G-Protein Coupled Receptors

Question 1

In what class of organisms are GPCRs found?

Answer 1

Eukaryotes
Largest known class of membrane receptors (in humans >38000 known GProteins)

Question 2

GPCRs are the target of how many modern medicinal drugs?

Answer 2

30-50%

Question 3

Examples of GPCR ligands

Answer 3

Light sensitive compounds, pheromones, hormones, odours, neurotransmitters

Question 4

GPCR Structure

Answer 4

7 transmembrane alpha helices

Question 5

G protein strcuture

Answer 5

Have the ability to bind GTP (active)(guanosine triphosphate) and GDP (inactive)
Some have a single subunit
All G proteins that associate with GPCRs are heterotrimeric (3 different subunits: alpha subunit, beta, and gamma)
Alpha and gamma are attached to the cell membrane by lipid anchors
Has GTPase activity

Question 6

What is the process of GPCR activation?

Answer 6

• Ligand binds to receptor
• Receptor undergoes conformational change
• Gprotein detaches from receptor due to conformation change
• GTP replaces GDP and Gprotein undergoes another conformational change
• The protein dissociated resulting in an activated alpha subunit, and an activated beta-gamma heterodimer
• Activated alpha subunit regulates membrane bound enzyme
• Membrane catalyses reaction that produces 2nd messenger
• 2nd messenger amplifies message of ligand through protein kinases (phosphorylates proteins)
• Biological response

Question 7

Downstream signalling pathway of G-alpha-s

Answer 7

• Stimulates adenylyl cyclase (uses ATP as substrate to produce cAMP as 2nd messenger)
• cAMP activates cAMP-dependent Protein Kinase A (PKA)
• PKA = 2 regulatory + 2 catalytic subunits
• cAMP binds to regulatory subunits > now active catalytic subunits are released
• Catalytic subunits enter nucleus > phosphorylates target protein (usually a transcription factor)
• This activates transcription factor so it can bind to promoter or regulatory sequence
• Transcription of target gene
• Protein phosphatases can also deactivate activated genes
• PKA upregulates phosphodiesterase which converts cAMP to 5’AMP thereby generating a negative feedback loop

Question 8

Downstream signalling pathway of G-alpha-i

Answer 8

• Inhibits adenyly cyclase enzyme
• Decreases cAMP and thus PKA

Question 9

Downstream signalling of G-alpha-q

Answer 9

• Stimulates phospholipase C to cleave the membrane lipid phosphatidylinositiol bisphosphate (PIP2) -> inositol triphosphate (IP3) + diacyl glycerol (DAG)
• IP3 binds to gated calcium channel in ER to release calcium
• (DAG + Ca2+) activate protein kinase C (PKC)
• PKC catalyses phosphorylation of cellular proteins
• Increase of intr. Ca inhibits the IP3 gated calcium channel

Question 10

What is calmodulin?

Answer 10

A calcium binding protein.
Upon release of Ca from the ER, Ca binds to calmodulin to enable it to bind to an enzyme and become activated
The calcium-calmodulin complex upregulates the Ca-ATPase pump to get rid of increased intr. calcium

Question 11

How is the GPCR signalling pathway stopped?

Answer 11

Ligand detaches > G protein converts GTP to GDP > alpha subunit recombines with beta-gamma subunit > signalling stops

Question 12

What is the GPCR Kinase (GRK)?

Answer 12

Small family of serine and threonine proteins
This is activated when the right ligand binds to the GPCR
GRK + PKA + PKC = phosphorylate the GPCR itself
The phosphorylated GPCR binds with high affinity to arrestin (prevents interaction of the receptor with the Gprotein)

Question 13

Example: how does the cholera toxin work?

Answer 13

Cholera alters G-alpha-s subunit so it can’t hydrolyse GTP (remains active)
Active adenylyl cyclase means more cAMP which causes a high water and Cl efflux into the gut, leading to sever diarrhea

Question 14

What is GEF?

Answer 14

Guanine Nucleotide Exchange Factor
Promotes the exchange of GDP for GTP

Question 15

What is GAP?

Answer 15

GTPase Activating Protein
Speeds up the hydrolysis of GTP to GDP +Pi

Question 16

What is desensitisation?

Answer 16

The process that blocks the receptors from turning on additional Gproteins. Takes place in 2 steps:
1. Cytoplasmic domain of activated GPCR is phosphorylated by a Gprotein-coupled Receptor Kinase (GRK)
2. Arrestins bind (compete for binding of heterotrimer Gproteins)

Question 17

Endocytosis of GPCRs

Answer 17

Arrestin, while bound to the GPCR, can also bind to AP2 (situated in clathrin-coded pits)
Interaction between the arrestin and clathrin coated pits promotes the uptake of phosphorylated GPCRs into the cell by endocytosis where:
- This leaves the GPCRs attached to endosomes, and sometimes the arrestin molecules can act as a scaffold for the assembly of various cytoplasmic signalling pathways (eg MAPK)
- Receptor can be moved to lysosomes where they’re degraded (cell will temporarily lose sensitivity to the ligand)
- Receptors may be dephosphorylated and returned to the plasma membrane (this is called resensitisation)

Question 18

What are Regulators of Gprotein Signalling (RGS)?

Answer 18

Regulate the speed of the hydrolysis of GTP -> GDP + Pi
Interaction of RGS protein increases the rate of hydrolysis of the beta subunit