17 - GPCRs and Second Messenger Signalling

Question 1

What can heterotrimeric G proteins be classified into?

Answer 1

The heterotrimeric Gproteins can be classified into three families
1. Gs family: activate (stimulate) adenylate cyclase
2. Gi family: inhibits adenylate cyclase (family includes Go and Gt)
3. Gq/11 family: acivat phospholipase C

Question 2

What does the Gs family do?

Answer 2

Activates adenylate cyclase which produces cAMP in the cell resulting in the activation of protein kinase A

Question 3

Features of the Gi family (5)

Answer 3

1. Gs is switched off by the Gi family
2. Generally if one activates Gs, another will activate Gi
3. Gt activates cGMP speicif PDE6 causing degradation of cGMP in the retina causing signalling from the retina to the brain
4. G0 which activates potassium channels
5. Gi can activate PI3 kinase gamma which activates PIP3 resulting in the activation of PKB and PDK1

Question 4

Features of the Gq/11 family (2)

Answer 4

1. No effect of cAMP signalling
2. Activate a phospholipase Cbeta which involves DAG and InsP3 causing the activation of protein kinase C and calcium release

Question 5

What do G proteins (Gq/11) activate?

Answer 5

Phospholipase C (PLC)

Question 6

What happens when Gq/11 activated PLC?

Answer 6

PLC then cleaves PtdIns (4,5)P2 (PIP2) to from inositol triphosphate (IP3) and diacylglycerol (DAG)
o DAG remains stuck to the membrane and IP3 is released into the cytosol

Question 7

What do DAG and IP3 do?

Answer 7

• DAG and IP3 both act as secondary messengers
• IP3 stimulates calium release from the endoplasmic reticulum
• DAG stimulates protein kinase C (PKC)
• Phospholipase C cleaves the phosphatidylinositol, leaving DAG and the IP3 on the bottom and is released

Question 8

What happens when a Gq coupled receptor is activated?

Answer 8

o this causes activation of the Gq alpha subunit which become GTP bound
o The GTP bound alpha subunit interacts with phospholipase Ca the cell membrane this activates the phospholipase C to cleave and produce DAG and IP3
o The Dag can now interact with PKC and this interacts with the C1 domain PKC and the C2 domain bind calcium
o The calcium is released fromIP3 gated calcium channels which are found in the ER and they span the ER membrane, the channels are opened after the IP3 interacts providing the calcium for the output to occur

Question 9

How many classes (isoforms) of protein kinase C (PKC)?

Answer 9

There are three different classes of PKC that have been discovered
1. conventional
2. new
3. atypical

Question 10

What does the conventional class of PKC involve

Answer 10

The conventional class involves PKC alpha, two betas and gamma
o these have got C2 domains and C1 domains which allow it to be activated, in the inactive form these fold in on the knase domain blocking the activity of the domain
o the C2 domain will bind calcium and the C1 will bind DAG, this causes the regulatory regions to flip away from the kinase domain by the movement of a hinge domain, allowing the kinase domain to bind ATP and become activated

Question 11

What does the new class of PKC involve?

Answer 11

New nPKC delta, epsilon, theta and eta
o these are activated by DAG
o they have C2 domain but it doesn’t bind calcium so well so it is activated by DAG alone

Question 12

What does the atypical class of PKC involve?

Answer 12

Atypical aPKC theta iota lamda
o they have a C1 domain but only a prtical C1 domain, and no C2 domain
o not activated by DAG but activated by other mechanisms

Question 13

What is PLC beta activated by?

Answer 13

The phospholipase C beta is activated by G proteins (Gq/11 activated through the alpha subunit, but Gi/0 activates through the beta gamma subunit
o G protein coupled receptors
o This then hydrolyses PIP2 into DAG and IP3

Question 14

What is PLC gamma activated by?

Answer 14

Receptor tyrosine kinases (RTKs)

Question 15

What is PLC epsilon regulated by?

Answer 15

PLC epsilom was discovered and this is regulated by the small G protein Ras which is usually activated by receptor tyrosine kinases

Question 16

What is PLC delta activated by?

Answer 16

PLC delta is activated by calcium which enters through calcium channels in cells

Question 17

What do all isoforms of PLC do when activated?

Answer 17

All isoforms once activated causes hydrolysis of PIP2

Question 18

Give examples of other cell responses regulated by GPCR/PLC signalling (target tissue (1), signalling molecule (2), and major response (3))

Answer 18

A. 1 - liver, 2 - vasopressin, 3 - glycogen breakdown
B. 1 - pancreas, 2 - acetylcholine, 3 - amylase secretion
C. 1 - smooth muscle, 2 - acetylcholine, 3 - contraction
D. 1 - blood platelets, 2 - thrombin, 3 - aggregation

Question 19

Why are PLCs important in wounding?

