CB Block 2 Notes

Define contact dependent signalling

The cell sending the signal is directly adjacent to the cell receiving the signal. 

Define paracrine signalling

A cell sends a diffusible signal to the local neighbourhood. 

Define synaptic signalling

Cells send electrical impulses over long distances via the axons

Define endocrine signalling

Cells send signals over long distances in the form of hormones that are produced by glands in one area of the body, and transported in the bloodstream to their site of action. 

What does fast effects from signalling involve

Fast outputs involve modifying proteins that are already present in the cell, for example by post-translational modifications. Typically, fast responses take seconds to minutes.

What does slow effects from signalling involve

Slow outputs require transcription and translation of new proteins, which is a process that takes much longer. Slow responses typically take minutes to hours

How does a generic signalling pathway operate

In a generic signalling pathway there is a signal of some sort that activates a receptor, which may or may not be membrane bound. This receptor then initiates a series of events within the cell that result in the desired effect. 

What is signal transduction

The relay of the information between receptor and effector molecule

How does the number of inhibitory steps affect overall effect of a signalling pathway.

The overall effect of ligand on the effect can be calculated by counting the number of inhibitory steps

Because two inhibitory steps cancel each other out (because you are inhibiting an inhibitor), you know that an even number of inhibitory steps result in net activation of the pathway, while an odd number of inhibitory steps result in net inactivation of the pathway.

How do small hydrophobic molecules signal in a cell

Intracellular receptors

The signal is often bound to a carrier protein while in the extracellular environment, but the signal is capable of crossing the membrane of the target cell on its own.

 Once inside the cell, the signal molecule binds to a receptor protein that directly exerts the function. This is the most direct path a signalling pathway can take, but it does require that the signal be membrane permeable.

Because the membrane is composed of phospholipids, the internal environment is highly hydrophobic. Only hydrophobic molecules can freely cross the membrane. 

What are examples of hydrophobic signalling molecules 

Steroid hormones, retinoids (vitamin A), vitamin D are ligands that diffuse freely through the membrane and bind to intracellular receptors.

What are nuclear receptors

Intracellular receptors generally cause changes in transcription by binding to nuclear receptors. 

Nuclear receptors have three major domains – a ligand-binding domain, a DNA-binding domain, and a transcription-activating domain.

Example of intracellular signalling: Cortisol   

In this example, the steroid hormone cortisol is released from the adrenal cortex, carried in the bloodstream, and crosses the membranes of target cells. 

Once in the cell, it encounter the glucocorticoid receptor, which consists of a ligand binding domain (LBD), a DNA binding domain (DBD), and an activation function domain (AF1). Binding of cortisol activates the receptor, causing it to dissociate from molecular chaperone complexes that hold it in the cytoplasm and allowing it to enter the nucleus. The DBD can then bind DNA and the AF1 domain can activate transcription.

Which molecules are membrane impermeable 

• Proteins 

• Small peptides 

• Ions

What are the 3 main extracellular receptors

3 main classes of receptor (in order of speed)

• ion channel-coupled receptors, 

• G-protein coupled receptors 

• and enzyme-coupled receptors

Example of extracellular signalling- Delta Notch pathway

Delta- transmembrane protein that is a signaling molecule. 

Notch- cell surface transmembrane protein receptor 

1. a transmembrane protein called Delta (ligand) binds to Notch (receptor) in the extracellular space

2. Binding to delta changes the conformation of notch, exposing a cleavage site 

3. cleavage of intracellular domain (ICD)

4. ICD acts as a transcriptional activator

5. This only works between adjacent cells

The extracellular portion of the protein is therefore cut off after ligand binding. The loss of this large piece of protein changes the conformation of the intracellular region of the protein, exposing another cleavage site. The internal portion of the protein is therefore cut away from the membrane, and can then move into the nucleus. Once in the nucleus, it interacts with proteins that bind to the promoters of target genes, and activates transcription. Ligand binding ultimately leads to transcription

What are Ion channel couples receptors 

This type of receptor is an ion channel that adopts a closed conformation in the absence of ligand. Ligand binding (or another form of activation) causes a conformational change, such that the regions of the protein that block the pore of the channel are moved out of the way, opening the channel for ion transport. Ions then flow down their electrochemical gradient, either moving in or out of the cell. 

