G protein coupled receptors 2

Question 1

What are some examples of signals activating the Gq class of G proteins

Answer 1

1. Vasopressin,
2. Acetylcholine neurotransmitter
3. Thrombin,

Question 2

What is vasopressin

Answer 2

1. hormone originating in the hypothalamus.
2. Acts on kidney to promote water reabsorption and blood pressure,
3. liver to promote gluconeogenesis.

Question 3

What is acetylcholine neurotransmitter

Answer 3

1. many functions.
2. Amylase secretion in pancreas
3. Muscle contraction in smooth muscle cells

Question 4

What is thrombin

Answer 4

1. made in liver goes into circulation

2. involved in blood clotting and platelet aggregation.

Question 5

What pathway do Gq proteins activate

Answer 5

1. Gq proteins activate the inositol phospholipid pathway

Question 6

Describe activation of inositol phospholipid pathway

Answer 6

1. Initial activation of the G protein is the same as for Gs, however, the target enzyme in the PM is phospholipase C-beta.
2. This hydrolyses PIP2 to form IP3 and DAG, with each then activating downstream signalling targets.
3. Note cross-talk with MAP-kinase pathway.
4. PIP2 (also PI and PIP) is also a substrate of PI3-kinase in the RTK/Ras/MAP kinase signalling pathway

Question 7

What does Phospholipase C-b

Answer 7

1. Phospholipase C-b cleaves PI(4,5)P2
2. When cleaved it forms diacylglycerol which remains associated with the membrane and activates protein kinase c
3. Inositol can act as small second messenger as no longer attached to plasma membrane

Question 8

Describe cross-talk between Gq and Ras/MAPK signalling pathways

Answer 8

1. C-kinase can phosphorylate raf and this changes the conformation and causes its activation
2. Raf is target for Ras and can bind to it and cause its activation
3. Cross-talk both can act on same target- Raf
4. Sometimes signals reinforce message or antagonise- more complex effects

Question 9

What does protein kinase C activate

Answer 9

1. Protein kinase C activates NFκ-B and Raf

Question 10

What is NFk-B

Answer 10

1. NF-κB is found in almost all animal cell types and is involved many cellular responses including, inflammation, proliferation and survival.
2. Mis-regulation of I-kappa-B is important in many cancers and the pathway is a target for several oncogenic viruses, e.g.
   a) T-cell leukemia virus type 1 (HTLV1),
   b) Kaposi sarcoma-associated herpesvirus (KSHV),
   c) Epstein bar virus (EBV).
3. NF-kB is a transcription factor

Question 11

When is NF-kB activated

Answer 11

1. PKC inactivates the inhibitor of NFk-B
2. Normally sequestered in cystosol so can’t act on gene activity
3. Held by Ik-B
4. When phosphorylated ik-B conformation changes
   5 . Recognised by ubiquitin and targeted for proteosome degradation
5. Releases NF-kB so can translocate into nucleus and activate specific genes

Question 12

What is key role of IP3

Answer 12

1. IP3 is a second messenger
2. Main role is to cause release of Ca2+ from ER stores
3. Diacylglycerol activates protein kinase C
4. IP3 opens IP3 gated calcium release channels
5. Allows calcium to flood out
6. Full activation of Protein kinase c involves activation with diacylglycerol and interaction with calcium

Question 13

What is required for full activation of protein kinase c and what experiment shows this

Answer 13

1. Both DAG and Ca2+ are required for full activation of PKC as demonstrated by treatment of cells with TPA and ionomycin.
2. TPA is a carcinogenic plant derivative phorbol ester compound.
3. The Ca2+ ionophore, ionomycin, is a small lipophilic molecule that can bind Ca2+ and transport it across membranes.
4. TPA is used in conjunction with chemical mutagens in experimental models for initial events in papilloma skin cancers.

