Cell Signalling

Front 1

Describe the 5 main modes of cellular communication, with examples.

Back 1

Cell-cell: requires direct contract i.e. by gap junctions or a ligand-receptor interaction, e.g. gap junctions in the heart ensure all the muscle contracts simultaneously

Autocrine: signals are released from a cell which stimulate either the same cell or travel a short distance to stimulate another cell of the same type, e.g. tumour cells

Paracrine: signals are released from a cell and travel a short distance to stimulate another cell of a different type, e.g. at the neuromuscular junction neurotransmitters are released from neurons which stimulate the muscle cell

Endocrine: hormones are released from cells into the blood stream and travel slowly over a short distance to stimulate another tissue - all tissues will be bathed in the hormone, but the response depends on the responsiveness of the tissue to the hormone

Electrical: AP travels down a neuron and then stimulates the release of neurotransmitters

Front 2

For what 3 reasons may the responses in different tissues be different to the same signalling molecule?

Back 2

Different membrane receptors

Same membrane receptor but different signalling pathway

Same membrane receptor and signalling pathway but different protein repertoires

Front 3

What are the 5 main types of receptor?

Back 3

Ionotropic: ligand-gated ion channels in the cell membrane, e.g. nACh receptors

Metabotropic: G-protein-coupled receptors in the cell membrane, e.g. beta adrenoceptors

Enzyme-coupled: coupled to an enzyme on the intracellular part of the receptor, or the intracellular part of the receptor has enzymatic activity, activation of receptor causes activation of enzyme, e.g. receptor tyrosine kinase

Cytoplasmic: inside the cell, e.g. guanylyl cyclase

Nuclear receptors: when activated, they alter gene transcription, e.g. steroid hormone receptor

Front 4

What are the 6 main classes of G-protein?

Back 4

G(s): alpha subunit stimulates adenylyl cyclase

G(olf): alpha subunit stimulates adenylyl cyclase in olfactory sensory neurons

G(i): alpha subunit inhibits adenylyl cyclase and beta-gamma subunit activates potassium channels

G(o): beta-gamma subunit activates potassium channels, deactivates calcium channels and stimulates phospholipase C (also alpha)

G(t): alpha subunit stimulates phosphodiesterases in rod cells

G(q): alpha subunit stimulates phospholipase C

Front 5

Describe the G(s) signalling pathway to adenylyl cyclase.

Back 5

1. Adrenaline binds to a beta-adrenoceptor, which is associated with a stimulatory G(s) protein.

2. This activates the G(s) protein, causing the alpha subunit to dissociate from it and activate the first effector adenylyl cyclase.

3. Adenylyl cyclase catalyses the coversion of ATP to the second messenger cAMP.

4. cAMP binds to the second effector protein kinase A, activating it.

5. This causes protein kinase A to phosphorylate target proteins.

6. To reverse the activity of cAMP, phosphodiesterases hydrolyse cAMP into AMP.

Front 6

What effect does adrenaline have on heart rate and contractility?

Back 6

It increases heart rate and contractility, so inhibitors of adrenaline are used to treat high blood pressure and panic attacks.

Front 7

What effect does adrenaline have on glycogen breakdown in the muscle and liver?

Back 7

It increases it.

Front 8

What effect does adrenaline have on smooth muscle in the bronchi?

Back 8

It causes smooth muscle relaxation, so agonists can be used to treat asthma.

Front 9

How does cAMP activate protein kinase A?

Back 9

1. 2 cAMP molecules bind to each of the 2 regulatory domains of protein kinase A

2. This causes the 2 catalytic domains do dissociate from the regulatory domains

3. The catalytic domains then bind to a target substrate, phosphorylating it

Front 10

What kinds of target substrates may protein kinase A phosphorylate?

Back 10

Another protein kinase, which is activated through phosphorylation

A substrate which sits in its active form and phosphorylation inactivates it

A substrate which is phosphorylated by protein kinase A to cause its movement to be localised to its target

A substrate which is phosphorylated by protein kinase A causing a conformational change in its binding site

Front 11

What is the function of phosphatases?

Back 11

Phosphatases counteract the effect of protein kinase A by dephosphorylating the target substrate - this usually means the target substrate is inactivated

Front 12

What specific amino acid sequence does protein kinase A phosphorylate at?

Back 12

Arg and Lys residues when they're upstream from Ser or Tyr

Front 13

Describe how adrenaline can cause an increase in cardiac muscle contractility.