Types of activation for ion channels 

• Ligand binding 

• Voltage activated (in nerve signals)

Example of ion channel receptors- Acetylocholine receptor

Acetylcholine binding causes alpha helices lining the pore to rotate outward, disrupting a ring of hydrophobic amino acids that block ion flow. K+ and Na+ can then flow down their concentration gradients into the cytoplasm, depolarizing the cell and potentially stimulating muscle contraction.

What are transient receptor potential channels

family of ion channels are involved in sensing physical forces, such as those involved in hearing, smell and pain sensing.  

What is the structure and function of TRP channels

They are tetramers made up of monomers with 6 transmembrane domains. The tetramer forms an ion channel that is block by the cytosolic N-terminus and C-terminus. This prevent influx of ions in the absence of mechanical stress. 

The precise mechanism of opening is not yet known. Two possible models include the stress on the membrane making lateral stress which pulls the channel open, or pressure on the intracellular domains pulling down on the transmembrane domains to remove the block.

How do G-coupled receptors pass on signal

• Ligand binding causes a conformational change allowing the inactive GPCR to interact with a GTP-binding protein called G-protein causing binding and activation

• Binding to the GPCR stimulated the G-protein to release its bound GDP and replace it with GTP

• This activates the G-protein

• This then causes dissociation of the alpha subunit from the beta-gamma subunits

• These two sides of the active G-protein goes on to activate downstream proteins.

The fact that G-proteins hydrolyse GTP to GDP means that the G-protein can switch itself off, aids in regulation 

What are the types of G-protein alpha subunit

G⍺S

mediates: activation of adenylyl cyclase to increase cAMP concentration 

example GPCRs: glucagon receptor, olfactory receptors, β-adrenergic receptors

G⍺i

mediates: inhibition of adenylyl cyclase to decrease cAMP concentration 

example GPCRs: cannabinoid receptors, dopamine receptors, opioid receptors

G⍺q/11

mediates: activation of phospholipase C-β to cleave phosphatidylinositol 4,5-bisphosphate

example GPCRs: serotonergic receptors, ⍺-adrenergic receptor, histamine receptor

G⍺12/13

mediates: activation of RhoGEF to increase activity of the Rho GTPase

example GPCRs: LPA receptor, fMLP receptor, angiotensin receptor

What are enzyme coupled receptors

• Slowest of the receptors 

•  These are proteins with enzymatic activity that is activated in response to ligand binding. 

• Often the signal is a dimer, and binding encourages dimerization of two or more monomers, activating enzymatic function.

• They are also usually a kinase (phosphorylation) 

• The enzyme may be an intrinsic part of the receptor protein or an associated enzyme. 

Classes of enzyme coupled receptors: TGF beta

TGFbeta - Binds to TGFbeta and BMP 

• They form heterotetramers of two Type I and two Type II receptors. Ligand binding stimulates type II receptors to phosphorylate type I receptors, activating their own kinase domains. TGFbeta signalling is involved in controlling cell growth, differentiation and movement.

Classes of enzyme coupled receptors: Cytokine receptors

Cytokine receptors- Binds to cytokines (Epo, prolactin)

• They are generally released upon cell trauma or infection. They circulate in the bloodstream but in much smaller concentration than hormones, and are released from a wide range of sources not just specific glands (as for hormones). Dimerisation of their receptors by binding ligand brings their associated enzymes (JAK) into close proximity, allowing them to transactivate each other by phosphorylation. Cytokine signalling is important for enhancing the immune response.

Classes of enzyme coupled receptors: Receptor Tyrosine Kinases

RTK (receptor tyrosine kinase) receptors - Binds to growth factors - For RTKs, dimerization either positions kinase domains in correct orientation for phosphorylation or causes conformational changes that activate the kinase domain. Receptors then phosphorylate the neighbouring receptor during transphosphorylation. This further activates the kinase domains for more extensive phosphorylation. These phospho-residues form binding sites for proteins that will relay the signal. 

Example of RTK binding: Insulin receptor 

Insulin binding causes a conformational change from an inverted V to the T form. This change in the external alpha domains is transmitted to the intracellular beta domains, bringing them into close proximity and allowing trans-autophosphorylation. This allows the recruitment of downstream molecules like the Insulin Receptor Substrate (IRS-1). 