Question 14

How does Ca2+ act as an intracellular messenger

Answer 14

1. Not just GPCR signalling but other factors can lead to lead to increased cytosolic Ca2+ concentration.
2. Ca2+ plays a role in (e.g.):
3. Triggering embryo development after fertilization
4. Muscle contraction
5. Secretion in nerves and other secretory cells
6. Effects are mediated by Ca2+ response proteins

Question 15

How are Ca2+ levels kept low

Answer 15

1. Ca2+ pumps on PM export Ca2+ maintaining low levels in resting cells
2. Organellar Ca2+ pumps help re-establish resting Ca2+ levels
3. Ca2+ binding proteins in cytosol reduce free Ca2+ levels
4. Storage in ER is the major mechanism used to maintain low cytosolic [Ca2+] and as a source for Ca2+ signalling.

Question 16

What do Ca2+ pumps on PM do

Answer 16

1. Ca2+ ATPase in the plasma membrane (all cells)
2. Na+ /Ca2+ antiport; mainly in cells that rely heavily on
3. Incoming sodium ion replaces outgoing calcium
4. Ca2+ signalling (secretory cells, neurons and muscle)

Question 17

How do organellar Ca2+ pumps help re-establish resting Ca2+ levels

Answer 17

1. Ca2+ ATPase in ER membrane

2. active Ca2+ import in mitochondrion

Question 18

When is storage of Ca2+ in mitochondrion used

Answer 18

1. Storage in mitochondria is mostly an emergency measured,

2. e.g. in cells with a damaged plasma membrane with very high cytosolic Ca2+.

Question 19

What does IP3 do

Answer 19

1. IP3 opens Ca2+ channels on ER membrane

Question 20

What are the typical Ca2+ levels, extracellular, resting cell, stimulated cell, damaged cell

Answer 20

1. Typical extracellular Ca2+ level ~10-3M
2. Resting cell- 10^-7 M
3. Stimulated cell- 5*10^-6 M
4. Damaged cell- >10^-5 M

Question 21

What can intracellular Ca2+ levels be visualized with

Answer 21

1. C2+ sensitive fluorophores:
2. Fura-2
3. Aequorin

Question 22

What causes a rapid burst of Ca2+ release

Answer 22

1. A rapid burst of Ca2+ release results from positive feedback
2. Ca2+ binds to the IP3-gated channels
3. Ca2+ binds to ryanodine channels, also on ER membrane
4. Both result in rapid release of Ca2+ into the cytoplasm

Question 23

What causes the reversal of the IP3 signal

Answer 23

1. Reversal of the IP3 signal involves negative feedback
2. Lipid phosphatase converts IP3 to IP2- removes phosphate
3. Lipid-kinase- phosphorylates IP3 to IP4
4. The enzymes that turn over IP3 are themselves activated by Ca2+ and the IP4 product interacts with, and stimulates Ca2+ ATPases that pump Ca2+ out of the cytosol.
5. In this way IP4 is part of a negative feedback loop.

Question 24

How do hepatocyte respond to different concentrations of vasopressin

Answer 24

1. Hepatocyte response to increasing concentrations of vasopressin
2. Liver cells in culture.
3. Different concentrations of vasopressin
4. Cytosolic Ca2+ levels measured using Ca2+-sensitive fluorophore.
5. Ca2+ spike amplitude is constant, but frequency increases with vasopressin concentration.
6. Cells are able to interpret ‘spikes’ in Ca2+ levels, without allowing [Ca2+] to become dangerously high, through decoding spike frequency.

Question 25

Why do Ca2+ levels oscillate

Answer 25

1. Intracellular Ca2+ levels oscillate due to the +ve and –ve feedback mechanisms 2. Positive feedback: Ca2+-induced Ca2+ release by (IP3 - and ryanodine channels) 3. Negative feedback: high Ca2+ stops further Ca2+ release through IP3 removal and (IP4 ) promotes Ca2+ removal from cytosol

Question 26

How does the frequency of Ca2+ oscillations influence response

Answer 26

1. Frequency dependent cell responses: 2. Release of pituitary hormones is pulsatile and pulses follow the frequency of Ca2+ spikes. 3. Some cells activate transcription of different sets of genes in response to specific frequencies of spikes.

