Membranes and Cell Signalling Coyler

Question 1

What is cross talk?

Answer 1

The influence of another pathway on a different one.

Question 2

Where and by who was cycling AMP discovered?

Answer 2

1959 by Sutherland.

Question 3

How is cyclic amp produced?

Answer 3

From ATP

Question 4

What does cyclic amp do?

Answer 4

Acts primarily through a protein kinase. Stimulates glucose mobilisation for glycogen, fight or flight response, triglyceride breakdown.

Question 5

Describe the production of cAMP?

Answer 5

Involves a receptor, a G-protein complex and the target/effector adenylate cyclase. When the ligand binds there is a conformational change allowing the G-protein to dock to the receptor. GDP is exchanged for GTP and the G-protein is activated and it’s three subunits fall apart. The alpha subunit moves independent of the beta and gamma subunits and performs work. The alpha subunit targets the effector and cAMP is produced from ATP.

Question 6

What are second messengers?

Answer 6

Primary signal usually has a chemistry that cannot enter the cell therefore secondary messengers are required to convey the signal inside. Can be stimulatory or inhibitory.

Question 7

What are PDE?

Answer 7

Phosphodiesterase, any enzyme that breaks a phosphodiester bond, 11 types.

Question 8

How do PDE classes differ?

Answer 8

All have similar catalytic pore for the similar catalytic function and differ in regulatory and targeting features.

Question 9

What does the work in the flight or fight response?

Answer 9

cAMP

Question 10

What does cAMP do in the flight and fight response?

Answer 10

Changes the contraction from slow shortening and lengthening of contractile to a vigorous and rapid process. Drives phosphorylation in key sites to bring about profound changes in cardiac output and heart rate.

Question 11

Why is control of fight or flight important?

Answer 11

To limit cAMP production to a short/small pulse

Question 12

What do pathogens do to the flight or fight response?

Answer 12

They short circuit the control systems. Eg. Cholera toxin or bordettella pertusis

Question 13

What are the control points of the fight or flight response?

Answer 13

Receptor is optimised for inactivity, receptor is internalised by the cell, G alpha hydrolyses GTP, GAP activates GTPase activity and therefore spreads the reaction to turn off the production of cAMP

Question 14

Describe the catalytic enterty of cAMP-dependent protein kinase?

Answer 14

Small globular protein with 2 domains (N and C terminal), has specific constant between substrate and protein. Two subunits for catalytic activity and one for control or regulation.

Question 15

What is the cAMP-dependent kinase deterred by?

Answer 15

Tyrosine at phosphorylation site: acidic residues at P-1, K at P+2 or F at P+4.
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Limited motif for the enzyme to read and activate on.

Question 16

What is IP3?

Answer 16

Phosphoinositol 1,4,5 triphosphate

Question 17

What are the roles of IP3?

Answer 17

Contraction in smooth muscles, fertilisation, aggregation of platelets (blood clots), secretion (cells of adrenal cortex).

Question 18

Where does IP3 come from?

Answer 18

The internal leaflet of the plasma membrane, it’s substrate is a lipid with a inositol head group which is linked to a glycerol backbone as phosphatidylinositol

Question 19

What are the two mechanisms of activation of phospholipase c?

Answer 19

Family of GPCRs which respond to light, acetylcholine and serotonin or platelet derived growth factor receptors

Question 20

What’s the difference in the mechanism of regulation between GPCR activation of PLC and cAMP?

Answer 20

Both the alpha and beta gamma subunits have activity to activate PLC but only the alpha subunit activates cAMP

Question 21

What is the mechanism of growth factor activation of phospholipase C?

Answer 21

Ligand binds to two platelet derived growth factors bringing the two kinase domains on the receptors close together which autophosphorylate one and other. Target residues are phosphotyrosine, SH2 domains recognise these. PlCgamma has a SH2 domain that is targeted to the PDGF.

Question 22

What inhibits PLCgamma?

Answer 22

PKA can accces other sites on PLCgamma and phosphorylate to inactivate.

Question 23

How can you destroy IP3?

Answer 23

Remove or add phosphate groups.

Question 24

What is IP3 receptor?

Answer 24

The IP3 receptor, a calcium channel

Question 25

What organelle primarily stores calcium?

Answer 25

The ER, has a calcium pump, a buffer and a IP3 receptor to discharge the calcium ions when the ligand is around (a channel so only let’s it go out).

Question 26

Describe the IP3 receptor?

Answer 26

Large size in order to tightly control opening and closing, 2700+ residues, 3 isoforms, assemble as a tetramer, highly selective only takes IP3 (1,4,5).

Question 27

How is the IP3 receptor regulated?

Answer 27

By multiple binding sites for luminal and cytosolic calcium, ATP, phosphorylation (PKA) etc.

Question 28

What does IP3 binding to a receptor cause?

Answer 28

The release of calcium.

Question 29

How is calcium concentration studied?

Answer 29

Two reporter groups: photo protein (aequorin) and fluorescent dyes bases on EGTA (Fura-2, Fura-3)

Question 30

What is capacitative calcium entry?

Answer 30

The calcium that comes from the blood across the plasma membrane

Question 31

What does calcium do?

Answer 31

Interacts with many protein to alter function sometimes directly and other times through regulatory proteins.

Question 32

Give some methods of direct alteration of function of proteins with calcium?

Answer 32

Channels, protease and Ligases.

Question 33

Give some methods of indirect alteration of function of proteins with calcium thorough regulatory proteins?

Answer 33

Calmodulin and annexins

Question 34

Describe calmodulin?

Answer 34

A ubiquitous, abundant protein. 16.8 kDa, 4 calcium binding sites with a particular structure.
Dung-bell structure with calcium bidding sites on either end and a site for calcium collation in the centre palm of the EF-hand structure.

Question 35

What does calmodulin affect?

Answer 35

Cyclic nucleotide metabolism, glycogen meta phloem, calcium transport, smooth muscle contraction

Question 36

What does calmodulin require to become active?

Answer 36

All 4 sites to bind calcium

Question 37

What happens when calmodulin is activated?

Answer 37

The long central helix buckles through a substantial conformational change, exposing a rich in methionine hydrophobic domain.

Question 38

What is the cluster of methionine residues called when calmodulin is activated?

Answer 38

A methionine puddle

Question 39

What is the simple motif that activated calmodulin interacts with and why?

Answer 39

A bipartite characteristic motif with positive charges followed by hydrophobic residues. It wraps around it to influence the protein.

Question 40

How is neurotransmitter release controlled?

Answer 40

Calcium dependent. Synapsin one is central to release and is at an interface to CaM kinase 2. Phosphorylation of synapsin by CaM kinase 2 releases it from the cytoskeleton which occurs when calcium is present.