What are co-receptors

Coreceptors are usually transmembrane proteins that act as secondary receptors for the ligand. In some cases they help a receptor hold on more tightly to the ligand, while in other cases they are directly involved in signal transduction. For example, in Wnt signalling, Frizzled is the receptor and LRP is a coreceptor. Frizzled and Wnt both bind to the Wnt ligand – Frizzled is GPCR that activated Dishevelled, while ligand-bound LRP can be phosphorylated on its cytosolic tail. Together, Frizzled and Wnt recruit axin. Both proteins are required to transduce the Wnt signal. 

What are the major mechanisms of signal transduction

• Protein binding/recruitment 

• GTP binding 

• Phosphorylation

• protein cleavage

• modifying phospholipids

• small messengers

What are the common protein binding domains

C2	second domain of PKC	binds membranes in the presence of Ca2+
PDZ	named for initials of founder proteins	binds to the C-terminus of a target protein
PH	Pleckstrin homology	binds phosphoinositides in membranes
PTB	Phosphotyrosine binding	binds phosphotyrosine
PX	Phox homology	binds phosphoinositides in membranes
SH2	Src homology 2	binds phosphotyrosine
SH3	Src homology 3	binds proline rich peptide sequences (PxxP)

What is a protein binding domain 

 Region of a protein with a defined fold, that is always consistent no matter the surrounding sequence.

 Domains are classified according to sequence and structure, NOT function. Function is therefore largely conserved among classes because of the similarity of structure, but can deviate from the classic definition in some cases.  

How are binding domains placed in the structure

Protein domains can be buried within the structure of a protein, or freely accessible on the surface of a protein. Many signalling events take advantage of this fact by changing the conformation of a protein to expose or hiding a binding site. 

Example: Activation of a receptor causing a conformational change

Activation of a receptor may cause a conformational change that allows it to be recruit proteins from the cytoplasm to the cell cortex. In this example, an activated RTK recruits the adaptor protein Grb2 to the membrane via a newly created phosphotyrosine. The conformational change Grb2 undergoes upon binding the receptor exposes SH3 protein binding domains that recruit Sos.

What are G-proteins

G-proteins exist in two states: active (GTP bound) and inactive (GDP bound).

G-proteins are GTPases – GTP hydrolysing enzymes – with extremely low activity.

How do GAPs (GTP activating proteins) aid G-proteins

 A helper protein called a GTPase activating protein is needed for productive GTP hydrolysis . This converts G protein from the active to inactive state. 

How do GEFs (Guanine nucleotide exchange factors) aid G-proteins

G-proteins bind tightly to their nucleotide co-factor – a GEF (guanine nucleotide exchange factor) to release the GDP. GTP can then spontaneously bind due to its higher concentration in the cell. This converts G protein from the inactive to active state. 

Example of G-proteins: Ras GTPase pathway

1. The activated receptor recruits the adaptor protein Grb2

2. Grb2 recruits Sos, a GEF for the RAS GTPase (G protein)

3. Sos promotes RAS’ exchange of GDP for GTP 

4. GTP-bound RAS activates downstream targets (usually enzymes of some sort, e.g. kinases)

How can serine, threonine or tyrosine be phosphorylated

Phosphate groups have two negative charges that can pull  regions of positive charge towards them. 

This changes protein function by either

• Creating or masking a binding motif

• Inducing conformational change

Kinases add the phosphate and phosphatases remove it.

What are the main types of kinase

Main kinases 

• Receptor tyrosine kinases (RTK)

• Map kinases

• Protein kinase A/B/C

What are some advantages of phosphorylation in signalling

Advantages of phopshorylayion 

• Rapid and reversible

• Many kinases which can be assembled into phosphorylation cascades, which provides opportunities for regulation

• A weak signal can be amplified

• It allows a threshold to be established for cell output

How does phosphorylation lead to degredation

Phosphorylation can also cause degradation of the protein. Phosphorylation can occur before binding to ubiquitin ligase which leads to polyubiquitinylation and then degradation by the proteasome. 

