Beta 2 Adrenoreceptor Regulation

Question 1

When adrenaline is circulating the body is comes into contact with with what receptors specifically in the liver and skeletal muscles?

Answer 1

Beta 2 adrenoreceptors

These are G protein coupled receptors!

Question 2

What do beta 2 adrenoreceptors in the body do? What are they coupled with? And what pathway do they activate

Answer 2

They detect adrenaline

They provide the bodies muscles with the energy to contract needed in a fight or flight scenario

Remember These beta 2 adrenoreceptors are G protein coupled receptors
The receptor is coupled with alpha and beta G protein sub units

The adrenoreceptor sends signals to the alpha subunit it is ATTACHED to. This then allows a signal to pass through the Gs pathway! The receptor’s alpha sub unit becomes ACTIVATED>

Question 3

So what happens when adrenaline binds to beta 2 adrenoreceptors?

And remember these receptors are G protein coupled receptors

Answer 3

When the adrenaline binds it causes a signal to be sent from the receptor to the alpha sub unit the receptor is bound to

On the alpha sub unit this causes a GTP to bind which is exchanged for a GDP.

When the GTP is BOUND to the alpha sub unit … the alpha and beta subunits can DISSOCIATE from each other and the receptor

These sub units can go onto work on downstream effectors

Question 4

When happens when the alpha sub unit from the receptor dissociates?

Answer 4

The alpha sub unit becomes activated. This is referred to as the stimulatory G alpha S sub unit

This works on the enzyme adenylate cyclase

This produces a second messenger cyclic AMP which works further downstream

Question 5

How does the beta 2 adrenoreceptor and the work of adenyl cyclase allow for signal amplification?

Remember this receptor is G protein coupled and works when adrenaline binds to it.

Answer 5

• while the beta 2 adrenoreceptor has an agonist bound it can activate multiple G proteins

While the alpha sub unit (the stimulatory G alpha S protein) is bound to adenyl cyclase, it allows for lots of cyclic AMP to be made

This is amplification!

Question 6

What does cyclic AMP do once it has been produce by adenyl cyclase after the GTP bound alpha S subunit from the adrenoreceptor has bound?

Answer 6

It associates with regulatory subunits found on protein kinase A

This causes the regulatory sub unit bound to the kinase to LEAVE the kinase

The kinase then becomes ACTIVATED

Question 7

What happens once the CAMP activates protein kinase A?

Answer 7

This kinase A then uses the energy of ATP to phosphorylate other kinases such as calmodulin phosphorylase kinase.

Question 8

What does calmodulin phosphorylase kinase do?

Note this is activated by kinase A

Answer 8

So once ACTIVATED This kinase is sensitive to intracellular levels of calcium and calcium which binds to calmodulin

The phosphorylation of this calmodulin P Kinase lowers it sensitivity to the calcium

This means it can become ACTIVATE (as in the kinase has activated it already and thus it is ready to become active) at lower levels of calcium

The role of this enzyme is to phosphorylate the enzyme phosphorylase P

Question 9

What does the enzyme phosphorylase P do?

Remember protein kinase A phosphorylates and makes calmodulin phosphorylase kinase become ACTIVATED

This calmodulin kinase then phosphorylates enzyme phosphorylase P

Answer 9

This enzyme metabolises the conversion of gylcogen to glucose 6 phosphate

This allows glucose 6 phosphate to be cleaved off of glycogen

This provides the energy in the liver and skeletal muscles for contraction. Via the production of ATP via glycolysis.

Question 10

Again what does the beta 2 adrenoreceptor do?

Answer 10

It responds to adrenaline

It activates a g protein

This g protein activates adenyl cyclase which produces cAMP. This cAMP is used to activate protein kinase A, then calmodulin phosphorylase kinase. This calmodulin remains activated until it detects calcium (low levels can be sensed due to phoshphorylation of kinase A)

This then is activated activating phosphorylase P.

This phosphorylase helps break down glycogen into glucose 6 phosphate

This is then broken down to provide the energy for muscle contraction.

Question 11

What are the two ways in which signalling from the beta 2 adrenoreceptor is limited?

(Note this would stop glucose metabolism from THIS pathway and stop muscle contraction in response to adrenaline)

Answer 11

1. GTPase action on the alpha sub unit can cause the GTP bound G alpha protein to then become GDP bound. In this form the alpha sub unit can no longer bind to the adenyl cyclase.
2. The beta arresting kinase can stop further signalling.

Question 12

How does the beta arresting kinase work? Which of the g protein sub units does it work with from the original beta 2 adrenoreceptor G protein complex?

(Note it acts as a negative feedback system)

Answer 12

When the beta 2 adrenoreceptor has been under prolonged stimulus the BETA gamma subunits which has detached from the original G protein coupled receptor complex goes onto recruit a receptor kinase

This kinase phosphorylates the carboxyl terminus of the beta 2 adrenoreceptor

This attracts a protein called beta arresting

This stops signalling to the alpha sub unit of the complex

Note: this happens after some time! In effect the receptor becomes DESENSITISED overtime!
The receptor no longer responds to the prolonged stimulus.