Question 27

Describe calmodulin structure

Answer 27

1. Calmodulin Mediates Many Responses to raised Ca2+ levels 2. Non enzymatic protein that is the key interpreter of calcium levels 3. Helical domain 4. At either end has folded structures which contain calcium binding sites

Question 28

How does calmodulin mediate responses

Answer 28

1. Calcium ions can bind to calmodulin binding sites 2. As long as two sites are occupied the molecules undergoes small conformational change 3. Enables it to interact with target protein- wraps around it 4. Target protein interaction induces a more dramatic conformational change in which calmodulin wraps itself around a regulatory domain, which in turn alters the conformation and activity of the target. 5. No intrinsic enzymatic activity. 6. Can bind and stimulate Ca2+ATPase (one of the target proteins) in plasma membrane (part of negative feedback).

Question 29

What role do Ca2+/calmodulin dependent kinases (CaM-kinases) play

Answer 29

1. CaM-kinases are serine/threonine kinases 2. Certain CamKs can activate CREB (cross-talk with Gs/cAMP signalling). 3. Response to increasing Ca2+is cell type specific.

Question 30

What CaM-kinases have narrow specificity

Answer 30

1. Myosin light-chain kinase; involved in smooth muscle contraction. 2. Phosphorylase kinase; 3. glycogen breakdown in liver (note cross-talk with Gs/cAMP/PKA pathway).

Question 31

What CaM-kinases have broad specificity

Answer 31

1. CaM-KII; 2. multifunctional and ubiquitous; 3. enriched in neurons that use catecholamine neurotransmitters (dopamine, adrenaline, noradrenaline).

Question 32

How does CaM-kinase II act as a molecular memory device

Answer 32

1. Way to sustain a response to a signal even after the signal has gone 2. Calcium calmodulin 3. Kinase- Inhibitory domain interacts with catalytic domain 4. Calcium calmodulin binds to inhibitory domain and allows the kinase to be released so it is partially active 5. Allows phosphorylation of kinase 6. Allows active conformation of kinase even in absence of calmodulin

Question 33

What does structure of Ca2+/Calmodulin-Dependent Protein Kinase II allow

Answer 33

1. Ca2+/Calmodulin-Dependent Protein Kinase II is a hexameric complex 2. Allows subtle control over activity of complex 3. Can activate and keep active different numbers of hexameric subunits

Question 34

What role does CaM-K II molecular memory function have in spatial memory

Answer 34

1. Role in vertebrate learning and memory 2. KO Mice lacking a brain-specific form of CAM-K II have specific defects in their ability to remember where things are (spatial memory) and are slower to develop a response to fear stimuli. 3. Activity of active CAM-K II in specific neurons likely alters long-term neuron function and ‘wiring’.

Question 35

How can CaM-K II as a Ca2+ spike frequency decoder

Answer 35

1. CaM-K II autophosphorylation and delayed inactivation can distinguish between different frequencies of Ca2+ oscillation 2. As CAM-K II activity reaches maximum it becomes increasingly difficult for phosphatases to compete. 3. Once all sub-units of each CAM-K II complex are phosphorylated full activity can be maintained in relatively low Ca2+ levels.

Question 36

Describe desensitisation of G protein-coupled receptors

Answer 36

1. All require phosphorylation of the receptor by either PKA, PKC or G protein coupled receptor regulatory kinase (GRK). 2. Interaction of the activated receptor with G-protein is reduced by phosphorylation serine/threonine residues on cyto loops, and blocked by binding of arrestin to the phosphorylated receptor. 3. In longer term if signal persists, Provokes internalisation of receptors

Question 37

Describe Cross-talk between Gq and Gs signalling pathways

Answer 37

1. Ca2+ and cAMP levels can influence each other a) e.g. cyclic AMP phosphodiesterase and adenylyl cyclase regulation by Ca2+ /calmodulin; b) e.g. cAMP/PKA can influence IP3-induced Ca2+ release. 2. Enzymes directly regulated by Ca2+ and cAMP can influence each other, a) e.g. some CaM-kinases are phosphorylated by PKA. 3. Proteins directly regulated by Ca2+ and cAMP can influence the same downstream target molecules, a) e.g. PKA and CaM-kinases often phosphorylate different residues on the same protein, such as CREB; b) e.g. phosphorylase kinase is regulated by PKA and by Ca2+ /calmodulin.