Example: phosphorylation leading to degredation

This is used in Wnt signalling to degraded beta-catenin. In the absence of signal, beta-catenin is phosphorylated by the destruction complex and undergoes degradation. 

Example: Protein cleavage in hedgehog signalling

In the hedgehog signalling pathway, degradation is used to alter the function of the effector molecule Ci. In the absence of ligand, Ci is cleaved by the proteasome into a smaller fragment that can act as a transcriptional repressor. In the presence of ligand, the Ci remains uncleaved and can act as a transcriptional activator. 

What is the basic structure of a membrane

Membrane structure

• Phosopholipid bilayer

• Hydrophobic tails cluster in the middle of the membrane

• Hydrophillic heads on the outside 

• Each of the different phospholipids have a different head group. 

How are different phospholipids arranged in the lipid bilayer

Different phospholipids cluster in different layers of the lipid bilayer. PC and SM are mostly in the outer leaflet, and PE, PS and PI are mostly in the inner membrane. This generates a charge difference, with the inner surface of the cytosolic side of the membrane being negatively charged

Example: signalling my modifying phospholipids in the membrane, Phosphatidylinositol 

he phosphatidylinositol molecule is a fatty acid tail, joined to glycerol and phosphate and inositol. Each of the carbons in the inositol ring can be phosphorylated, and some signalling pathways involve converting in between different phosphorylated states. This depends on the availability of the kinases and phosphatases, which are located in different regions of the cell. This means each organelle’s membrane has a different content of phosphoinositides. 

The modification of phosphatidylinositols is best studied, but phosphatidylcholine and sphingomyelin may also be involved in signalling pathways. 

How do PI3Ks (Phosphoinositide 3-kinases) affect the inositol carbon ring

Phosphoinositide 3-kinases (PI3Ks) add phosphate to position 3 of the inositol carbon ring

PI3K is an enzyme that adds phosphate groups to position 3 of the carbon ring to generate new binding sites for protein binding domains like PX and PH. 

Example: PI3Ks in signalling pathways

Example Akt (protein kinase B) pathway:

An activated RTK recruits PI3K and activates it. PI3K can then phosphorylate PIP2 to PIP3, creating a binding site for PDK1 and Akt. PDK1 then phosphorylates Akt.

Akt (protein kinase B) has many targets in the cells, changing metabolism, protein synthesis and cell survival. 

How does cleavage of PI(4,5)P2 relay signals

Instead of being phosphorylated, PI(4,5)P2 can also be cleaved into:

Diacylglycerol- Diffuses in the membrane

Inositol 1,4,5-triphosphate- Diffuses in the cytoplasm 

This process is catalyzed by phospholipase C

Example: activation of phospholipase C

Both GPCRs and RTKs can activate Phospholipase C. 

Eg. 

• The GPCR activates a trimeric G protein, which in turn activates phospholipase C (PLC). 

• PLC then cleaves PIP2 into Inositol 1,4,5-triphosphate (IP3) and Diacylglycerol (DAG). 

• DAG diffuses in the membrane and can bind to Protein Kinase C (PKC). 

• IP3 moves to the endoplasmic reticulum when it binds to a gated Calcium channel. This opens the channel, releasing Ca2+ into the cytoplasm, where it can bind to PKC.

•  Both DAG and the Ca2+ are needed to activate PKC. 

What are small messengers in signal transduction

Second messengers are small molecules that are made in response to receptors. There are many different examples, but they usually amplify a signalling cascade. 

The localised production of second messengers can ensure a localised target response.

Examples

• Calcium ions 

• Cyclic AMP 

• Inositol 1,4,5, triphosphate

• Diacylglycerol

Example: Gated calcium ion channels open to allow calcium ions to flow down concentration gradients

• The calcium concentration in the cytoplasm is low compared to the extracellular space and ER lumen. 

• There is a 10-100X increase in cytosolic calcium concentration upon opening of the calcium pumps

• Plasma membrane channels are opened by membrane stretch, membrane depolarisation and extracellular signals

• ER channels are opened by IP3 in the cytoplasm, and extracellular signalling

• Calcium pumps move cytosolic calcium into the ER or out of the cell

How do Calmodulin-dependent kinases (CaM-Kinases) mediate cellular calcium response

Changes in calcium concentrations can activate calcium dependent proteins. 