Question 13

What does the G alpha Q protein do?

(Note this G protein is not exclusive to the beta 2 adrenoreceptor as it works on other G protein coupled receptors!

Answer 13

It controls the activity of phospholipase C which is a lipase that breaks down lipids

These types of second messengers are derived from membrane lipids

The breakdown of these membrane lipids create second messengers like IP3 and DAG

NOTE beta gamma sub units may also have a similar role!

Question 14

What receptors use Lipid derived signalling ?

Answer 14

Many!

For example the lipid derived messenger IP3 can interact with calcium, helping signalling from ligand gate ion channels for example.

Question 15

What are the three types of lipid membranes broken down to make secondary lipid messengers? And what is the main one?

Answer 15

PIP2 - phosphatidylinositol 4,5 bisphosphate

PC

Sphingomyelin

Question 16

What is the structure of PIP2 and what enzymes break it down into lipid derived second messengers?

(Remember cAMP is a type of second messenger too)

Answer 16

• this PIP2 is made up of phosphatic acid, diacylglycerol and IP3, arachidonic acid

These are important second messengers!

Enzymes such as phospholipase C, D and A2 are the main enzymes which break down this membrane phospholipid into second messengers

Question 17

In particular what breaks down the bond between PIP2 and IP3? And what does the IP3 do?

Answer 17

Phospholipase C (remember this is regulated by G alpha Q proteins) breaks the phosphodiester bond between PIP2 and the IP3. This allows it to be liberated.

It can go onto affect effector proteins some distance away from its production

NOTE - phospholipase D can also liberate the IP3 but this causes the IP3 not to be a triphosphate! This causes the IP3 to be phosphatic acid instead!!

Question 18

What is a good characteristic of the IP3 lipid derived second messenger?

Answer 18

It is hydrophilic - thus it dissolves well in the cytoplasm (it isnt scared of water)

Question 19

What enzyme liberates DAG from PIP2? And whats a characteristic of DAG?

Answer 19

Phospholipase C

DAG is hydrophobic. It doesn’t dissolve in the water of the cytoplasm as well so it mainly stays around the plasma membrane.

Question 20

What does phospholipase A2 do to PIP2, the membrane phospholipid?

Answer 20

It liberate arachidonic acid

And remember this is used by the COX enzymes attached to the ER in the cell, used for calcium signalling?

Question 21

Summary of steps in lipid liberation to make lipid derived second messengers?

Answer 21

A receptor activates G alpha Q

This then liberates a G beta gamma sub unit from a G protein coupled receptor
(Remember these receptors typically have an alpha and beta sub unit)

This then act on phospholipases

By activating this enzyme, they generate a second messenger such as Ins P3 (IP3) or DAG

This regulates effector function downstream

Question 22

The G alpha Q’s most common pathway involves the breakdown of PIP2 into DAG and IP3 which are second messengers. What do these second messengers do?

Answer 22

DAG the second messenger stimulates Protein kinase C (this is not the same kinase that activates calmodulin phosphorylase kinase, this is kinase A._

Kinase C works to phosphorylate proteins in the plasma membrane not in the G protein S pathway!!

IP3 woks on Ca2+ channels and IP3 receptor.

Note PI3 kinases can also break down PIP2 into protein kinase C pH domain proteins

Question 23

Note - the enzymes involved in breaking down the phospholipids in the plasma membrane differ between different G protein pathways. However they all have the same properties

What are some similar properties?

Answer 23

They have similar catalytic domains which break down the phosphodiester bond which generates DAG and IP3

They all have regulatory domains

They have PH domains

Some have calcium binding domains - showing calcium binding is important

Question 24

What class of enzyme does the G alpha Q protein work on and what part of the enzyme recognises it?

Answer 24

• the phospholipase C beta 1 -4 isoforms
• these enzymes have extended carboxyl terminals

These recognise the G alpha Q sub units

Question 25

What is important to note about the different isoforms (versions) of the enzymes which break down the phospholipids in the plasma membranes?

Answer 25

The amount of these enzymes vary in different compartments of the cells in the plasma membrane

And they vary in different locations.

Question 26

What is the difference between PKA and PKC (enzymes)

Answer 26

They are structured very similarly

Differences:

PKA is regulated by cyclic AMP

Protein kinase C is regulated by DAG

Similar? Both of these kinases phosphorylate proteins at the site of serine and threonine kinases.

Question 27

What are protein kinase C’s (not A) activated by?

Answer 27

DAG on the enzymes C1 domains

Or calcium 2+ on the C2 domain

Question 28

Where does protein kinase C work and why is this obvious when we consider that DAG stimulates it?

Answer 28

Remember DAG is hydrophobic. It doesnt dissolve in the ctyoplasm of the cell and stays near the membrane.

