyclin.

Question 30

What are the functions of scaffold proteins?

Answer 30

They different signalling pathways to avoid cross-talk.

Even if 2 signalling pathways are the same, if they have different scaffold proteins then this can still lead to different results.

Question 31

Describe enzyme PI-3-kinase.

Answer 31

The substrate of this are lipids, in particular phospholipids.

These enzymes can be activated not only by RTKs, but also GPCRs, therefore it’s a key enzyme in signal transduction in eukaryotic cells.

Question 32

Describe how some parts of the intracellular signalling pathway can be in common.

Answer 32

In some cases, the activation of 2 different types of receptors can end up with the same type of proteins.

Therefore in the eukaryotic cells, there may be a continuous cross-dock of different signals arriving at the same type. Some can counteract while others can amplify each other.

Question 33

Describe how some enzyme-coupled receptors associate with cytoplasmic tyrosine kinases.

Answer 33

Some receptors that lack a kinase domain associate with cytoplamic tyrosine kinases. They phosphorylate many proteins, often the receptors themselves upon the binding of a ligand.

Question 34

How does cytokine receptors work?

Answer 34

When a cytokine binds to the ligand binding site of cytokine receptors, its arrangement is altered and 2 JAKs are brought into close proximity.

JAKs phosphorylate each other, then the tyrosine on the cytoplamic tails of cytokine receptors.

This creates docking sites for STATs, which is also phosphorylated amd associate before moving into the nucleus, forming a transcription regulatory complex with other proteins.

Question 35

What is the role of tyrosine phosphotases?

Answer 35

To reverse tyrosine phosphorylation.

Question 36

Describe the features of tyrosine phosphotases.

Answer 36

Most display exquisite speficity for substrates and removes phosphate groups from selected phosphotyrosines on a subset of genes.

Question 37

What are nuclear receptors?

Answer 37

They are ligand modulating transcription regulator that work inside the cell and receives signals from hydrophobic signals that can enter the cell.

Therefore no transmembrane proteins are needed.

Examples include steroid hormones, thyroid hormones, retinoids and vitamin D.

Question 38

How does nuclear receptors work?

Answer 38

After the signal binds to specific intracellular receptor proteins, the ability of these proteins to control the transcription of specific genes is altered.

Nuclear receptors bind to specific DNA sequences adjacent to genes regulated by ligand.

They effectively serve as both intracellular receptors and effectors.

Question 39

Describe the structure of nuclear receptors.

Answer 39

3 major domains: TA (Transcription Activating), DB (DNA Binding) and LB (Ligand Binding).