The calmodulin-dependent kinases act as mediators. 

The calmodulin binds calcium via the EF hand motifs. although calmodulin has no enzymatic activity by itself, it binds to the CaM-kinase and activates it.

 CaM kinase then undergoes autophosphorylation to fully activate the protein and transduce signal downstream.

What are the main types of CaM-Kinases

Kinase	Type	Targets	Physiological role
CaMMKK	multiple substrates	CaMKI, CaMKIV	Gene transcription, Apoptosis
CamKI	multiple substrates	Synapsin 1  CREB	Vesicle mobilisation Gene transcription
CaMKII	multiple substrates	AMPA/NMDA receptors L-type Ca2+ channels	Synaptic plasticity Regulation of ion channels Gene transcription
CaMKIV	multiple substrates	CREB, CBP SRF, HDAC4 Oncoprotein 18	Gene transcription
CaMKIII	specific substrates	Elongation factor 2	Protein translation
Myosin light chain kinase	specific substrates	Regulatory light chain of myosin	Muscle contraction Intracellular transport
Phosphorylase kinase	specific substrates	Glycogen phosphorylase	Glycogen metabolism

Example: Ca2+-activated Synaptotagmins promote fusion of vesicles to the cell membrane

CaM kinases are involved in fusing vesicles to the cell membrane

1.  A presynaptic vesicle is docked at the membrane by SNARE proteins.

2.  An action potential reaches the synapse and opens calcium channels, causing influx of calcium ions. 

3. When calcium binds to synaptotagmin, it causes it to bind to the SNARE proteins and the phospholipid membrane, bringing the vesicle closer to the plasma membrane. 

4. It promotes formation of the fusion pore and release of neurotransmitter.

How is cAMP synthesized and degraded

cAMP is synthesised by adenylyl cyclase, and can be degraded by cAMP phosphodiesterase.

What are some effects of cAMP in the cell

cAMP has many different function and different cell types respond differently to increased cAMP.The effects of cAMP are mostly mediated by activation of Protein Kinase A (PKA). 

cAMP binding releases PKA’s catalytic subunit and allows it to phosphorylate targets such as some nuclear receptors, CREB (cAMP responsive element binding protein), a Ca2+ channel in the ER amongst others.

What are the functions of the 3 protein kinases

Protein kinase A = Regulates cAMP effects 

Protein kinase B = Akt 

Protein kinase C = Calcium release

What is the function of positive feedback loops in signalling

Positive feedback loops are a way to amplify the effect of a small amount of input to achieve a maximal level of output. As long as the pathway has been activated by signal once, then the signal can be sustained even when ligand is removed. 

What are some examples of how positive feedback can be utilised in signalling

Eg. Component B is capable of activating component A – therefore, whenever the A activates B, it has a side effect of also activating more A, and thus more B. 

Eg. enzyme E is activated by phosphorylation. Once active, E can phosphorylate more E, amplifying the signal. 

Example: Ca2+-induced Ca2+ release is a form of positive feedback

1. An initial signalling event triggers opening of the Ca2+ channels, either in the ER membrane or the plasma membrane. 

2. This leads to a local increase in the cytosolic calcium level. 

3. Since adjacent ER calcium channels are sensitive to calcium, they open when there is a small increase in concentration, further increasing the concentration.

4. This reinforces the initial increase. 

How is negative feedback used in signalling

In negative feedback, activation of a downstream step activates an upstream step. Negative feedback shortens the response duration and in some cases intensity.

What are some examples of how negative feedback can be utilised in signalling

Eg. in this case B inactivates A, ultimately reducing activation of B. 

Eg. Enzyme E is activated by signal, then activates an inhibitor protein (I) which inactivates E. This switches the pathway off again. 

How do short delays in negative feedback loops affect the signal

• Short delays cause a strong response to stimulus that rapidly decays, even when stimulus persists. 

Eg. The activity of E will increase when signal is present. Shortly afterwards, the activity of E will drop but still remain higher than in the absence of signal because there is still signal present to activate E again. Once signal is lost, the negative feedback loop shuts down signalling and E becomes inactive.

How do long delays in negative feedback loops affect the signal

• With a long delay, negative feedback can cause oscillations. 