Back 13

1. Adrenaline binds to a beta1-adrenoceptor, in the sarcolemma of cardiac cells - this receptor is associated with a G(s) protein

2. This activates the G(s) protein, causing the alpha subunit to dissociate and bind to adenylyl cyclase, which catalyses the conversion of ATP to cAMP

3. cAMP binds to the regulatory domains of protein kinase A, causing the catalytic domains to dissociate

4. The catalytic domains phosphorylate L-type calcium channels in the sarcolemma, activating them and causing an influx of calcium - this increases the intracellular concentration of calcium, which causes an increase in the strength of muscle contractions (positive inotropy)

5. The catalytic domains phosphorylate RyR2 receptors in the membrane of the sarcoplasmic reticulum, causing an increase in release of calcium from the sarcoplasmic reticulum - this also increases the intracellular concentration of calcium, which causes an increase in the strength of muscle contractions (positive inotropy)

6. The catalytic domains phosphorylate phospholamban, which is associated with the sarcoplasmic reticulum calcium-ATPase pump - the unphosphorylated phospholamban inhibits the pump, but when phosphorylated it causes the pump to be activated, which brings more calcium into the sarcoplasmic reticulum - this causes quicker relaxation of the muscle, which enables a second contraction to occur more quickly (positive lusitropy)

7. The catalytic domains phosphorylate troponin I, which is a calcium sensitive molecule that supports contraction - phosphorylation causes a reduction in its calcium sensitivity, causing quicker relaxation of the muscle, which enables a second contraction to occur more quickly (positive lusitropy)

Front 14

Why is there a difference in the effect of adrenaline binding to beta1-adrenoceptors and adrenaline binding to beta2-adrenoceptors?

Back 14

Adrenaline binding to both beta1 in cardiac muscle and beta2 in lung smooth muscle cause an increase in the activation of adenylyl cyclase, cAMP and protein kinase A

In cardiac muscle, the increase in intracellular calcium due to the phosphorylation of L-type calcium channels and RyR2 leads to calcium binding to calmodulin - calcium-calmodulin binds to myosin light chain kinase, activating it and causing it to phosphorylate myosin light chain which causes contraction

In lung smooth muscle, there is a different protein repertoire, so protein kinase A phosphorylates other proteins - protein kinase A phosphorylates myosin light chain kinase, inactivating it by decreasing its affinity for calcium-calmodulin, so it doesn't phosphorylate myosin light chain - this causes relaxation

Front 15

Describe how protein kinase A affects metabolism in the liver.

Back 15

1. Protein kinase A phosphorylates phosphorylas kinase, which phosphorylates glycogen phosphorylase

2. This causes glycogen breakdown to glucose-1 phosphate

3. Protein kinase A also passes into the nucleus and phosphorylates transcription factors e.g. CREB, which wfahen activated binds to CRE regions of DNA that code for enzymes involved in gluconeogenesis

Front 16

Describe how G(q) signalling to phospholipase C occurs.

Back 16

1. Adrenaline binds to an alpha1-adrenoceptor, which is associated with a G(q) protein

2. This causes the exchange of GDP for GTP, causing the alpha subunit to dissociate from the protein

3. The alpha subunit binds to phospholipase C, activating it

4. Phospholipase C hydrolyses a lipid on the membrane called PI45P2, producing the second messengers IP3 and DAG

5. DAG stays bound to the membrane and activates protein kinase C

6. IP3 diffuses throughout the cell and binds to receptors in the membranes of calcium-store organelles to release calcium into the cytosol, increasing the intracellular concentration of calcium

7. Calcium can also activate protein kinase C

8. Calcium also binds to proteins such as calmodulin, which can have downstream effects e.g. binding to myosin light chain kinase that phosphorylates myosin light chain and causes contraction in cardiac muscle

9. Protein kinase C, which has been activated by DAG and calcium, phosphorylates target substrates

Front 17

What effect does G(q) signalling to phospholipase C have in smooth muscle?

Back 17

It causes contraction i.e. vasoconstriction

Front 18

What effect does G(q) signalling to phospholipase C have in salivary gland cells?

Back 18

It causes exocytosis i.e. secretion

Front 19

Describe the structure of protein kinase C and how it is activated.

Back 19

It has 2 Cys-rich repeats for DAG binding, a calcium-activated lipid-binding domain for calcium to bind and localises the protein to the membrane, and 3 phosphorylation sites which must be phosphorylated and therefore activated prior to the arrival of a signal - this is done through a post-translational modification

Front 20

Describe the function of the pseudosubstrate region in the activity of protein kinase C.

