16 - Introduction to GPCR Signalling

Question 1

What Nobel Prizes were awarded regarding the importance of GPCRs?

Answer 1

• The Nobel prize in physiology or medicine, 1971 to Earl W. Sutherland, Jr, “for his discoveries concerning the mechanisms of the action of hormones”
• The Nobel prize in physiology or medicine, 1994 to Alfred G. Gilman and Martin Rodbell “for their discovery of G-proteins and the role of these proteins in signal transduction in cells”
• The Nobel prize in Chemistry, 2012 to Robert J. Leftkowitz and Brian K. Kobilka “for studies of G-protein-coupled receptors”

Question 2

How many genes in the human genome code for GPCRs?

Answer 2

Almost 800 genes - slightly over half of these are the olfactory receptors that enable us to smell

Question 3

How many classes can GPCRs be grouped into and what are they?

Answer 3

6 classes:
o Class A- Rhodopsin-like
o Class B- Secretin receptor family
o Class C- Metabotropic glutamate/pheromone
o Class D- Fungal mating pheromone receptors
o Class E- Cyclic AMP receptors
o Class f- Frizzled/smoothened

Question 4

What are GPCRs grouped based on?

Answer 4

Sequence homology and functional similarity

Question 5

What are GPCRs targets of?

Answer 5

GPCRs are the targets of over half of the drugs currently used

Question 6

Give an example of a GPCR that drugs target

Answer 6

The beta2 adrenergic receptor is the target of anti-asthma drugs, including salbutamol (Ventolin)
o Agonist to adrenaline receptors
o Mimics fight or flight which open the bronchi

Question 7

What is the basic structure of GPCRs?

Answer 7

• they have a very common transmembrane structure so all 800 genes code for receptors which have these seven transmembrane passes (each of these passes is helical), and extracellular domain
• the amino acid sequence weaves in and out of the membrane creating intracellular and extracellular loops and then we have the intracellular terminal region
• lipid anchors link the tail of the receptor to the plasma membrane, and on the extracellular domain we have glycosylation

Question 8

What is the basic structure of GPCRs?

Answer 8

• they have a very common transmembrane structure so all 800 genes code for receptors which have these seven transmembrane passes (each of these passes is helical), and extracellular domain
• the amino acid sequence weaves in and out of the membrane creating intracellular and extracellular loops and then we have the intracellular terminal region
• lipid anchors link the tail of the receptor to the plasma membrane, and on the extracellular domain we have glycosylation

Question 9

What do kinases and G proteins do to GPCRs?

Answer 9

Kinases phosphorylates the intracellular loops & G proteins contact with multiple areas of the intracellular portions

Question 10

How are the helical regions arranged?

Answer 10

The 7 helical regions cluster together in a circular orientation

Question 11

What is the common feature of GPCRs?

Answer 11

The common feature of GPCRs is they activate heterotrimeric G proteins
o Have three subunits: the alpha, beta, and gamma
o Beta: propellar shape
o Gamma: forms a peptide and interacts with the beta subunit (beta gamma subunit)
o The alpha subunit can dissociate easily- binds GDP and GTP

Question 12

What do small G proteins (like Ras) do?

Answer 12

Small G proteins-like Ras- bind GTP (on) and GDP (off)- not regulated by GPCR directly

Question 13

Regulation of G-proteins by GPCRs

Answer 13

• when a signalling molecule interacts with the receptor, e.g., adrenaline interacting with the adrenergic receptor, it causes the receptor to interact with the alpha subunit, resulting in GTP to change to GDP causing activation of the subunit and dissociation of the beta gamma subunit
• these can now interact with effector molecules (attached to the cell membrane)

Question 14

Give an example of the regulation of G proteins

Answer 14

In this particular situation, the alpha subunit is acted on to exchange GDP for GTP which requires a number of enzyme activities for example the GPCRs themselves have catalytic activity in the cytoplasmic tail (GEF activity) on alpha subunits
* There are 16 types of alpha subunit which regulate a different effector
* To switch off the activated alpha subunit, GAP activity is required§

Question 15

What do GAP and GEF stand for?

Answer 15

• GTPase-activating protein
• Guanine nucleotide exchange factor

Question 16

What do GAP and GEF do?

Answer 16

The combined effect of GEF and GAP proteins causes a molecular switch inside the cell

Question 17

What happens when there’s inactive G protein and GPCR, and the GPCR is activated?

Answer 17

If we have an inactive G protein and GPCR, once the GPCR is activated, this activates the GEF activity for the receptor allowing GDP exchange for GTP, now the alpha subunits and beta gamma subunits can bind to effectors

Question 18

What activity do RGS (regulator of G-protein signalling) have?

Answer 18

RGS proteins have GAP activity which stimulates the GTPase activity of the alpha subunit, hydrolysing the GTP to GDP, losing a phosphate, this is the switch off

Question 19

What activity do G proteins themselves have?

Answer 19

The G protein themselves, the alpha subunit has GTPase activity which hydrolyses the GTP on the alpha subunit to switch it off

Question 20

What needs to be switched off for the inactivation of GPCRs?

Answer 20

• G proteins
• receptor signalling

Question 21

Stages of GPCR inactivation

Answer 21

The inactive receptor binds a ligand, when this happens, in addition to activation of G proteins, there is also a family of enzymes which is activated known as GRK which phosphorylate the intracellular portion of the protein coupled receptor which then serves as a docking site for arrestin
o Arrestin interacts with the phosphorylation sites and this blocks any further activation of alpha subunits
o Arrestin causes the GPCR to be endocytosed so itthen comes away from the cell surface and is internalised within the cell
o Arrestins trigger a number of downstream signalling pathways

Question 22

Arrestin and crosstalk with RTKs (receptor tyrosine kinases)

Answer 22

• Once a GPCR is activated it activates a G protein for example Gs which stimulates adenylate cyclase which then also triggers the activation of GPCR kinase which phosphorylates the tail
• Arrestin then interacts with the tail of the phosphorylated receptor which can triger direct stimulation of gene expression or it can trigger the activation of the ErkMAP kinase pathway