Eg. 

1. When a negative feedback loop that acts after a long delay is present, the activity of E will increase when signal is present. 

2. During the longer delay, a lot of E has time to be activated. 

3. Once the delay is over, the active E will be able to activate a lot of the phosphatase I. 

4. Then I can inactivate most of the enzyme E, causing a significant drop in activity. 

5. Because there is less active E, the phosphatase I is no longer phosphorylated and becomes inactive 

6. But if there is still signal present, E can then be activated again, and the loop begins anew. 

7. Once signal is lost, the negative feedback loop shuts down signalling and E becomes inactive, and is not activated once more.

What is receptor internalisation

Internalising the receptors into vesicles to switch off a pathway. 

How does receptor internalisation occur

Vesicles such as endosome have a different pH to the extracellular space. This can cause conformational changes that release the ligand from the receptor, leaving receptor ready to be recycled back to the membrane. 

What happens if the release of the ligand is not promoted in the endosome

If the release of the ligand is not promoted in the endosome, other measures are needed, since the endocytosed receptor is still signalling. These receptors are delivered to the compartment called the multivesicular body (MVB). MVBs have internal vesicles formed from invaginations of their own limiting membrane. Once in the internal vesicle of the MVB, the receptor’s active cytosolic portion is sequestered from the cytosol. This physical separation stops signalling.

Receptors in MVBs can either be delivered to the lysosome for destruction, or can be recycled to the plasma membrane.

What are some examples of other methods of switching off signal transduction

In addition to receptor internalisation, there are many ways to switch a signalling pathway off again. And step that inactivates one of the signal transduction molecules is a form of negative feedback. This may include the removal or addition of covalently added groups, activation or inactivation of enzyme function, etc

Examples of inactivation: 

• kinases adding inhibitory phosphates

• phosphatases removing activatory phosphates

• phosphodiesterases degrading cAMP

• GAPs promoting GTP hydrolysis 

How can positive and negative feedback loops be combined

In most cases, a pathway will have multiple mechanism of feedback. In CICR, signalling opens the Ca2+ channels, causing release of more Ca2+ from the ER. This higher calcium concentration inhibits the calcium channel, preventing further increases. Ca2+ is actively pumped out of the cytoplasm to restore the pathway to baseline. This is an en example where we have early amplification followed by delayed negative feedback.

Example: Combining positive and negative feedback loops: Calcium induced calcium release

1. Signalling opens calcium channels in either the ER or plasma membrane

2. Moderate increase in cytosolic calcium causes neighbouring channels to open, increasing the calcium and the response.

3. Large increase in cytosolic calcium inhibits the calcium channel, preventing the calcium from rising further

4. Calcium is pumped back into the ER or out of the cell by the ATP-dependent calcium pump, resetting the pathway

Early positive feedback amplifies and reinforces the signal, then delayed negative feedback

How could you make sure that your negative feedback loop is slower to be activated than your positive feedback loop?

Use one of the early signal transduction proteins for positive feedback and require additional protein activation steps for the negative feedback loop

Or use a transduction protein for positive feedback and make a new protein for negative feedback that replies on transcription activated from the pathway

What are scaffolds

Proteins that bind to multiple proteins from the same signalling pathway. They have no function other than structural (i.e. they do not activate the signal transduction molecules). 

How do scaffolds work in signalling

By bringing together groups of interacting signaling molecules they ensure a cell is primed for response. As soon as the upstream component is activated, it is able to rapidly activate proteins in sequence. This high local concentration increases the speed as proteins don’t have to search for a partner, and increases specificity because only the relevant proteins are held in the scaffold. 

Example: Mitogen Activated Protein Kinase (MAPK) modules in RTK signalling

RTK signalling pathway

A GTPase activates a phosphorylation cascade. The generic names for the kinases involved are MAPKKK, MAPKK, and MAPK. but different family members can be activated by different ligands. 

Which signalling pathway is triggered when growth factors activate the RTK

Growth factors 

When growth factors activate the RTK, RAS activates the RAF-MEK-ERK series of kinases. These are held together by the KSR (kinase suppressor of Ras). 

Which signalling pathway is triggered when cellular stress activate the RTK

When cellular stress activates the RTK, then MLK3-MKK7-JNK are activated. These are held together by JIP1. 