Back 20

1. When unactivated, the pseudosubstrate region acts as a substrate and sits in the catalytic cleft of protein kinase C, inhibiting the binding of a real substrate

2. When calcium binds to the calcium-activated lipid-binding domain, this enables DAG to bind to the 2 cys-rich repeats

3. This localises protein kinase C to the membrane

4. As this localisation occurs, the pseudosubstrate region moves out of the catalytic cleft, allowing the real substrate to bind and become phosphorylated

Front 21

How does smooth muscle contraction occur?

Back 21

1. Adrenaline binds to the alpha1 adrenoceptor, eventually causing the production of IP3

2. IP3 binds to intracellular calcium stores, causing the release of calcium

3. This causes the formation of calcium-calmodulin, which binds to myosin light chain kinase

4. This phosphorylates myosin light chain, causing contraction and thus vasoconstricton

Front 22

Describe how the balance of adrenaline binding to adrenoceptors in smooth muscle affects the physiological outcome.

Back 22

Adrenaline binding to alpha1 adrenoceptors causes contraction

Adrenaline binding to beta2 adrenoceptors causing relaxation

If a low concentration of adrenaline is applied, signalling via beta2 adrenoceptors dominates, so there is a greater production of cAMP and vasodilation occurs

If a high concentration of adrenaline is applied, signalling via alpha1 adrenoceptors dominates, so there is enough release of calcium to overcome the effects of cAMP so vasoconstriction occurs

Front 23

Describe how adrenaline causes exocrine solution in the salivary gland.

Back 23

1. Adrenaline binds to a muscarinic receptor, which is associated with a G(q) protein

2. This causes the exchange of GDP for GTP, which causes the alpha subunit to dissociate from the protein

3. The alpha subunit binds to phospholipase C, which causes the production of IP3

4. IP3 binds to a receptor in the endoplasmic reticulum membrane, causing the release of calcium into the cytosol

5. Calcium stimulates the fusion of amylase-containing vesicles with the cell membrane, causing the secretion of amylase into the surrounding tissues

6. Calcium also binds to calcium-activated chloride channels, which causes the efflux of chloride into the lumen of the secretary organ, which allows osmosis to occur which facilitates secretion

Front 24

What is the effect of alpha2-adrenoceptor activation?

Back 24

Alpha2-adrenoceptors are associated with G(i) proteins, so they inhibit adenylyl cyclase

Front 25

What is the effect of alpha2-adrenoceptor activation in smooth muscle?

Back 25

Causes contraction, so supports alpha1-adrenoceptors and acts against beta2-adrenoceptors

Front 26

What ion channels do G(q) proteins have an effect on?

Back 26

Calcium binds to calcium-dependent potassium channels, which causes potassium efflux and repolarises neurons

Phospholipase C binds to calcium-dependent calcium channels, which causes calcium influx and secretion in salivary gland cells

Front 27

What ion channels do G(olf) proteins have an effect on?

Back 27

cAMP binds to cAMP-mediated sodium channels, causing sodium influx that depolarises olfactory sensory cells

Front 28

What ion channels do G(t) proteins have an effect on?

Back 28

Reduced cGMP prevents binding to cGMP-mediated sodium channels, which prevents sodium influx and causes hyperpolarisation in rod cells

Front 29

What ion channels do G(i) proteins have an effect on?

Back 29

The beta-gamma subunit binds to inwardly rectifying potassium channels, which causes potassium influx that hyperpolarises neurons and cardiac myocytes

Front 30

How does the G(t) protein enable light to be detected by rod photoreceptors?

Back 30

1. In the light, rhodopsin protein in the membrane is bleached, which causes the exchange of GDP for GTP on the G(t) protein, which is also called transducin

2. The alpha subunit dissociates and binds to phosphidiesterases

3. These break down cGMP to form GMP, thus reducing the intracellular concentration of cGMP

4. There is therefore less cGMP in the cytosol to bind to cGMP-dependent sodium channels

5. Less sodium enters, hyperpolarising the rod cell

Front 31

Describe how GPCRs are downregulated.

Back 31

1. An agonist binds to the GPCR, activating it and causing the G-protein to become dissociated

2. The GPCR then becomes phosphorylated by G-protein receptor kinases at intracellular phosphorylation residues

3. Arrestin then binds to the receptor, desensitising the receptor so it can no longer bind G-proteins

4. Arrestin also stimulates the internalisation of the receptor

5. The internalised receptor then becomes dephosphorylated and the arrestin stops binding, enabling the receptor to be re-inserted into the membrane

Front 32

How can protein kinase A or C downregulate GPCRs?