Answer 19

Prostaglandins which are produced in the local region of wounding, often stimulate pain response and can stimulate platelet aggregation

Question 20

PLC pathway in wound healing

Answer 20

Certain prostaglandins activate Gs coupled receptors but other prostaglandins can activate Gq coupled receptors, so once they interact with the prostaglandin receptor this stimulates phospholipase C beta which produces DAG and calcium mobilisation to activate PKC
* This feeds into the Raf MAP kinase pathway to stimulate transcription
* One enzyme produced is COX2 which stimulates the conversion of AA into prostaglandins which act on the receptor
- feed forward loop as long as there is wounding
Another root of singalling in wounds is the production to TGFalpha which interacts with the EGF receptor to stimulate ERKMAPK signalling
o When the EGF receptor is activated, the phospholipase Cgamma is activated to produce DAG and calcium mobilisation etc. causing the feed forward loop

Question 21

What happens when the PLC feed forward loop is not turned off?

Answer 21

If this loop is not resolved then continuous cell growth is promoted beyond reasonable limits and this is what happens in cancer
o There are cetain types of carcinogenic agents, e.g, phorbol esters of which TPA is an example
- this minics DAG and interacts with the cell memebrane and the c1 domain of PKC
- so TPA continuously stimulates the ERKMAP kinase pathway

Question 22

What do G proteins regulate/activate?

Answer 22

Gs coupled receptors were the first ones discovered and they activate a family of enzymes called adenylate cyclases
o Adenylate cyclases have two clusters of helical regions (2x6), these are linked by a C1 domains and in their cytoplasmic portion the have a C2 domain and binding of TP which is converted to cAMP upon the stimulation of the adenylate cyclase by active GTP bound GalphaS

Question 23

How many isoforms of adenylate cyclase are there?

Answer 23

There are 9 different isoforms of adenylate cyclase now know and all of them can be acitvated by Gs

Question 24

What can the different isoforms of adenylate cyclase be activated by?

Answer 24

All of them can be activated by the drug forskolin which can directly interact with adenylate cyclase and causes ATP to be converted into cAMP
o Mimics beta adrenergic receptors in the lungs in other countries as a drug (in Japan)

Question 25

How do the different classes of adenylate cyclase respond to different G proteins?

Answer 25

o The different classes show various responsiveness to Gi, some are not inhibited by Gi
o These isoforms are regulated differentially by Gbetagamma, Galphai and calmodulin, but all are regulated by forskolin and all are stimulated by GalphaS

Question 26

Synthesis of cyclic nucleotides

Answer 26

1. ATP is activated by adentylate cyclase, resulting in the loss of two phosphates and he formation of a phosphodiester linkage to the ring structure to from a cAMP
2. Once it is stimulated by adenylate acyclase and cAMP is produced it can activate downstream effectors and then is broken down by a large family of phosphodiesterase enzymes to make %’ adenosine monophosphate
3. The phosphodiesterase hydrolyses 3’ ester bond releasing the bond and producing 5’AMP which can then no longer interact with downstream effectors

Question 27

Provide a summary of cyclic AMP signalling

Answer 27

• Typically when you activate G protein coupled receptors we get activation of GalphaS which is GTP bound, it interacts with adenylate cyclase which converts ATP tolots of cAMP
• The outpiuts can be either interaction with classifally PKA which binds two molecules of cAMP releasing the catalytic unti to phosphorylate downstream targets
• In 1998 EPAC was discovered which binds cAMP which activates it, once activated it serves as a guanine nucleotide exchange factor for a small G protein Rap which then stimulates downstream singalling
  • EPAC is not a kinase in the way that PKA and PKC are

Question 28

Give examples of cell responses mediated by cAMP (1 - target tissue, 2 - hormone, 3 - major response)

Answer 28

A. 1 - thyroid gland, 2 - thyroid-stimulating hormone (TSH), 3 - thyroid hormone synthesis and secretion
B. 1 - adrenal cortex, 2 - adrenocorticotrophic hormone (ACTH), 3 - cortisol secretion
C. 1 - ovary, 2 - luteinisinghormone, 3 - progesterone secretion
D. 1. muscle, 2 - adrenaline, 3 - glycogen breakdown
E. 1 - bone, 2- parathormone, 3 - bone resorption
F. 1 - heart, 2 - adrenaline, 3 - increase in heart rate and force of contraction
G. 1 - liver, 2 - glucagon, 3 - glycogen breakdown
H. 1 - kidney, 2 - vasopressin, 3 - water resorption
I. 1- fat, 2 - adrenaline, ACTH, glucagon, TSH, 3 - triglyceride breakdown