Which signalling pathway is triggered when osmotic stress activate the RTK

And in the case of osmotic stress, RAC activates MEKK3-MKK3-p38, held together by OSM. The scaffold protein makes sure that each kinase is closely associated with the relevant neighbour, preventing accidental activation of the wrong kinase. 

What are lipid rafts

are regions of the plasma membrane that are enriched with certain membrane proteins. Since the enrichment or exclusion of proteins from the raft is selective, receptors can be clustered together into a raft domain

What is the structure of cholesterol

• Cholesterol is a sterol molecule with a polar head group, steroid ring structure and a non-polar hydrocarbon tail. 

• The polar head group is able to interact with the polar head groups of neighbouring phospholipids 

• The hydrophobic tail inserts into the bilayer along with the hydrocarbon tails of the phospholipids. 

What is the structure of cholesterol in the membrane

• Once in the membrane, cholesterol increases attractive forces between phospholipids to increase packing. 

• It also reduces permeability by preventing diffusion past the first region of the carbon tail. 

• And it stops phospholipids becoming too tightly packed at lower temperatures. A

• ddition of cholesterol to an artificial membrane causes formation of lipid rafts, consistent with its role in vivo, where it stiffens that region of the membrane.

It increases packing, fluidity and reduces permeability

Example of clustering receptors in signalling speed- Insulin and PI3K

• The insulin receptor activates PI3K to allow phosphorylation of PI(4,5)P2 and recruitment and activation of Akt. 

• A high concentration of receptors (as indicated by the black box) increases the speed at which receptor dimers are formed and activated. 

• This allows activation of the receptor and signal transduction is achieved more quickly. 

• The pathway also increases raft size, in a positive feedback loop. In addition, inhibitory proteins can be excluded from the raft region, favouring activation.

How does the absence of a lipid raft affect signalling speed

In the absence of lipid rafts, the receptors can diffuse more extensively, meaning it takes longer to form the dimer. Also, the inhibitor has not been excluded from the region, so any phosphorylation that occurs can be reversed. The lipid raft increases the speed of signalling. 

Example: the primary cilium is the site of hedgehog signalling in vertebrates

Proteins can be transported into and out of the cilium in response to signalling. For example, in the hedgehog signalling pathway, the hedgehog receptor is called patched1 - it is concentrated in the cilium membrane. Upon binding to ligand, the patched1 protein is transported out of the cilium and internalised into vesicles. The transmembrane protein Smoothened is then trafficked to the cilium membrane in response, where it can activate downstream signalling. The concentration of patched1 in the cilium increases the speed of signalling. 

What is the primary cillium

The primary cilium-  is a microtubule based structure that projects from the surface of the cell, and its structure does not allow free diffusion between its membrane and the rest of the plasma membrane. The cilium is a major site of signalling in cells, because many receptors are clustered in the cilium membrane. this achieves a similar effect to lipid rafts, by clustering receptors and increasing rates of activation. 

Generic example of signalling crosstalk

For example, a cell receiving A, B and C may know to survive, while addition of D and E on top of that tells a cell to grow, but addition of F and G tells it to differentiate. 

What are some common components of intracellular signalling

• Protein interaction motives

• Kinases

• Phosphatases

• GTPases

How are the signalling pathways intitated by GPCRs and RTKs integrating into a network

1. Their main kinase targets PKA, PKC, CaM-kinase, MAP kinase and PKB have many common targets, that either need to be or can be phosphorylated by more than one of these key kinases.

2. The kinase targets such as PKA or PKC can phosphorylate and thereby regulate members of other pathways.

3. PLC is a common component of both GPCR and RTK signalling.

Which GPCRs do alpha1- adrenergic receptors bind to and what is the function and effect

• Coupled to Gq/11 protein

• Activates phospholipase C

• Opens ER calcium channels leading to smooth muscle contraction

• Glyconeogenesis in the liver and adipose cells

• Secretion from sweat glands

Which GPCRs do beta adrenergic receptors bind to and what is the function and effect

