GPCRs 2

Question 1

Cellular Ca2+ levels:

Answer 1

Resting cells maintain cytoplasmic Ca2+ levels at amounts approximately 12,000 lower than extracellular levels.

One reason for this may be the requirement for phosphate which is used in biosynthesis and energy pathways. Ca2+ phosphate is insoluble.

The low cytoplasmic Ca2+ concentration also provides the ability to use transient changes in Ca2+ levels for signaling.

Ca2+ only diffuses short distances before be bound by other factors in the cytosol

Ca2+ stores exist in the endoplasmic reticulum and mitochondria.

Question 2

What are the two major mechanisms that exist to export Ca2+?

Answer 2

1, PMCA 
(plasma membrane Ca2+ ATPase)
High affinity for Ca2+
Low transport rate
Effective at maintaining low Ca2+

2. NCX 
(Na+/Ca2+ exchanger)
Low affinity for Ca2+
High transport rate
Effective at clearing high intracellular Ca2+

Question 3

Advantages of Ca2+ as a 2nd messenger?

Answer 3

1. Fast (Low Ca2+ levels in the cytoplasm relative to concentrations in intracellular Ca2+ stores or outside the cells means that opening ion channels in membranes can result in a very fast increases in cytoplasmic Ca2+ levels.)
2. Reversible (Ca2+ can be pumped back into intracellular stores from the cytoplasm. As it is quickly reversible repeated stimulation is possible. This allows repeated Ca2+ waves of oscillations; this allows for an increased ability to encode information in the signal. It also prevents a need for prolonged elevation in Ca2+ which may harm the cell.)

Question 4

How do GPCRs trigger Ca2+ signalling?

Answer 4

Ligand > GPCR > Heterotrimeric G Proteins > Activate Phospholipase C (Beta) > Interacts with a lipid on the membrane to produce both IP3 and DAG > IP3 stimulates IP3R channels on the ER or SR to release Ca2+ .

GPCRs, via Gq release, trigger the activation of PLCbeta.
PLCbeta produces both IP3 and DAG.
IP3 stimulates IP3R channels on the ER or SR to release Ca2+ .

Question 5

Phospholipases:

Answer 5

Cleave specific bonds in phospholipids
4 main groups based on which point they cleave the phospholipid.
Phospholipase C (PLC) is a subtype involved in GPCR signalling

Question 6

Phospholipase C (PLC):

Answer 6

Different types of phospholipids exist in membranes with different head groups, e.g. phosphatidylinositol (PI)

The inositiol ring of PI can be phosphorylated.

PLC converts phosphatidylinositol 4,5-bisphosphate (PIP(2)) to inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG).

6 subfamilies (Beta, gamma, delta, epsilon, zeta, eta)

Not all PLCs are activated by GPCRs

IP3 / DAG pathways can be activated independently of GPCR signalling.

DAG is retained in the membrane where it can activate DAG binding proteins.

IP3 is released into the cytoplasm where it can stimulate the release of Ca2+ from intracellular stores.

Question 7

IP3 stimulated Ca2+ release:

Answer 7

IP3 acts by stimulating IP3 receptors in the membrane of the endoplasmic reticulum (scaroplasmic reticulum or SR in muscle)

SR stimulation allows Ca2+ release from the intracellular stores.

IP3-R contain a transmembrane a-helical bundle, a cytoplasmic domain and exist as tetramers

Regulated by both IP3 and Ca2+

Question 8

Ca2+ release:

Answer 8

Ca2+ release from the ER occurs via IP3 receptor channels.

Ca2+ diffusion in the cytoplasm is limited by the binding of Ca2+ to various protein in the cytoplasm.

Ca2+ is transported back into the ER via “sarco-endoplasmic reticulum Ca2+ ATPases” (SERCAs).

Triggering Ca2+ release requires the amount of Ca2+ transported by the IP3 receptor to exceed that removed by the SERCA receptors.

Question 9

3 forms of Ca2+ release:

Answer 9

Blips (Low IP3)
Puffs (Intermediate IP3)
Waves (High IP3) (Propagation of channel opening along the membrane of the ER)

These different types of release are made possible by the way in which IP3 and Ca2+ regulate the IP3 receptor:
IP3 initially stimulates Ca2+ release but prolonged occupancy inhibits release
Increased Ca2+ concentrations in the cytoplasm, below a certain threshold, stimulate Ca2+ transport by the IP3 receptor

Question 10

Ca2+ entry from extracellular sources:

Answer 10

In addition to release from intracellular stores, Ca2+ can also enter the cytoplasm from outside the cell.

In the context of GPCR signaling this can occur via two mechanisms:

1. Store operated Ca2+ entry
2. Direct activation of plasma membrane Ca2+ Channels

Question 11

Ca2+ binding domains:

Answer 11

Ca2+ can bind to various proteins via specific Ca2+ binding domains:

1. C2 domain
2. EF hand domains

Question 12

Calmodulin:

Answer 12

Calmodulin is the prototypical EF hand protein

Calmodulin as 2 pairs of EF hands separated by a linker region (linker region is unstructured without Ca2+)

Can bind up to 4 Ca2+ ions; binding induces a conformation change in calmodulin

This allows calmodulin to interact with specific alpha helical structures in calmodulin binding proteins

Question 13

DAG can activate intracellular signalling:

Answer 13

DAG is generated by the action of PLC

It remains in the membrane

DAG is recognized by “C1” domains

C1 domains can be expressed alone or in tandem

Divided into ‘typical’ C1 domains that bind DAG and ‘atypical domains’ that have lost the ability to bid to DAG

Question 14

PKC Isoforms:

Answer 14

4 Main groups:

• Conventional
• Novel
• Atypical
• PKN

Question 15

cPKC activation requires phosphorylation, Ca2+, and DAG:

Answer 15

1) Phosphorylation:
PKC is phosphorylated on its C-terminus by the mTORC2 kinase complex

This creates a binding site for another kinase, PDK1, which phosphorylates the activation loop of PKC

At this point PKC is still inactive, as the pseudosubstrate motif inhibits activity

2) Membrane recruitment and activation:
Ca2+ binds to the C2 domain

The C2 domain then binds to PIP2 or PS lipids in the membrane

The C1 domain then binds diacyl glycerol in the membrane

The conformational change on DAG binding activates the PKC kinase domain

Question 16

C2 Domain:

Answer 16

C2 domains are approximately 130 amino acids mainly consisting of b-sheets.

Found in over 100 proteins, mostly with roles in signal transduction

Functions as a phospholipid binding domain

Some C2 domains (eg those in cPKC) require Ca2+ ; the binding of 2 – 3 Ca2+ in the loop regions of the C2 domain create a binding site for lipids with acidic or polar head groups.

Other C2 domain bind phospholipid in the absence of Ca2+

Question 17

EF Hand Domain:

Answer 17

EF hand is approximately 40 amino acids forming a helix loop helix motif

EF hands are normally present in pairs; binding of Ca2+ can be co-operative.

There are some exceptions (eg calpain) where there are 5 EF hands in one polypeptide – in this case the 5th EF hand mediates dimerization.

Two main types: in one Ca2+ binding to EF hands can induce a conformational change in the protein that can result in activation of other proteins or protein domains, in the other Ca2+ binding does not induce a significant conformational change. This latter group may exist to create a Ca2+ buffering system in the cell