Thus protein kinase C moves towards the membrane of the cell and works there

Question 29

How to track protein kinase A (involved in the beta 2 adrenoreceptor pathway for muscle contraction amongst others) and protein kinase C (stimulated by DAG controls the function of other proteins)

Answer 29

At the end of the carboxy terminus of the enzyme, tag a fluorescent protein like GFP

Then use microscopy to track

Question 30

Instead of having a receptor activated for the phospholipid membrane to be broken down to produce DAG and recruit protein kinase C, what can we do to by-pass this and recruit kinase C to the membrane?

Answer 30

Use a drug called PMA which mimics DAG

Putting it onto the cell drags the protein kinase C to the plasma membrane without the need of a receptor being stimulated

Note when the receptor stimulation is what is causing the kinase C to move to the plasma membrane via PIP2 and DAG, recruitment of PKC to the membrane is transient. Stimulation of the receptor can be turned off at any point and DAG can be broken down

This drug PMA isnt broken down unliked DAG. So it constantly will recruit protein kinase C

Question 31

How does the protein kinase C turn itself off?

Answer 31

Both the PKC and PKA have an inhibitory peptide

When the kinases are at rest and arent active - an inhibitory peptide folds over the enzymes catalysing domains which stops them from phosphorylating and breaking things down

The inhibitory peptide which are PART of the kinase has an alanine attached, stopping the enzyme from being phosphorylated and breaking anything down.

When the kinase is turned on its inhibitory peptide doesnt cover the catalytic site.

So when its time for the kinase to be turned off, this inhibitory peptide covers the peptide binding sites hole.

Question 32

What does DAG do to the inhibitory peptide which usually blocks protein kinase C’s catalytic site?

Answer 32

The DAG can push out the inhibitory peptide, so the DAG can replace it and turn on the protein kinase C

Question 33

Why is there a competition for turning on / off the protein kinase C? /A

Answer 33

This is because substrate such as DAG and inhibitory peptide compete for the catalytic site of both enzymes to turn them on / off

Question 34

What do protein kinase C’s do?

Answer 34

Phosphorylate many plasma membrane proteins
This may affect activity of downstream effectors

Protein kinase C can phosphorylate phospholipase C beta for instance and turn it off.

Protein kinase C may also phosphorylate other GPCRS. This may cause some pathways to be turned off or on.

Question 35

What does MONK 13 do?

Answer 35

This has a C1 domain which recognises DAG

This controls the docking of secretory vesicles in the plasma membrnae

Question 36

Mutations in the pip2 enzymes?

Answer 36

Lowe syndrome

• issue cause by not removing the 4 phosphates from PIP2

This causes a build up of pip2 in the membrane = aberrant signalling

This can affect they eyes and cause glaucoma - may also affect kidneys and causes retartdation

Question 37

What is the IP3 receptor and what does it do?

Answer 37

It is a ligand gated ion channel - ip3 controls this

Found in the ER membrane

The IP3 can bind to this receptor and cause the calcium to move out of the ER

Question 38

How do high levels of calcium not crystalise in the ER? And how does calcium get into the cytosol from the ER!!! (Note calcium can also enter the cytosol from the plasma membrane)

Answer 38

Proteins bind to the calcium inside of the ER stopping it from crystalising and increasing levels.

Note calcium levels in the cytosol are very low. This allows for a concentration gradient into the cytosol

Calcium moves out through IP3 receptor and the calcium pumps

Question 39

Two proteins involved in sequestering calcium in the ER

Answer 39

Calsequesterin and calreticulin

Question 40

How can calcium enter the cytosol?

Answer 40

Through LIGAND GATED and VOLTAGE gates channels in the plasma membrane

Through ligand gated channels in the ER

Question 41

What are ORAI channels sensitive to? And what is the STIM protein

Answer 41

Calcium levels in the ER

When levels decrease this is called store emptying

When the ER wants calcium levels to increase STIM proteins in the ER membrane detect this lowness and interact with ORAI channels in the plasma membrane - this opens the channel (in the plasma membrane)

This causes calcium to enter the cell and then the ER for store filling

Question 42

Calcium receptors in plasma membrane vs ER:

Answer 42

plasma membrane:
L type
ATP activated

ER:
IP3
Type 1 ryanodine

Question 43

Ryanodine receptor?

Answer 43

Found on the ER

Calcium release

Gated by calcium - it allows calcium to enter the cytosol when it detects calcium entering through the plasma membrane

In skeletal muscles this ensures there is enough calcium for a contraction

Question 44

Densitisation of IP3 receptors to IP3? And type 3 IP3 receptors?

Answer 44

As ip3 binds to ip3 receptors the receptors become more sensitive to ip3

You get speeding up of gating in the channels

However when ip3 is too high you get desensitisation you get transient signalling of the ip3 receptor

Type 3 ip3 receptor? Produces a prolonged and not a transient signal
- important for long muscle contraction

Question 45

Affect of GPCR mutations?

Answer 45

Diseases.

Which may affect effectors downstream - dont remember these just the idea that GPCRS activate effectors downstream