• Coupled to Gs protein

• Activates adenylyl cyclase

• Increase cAMP concentration

• cardiac muscle contraction

• Smooth muscle relaxtion

• Glyconeogenesis in the liver

• Secretion from stomach and kidney

Which GPCRs do alpha2- adrenergic receptors bind to 

• Coupled to Gi protein

• Activates phosphodiesterase 3

• Decrease cAMP concentration by inhibiting adenylyl cyclase

• Decreased secretion of insulin in pancreas

• Glucagon release from pancreas

• Decreased noradrenaline release

How does the cells environment affect strength of signalling

Different cells live in different environments. They may have exactly the same receptor, but different receptors may be sensitive to different levels of signal – 

Eg. the green receptor can be respond at a lower ligand concentration than the purple receptor. In a cell with both receptors, both will signal when ligand is high, only green will signal when ligand is moderate, and neither will signal when ligand is low. 

Example: mTOR as a central regulator of cell growth

Mammalian target of rapamycin (mTOR) is a central regulator of cell growth

mTOR is activated in response to nutrients and insulin signalling, and inactivated in response to cellular stress and inflammation 

mTOR regulates growth by controlling protein synthesis, respiration and autophagy

Example: The signal transduction molecules of one pathway can be used to regulate those of a different pathway. MAPK pathway and the PI3K pathway.

They are both activated by partially overlapping agonists although they use different receptors.
In the MAPK pathway, the receptor recruits the adaptor protein GRB2, which brings the GEF (SOS) to the membrane, where it activates RAS. RAS activates RAF, which phosphorylates MEK, which phosphorylates ERK, to promote cell survival, growth and proliferation. 

The PI3K pathway uses an adaptor to recruit PI3K which phsophorylates PIP2, ultimately activating Akt (PKB), which phosphorylates mTOR and promotes growth and proliferation. There are several sites of crosstalk between these pathways:

1. RAS can activate PI3K

2. RAS can inactivate the adaptor protein

3. PI3K can inactivate RAF

4. ERK can inhibit TSO2, which inhibits mTOR (hence ERK activates mTOR)

5. Akt also cross talks to other pathways, including NF-KB and Wnt signalling, which initiates apoptosis and promote proliferation respectively

How is pathway activity measured

Measurements of pathway activity are called ‘read outs’, and are experimentally tractable ways to measure activity. They rely on a detectable change. 

They may include

• activation of a receptor

• Changes to relay molecules

• outputs of the pathway (protein or transcription)

To measure whether a pathway is actively signalling or not, you must first identify what actually changes when a pathway is active compared to when it is inactive. 

How does the MAPK pathway change when it is active/inactive

Some proteins are phosphorylated when the pathway is active but not when the pathway is inactive. Others change location within the cell. There are also differences in the cellular output, for example changes in cell structure or transcription levels. 

What is immunoblotting

1. Proteins are separated according to size by gel electrophoresis 

2. Proteins are transferred to a nitrocellulose membrane to stop diffusion

3. Secondary antibody recognizes primary antibody and is conjugated to a flurophore or enzyme 

4. Primary antibodies are used to stain bands for specific proteins on the membrane 

Example: DUSP6 levels and beta-catenin in immunoblotting

Refer to image on docs

immunoblot showing DUSP6 levels before and after treatment with EGF, which is a ligand for the RTK. 

DUSP6 is transcribed in response to active signalling. Active signalling has a stronger band than the inactive signalling indicating there is more DUSP6 present when signalling is active. The levels of GAPDH are provided as a control and show a consistent amount of protein in each lane. 

Immunoblot showing beta catenin levels before and after treatment with an increasing dose of ligand for the Wnt receptor. Beta-catenin is degraded when the pathway is inactive. Active signalling samples have a stronger band than the inactive signalling indicating there is more beta-catenin present when signalling is active. The levels of GSK3 are provided as a control and show a consistent amount of protein in each lane. 

What are reporter genes

Reporter genes - used in pathways which activate transcription 

How do report genes function

It takes the signalling response element – the piece of DNA that the transcription factors bind to as the last step in signalling – and puts it next to a reporter gene. 

The reporter gene usually encodes a protein that can be easily detected, such as a fluorescent protein like GFP, or an enzyme like luciferase that catalyses a reaction that releases light. The reporter protein has no actual role in signalling – it is entirely used to measure the pathway.