Back 32

They phosphorylate the receptor and have the same function as arrestin - they cause it to become desensitised and internalise it

Front 33

Describe how receptor tyrosine kinase activation leads to downstream signalling.

Back 33

1. A molecule such as a growth factor binds to the receptor, causing it to dimerise with another receptor

2. This causes the autophosphorylation of the receptor at 4 specific tyrosine residues on the cytosolic part of the receptor, activating it to give it enzymatic kinase activity

3. This causes the recruitment of signalling complexes to the receptor, where they detect and bind specific residues on the cytosolic part of the receptor

4. The signalling complexes are then activated through phosphorylation by the tyrosine kinase activity of the receptor

5. The signalling complexes may be enzymatic upon activation, or they may be adaptor proteins so recruit other molecules to give them enzymatic activity - both of these result in downstream signalling

Front 34

With regards to receptor tyrosine kinases, what does the SH2 domain of signalling complexes bind?

Back 34

Phosphotyrosines

Front 35

With regards to receptor tyrosine kinases, what does the PTB domain of signalling complexes bind?

Back 35

Phosphotyrosines

Front 36

With regards to receptor tyrosine kinases, what does the SH3 domain of signalling complexes bind?

Back 36

Proline-rich receptor regions

Front 37

With regards to receptor tyrosine kinases, what does the PH domain of signalling complexes bind?

Back 37

Phospholipids in the membrane

Front 38

Describe the function of src as a signalling protein.

Back 38

It has an SH2 and SH3 domain and binds to receptor tyrosine kinase via SH2 - it has intrinsic enzymatic activity

Front 39

Describe the function of phospholipase C gamma as a signalling protein.

Back 39

Binds to receptor tyrosine kinase via SH2 and to membrane phospholipids via PH - it has intrinsic enzymatic activity

Front 40

Describe the function of STAT-1 as a signalling protein.

Back 40

A transcription factor - when phosphorylated it dimerases and moves to the nucleus to initiate transcription - it is phosphorylated by src and has intrinsic enzymatic activity

Front 41

Describe the function of GAP-1 as a signalling protein.

Back 41

It is a Ras-GTPase activating factor, meaning it regulates the function of G-proteins by stimulating GTPases which exchange GTP for GDP, thus inhibiting the activation of G-proteins - it binds to receptor tyrosine kinase via SH2 or to the membrane via PH and has intrinsic enzymatic activity

Front 42

Describe the function of Grb-2 as a signalling protein.

Back 42

It is an adaptor protein, so recruits other molecules to give it enzymatic activity

It is closely associated with Sos via its SH3 domain - Sos is a Ras guanine nucleotide exchange factor, which opposes the activity of GAP-1 by exchanging GDP for GTP, therefore activating G-proteins

Front 43

How are adaptor proteins used in the insulin receptor pathway?

Back 43

1. Insulin binds to the already dimerised insulin receptor, stimulating the receptor tyrosine kinase activity of the receptor

2. This causes the receptor to be autophosphorylated on the cytosolic residues

3. An adaptor protein is recruited called the insulin receptor substrate

4. The insulin receptor substrate is phosphorylated, causing it to recruit signalling molecules such as the lipid kinase PI3 kinase

5. PI3 kinase phosphorylates the lipid PIP2 to produce PIP3

6. PIP3 phosphorylates protein kinase B, which binds to the glucose transport vehicle GLUT4

7. This causes GLUT4 to be translocated to the membrane where it is inserted

8. Glucose can then pass into the cell through the channel along a concentration gradient

Front 44

The signalling molecules in which 2 pathways are frequently mutated in cancer?

Back 44

MAP-kinase and PI3 kinase pathways

Front 45

Give 4 examples of receptor tyrosine kinases.

Back 45

Insulin receptor

Vascular endothelial growth factor receptor

Insulin-like growth factor receptor

Epidermal growth factor receptor

Front 46

In which 2 ways may PI3 kinase activate its effectors?

Back 46

May induce a conformational change to give it increased enzymatic activity, or increase its affinity for its substrates

May translocate the effector to the membrane to increase the concentration of effector and its binding substrates in one area

Front 47

Describe the MAP-kinase cascade.

Back 47

1. A ligand binds to a receptor tyrosine kinase, causing it to dimerise and gain tyrosine kinase activity

2. This results in autophosphorylation and the recruitment of Grb-2, which binds to the cytosolic part of the receptor via SH2 domains

3. Grb-2 is closely associated with Sos, and the phosphorylation of Grb-2 causes the activation of Sos

4. Sos is a Ras guanine nucleotide exchange factor, which stimulates the binding of GTP to Ras

5. This initiates a pathway of phosphorylation which leads to the phosphorylation of transcription factors in the nucleus at specific amino acids

6. This allows cell division to occur

Front 48

Describe what cytokine receptors are.

Back 48

They are a type of membrane receptor associated with janus kinase enzymes, which are intracellular non-receptor tyrosine kinases, that induce signals via the JAK-STAT pathway to affect transcription

Front 49

How does the JAK-STAT pathway work?

Back 49

1. Cytokine binds to a cytokine receptor, causing cross-links to form between adjacent receptors, forming a dimer

2. This causes the janus kinase enzymes on the adjacent receptors to phosphorylate each other on their tyrosine residues

3. This results in the recruitment of proteins such as STAT-1 and STAT-2

4. When STAT-1 and STAT-2 bind, they are phosphorylated by janus kinase on their tyrosine residues

5. This causes them to dimerise via their SH2 domains and dissociate from the receptor

6. The phosphorylated STAT dimer is then translocated to the nucleus, where it functions as a transcription factor

Front 50

What type of receptor is a beta1-adrenoceptor?

Back 50

G(s)-protein-coupled receptor

Front 51

What type of receptor is a beta2-adrenoceptor?

Back 51

G(s)-protein-coupled receptor

Front 52

What type of receptor is an alpha1-adrenoceptor?

Back 52

G(q)-protein-coupled receptor

Front 53

What type of receptor is an alpha2-adrenoceptor?

Back 53

G(i)-protein-coupled receptor

Front 54

What type of receptor is a muscarinic receptor?

Back 54

G(q)-protein-coupled receptor

Front 55

What type of receptor is the receptor tyrosine kinase?

Back 55

A receptor with an enzymatic domain

Front 56

What type of receptor is the insulin receptor?

Back 56

A receptor with an enzymatic domain (receptor tyrosine kinase)

Front 57

What type of receptor is the epidermal growth factor receptor?

Back 57

A receptor with an enzymatic domain (receptor tyrosine kinase)

Front 58

What type of receptor is the vascular endothelial growth factor receptor?

Back 58

A receptor with an enzymatic domain (receptor tyrosine kinase)

Front 59

What type of receptor is the insulin-like growth factor receptor?

Back 59

A receptor with an enzymatic domain (receptor tyrosine kinase)

Front 60

What type of receptor is the cytokine receptor?

Back 60

A receptor associated with an enzyme

Front 61

What type of a receptor is guanylyl cyclase?

Back 61

Cytoplasmic/intracellular receptor

Front 62

Describe how guanylyl cyclase signalling occurs?

Back 62

1. Guanylyl cyclase may be associated with a receptor, in which case a ligand binding to the receptor will activate the guanylyl cyclase

2. Alternatively, nitric oxide may pass through the membrane and activate guanylyl cyclase directly in the cytoplasm

3. When guanylyl cyclase is activated, it aids the coversion of GTP to cGMP

4. The cGMP may be converted back to 5-GMP and then GTP by phosphodiesterases

5. The cGMP may also bind to protein kinase G, which is then activated and phosphorylates target proteins

6. Phosphatases counteract the effect of protein kinase G by dephosphorylating target proteins

Front 63

How does the activation of protein kinase G by cGMP cause smooth muscle cell relaxation?

Back 63

Protein kinase G phosphorylates calcium channels, inactivating them, which causes a reduced influx of calcium

Protein kinase G phosphorylates potassium channels, activating them, which causes an increased efflux of potassium that hyperpolarises the cell

Protein kinase G phosphorylates myosin light chain phosphatase, activating it, which causes it to dephosphorylate myosin light chain, preventing contraction

Front 64

What type of receptor is the cortisol receptor?

Back 64

Nuclear receptor

Front 65

How do cortisol receptors function?

Back 65

1. Cortisol receptors exist in the cytoplasm and have an effect on the nucleus, but in their inactive state they have a chaperone protein associated with them that prevents them from entering the nucleus through tiny nuclear pores

2. A small signalling molecule passes through the membrane into the cytosol and binds to the cortisol receptor

3. This causes the removal of the chaperone protein from the cortisol receptor, making it small enough to enter the nucleus

4. The receptor is translocated to the nucleus, where it acts as a transcription factor through its interactions with specific sequences of DNA

Front 66

What type of receptor is aldosterone?

Back 66

Nuclear receptor

Front 67

What type of receptor are anabolic steroids?

Back 67

Nuclear receptor