G-proteins

Question 1

G-proteins activation (GTP-hydrolysing enzyme/GTPase)

Answer 1

Guanine nucleotice Exchange Factors (GEFs) are activated in GTP-bound state
- GTP hydrolysed into GTP by GTPase-activating Factors (GAFs)

Question 2

2 classes of G-protein superfamily

Answer 2

1) Heterotrimeric (associated with GPCRs)

2) Small GTP-binding proteins

Question 3

G protein Coupled Receptor (GPCR) structure

Answer 3

7 TM domains

• extracellular part for ligand binding
• intracellular parts aid in binding to heterotrimeric G proteins

Question 4

GPCR evolution

Answer 4

Highly diverse (yeast to man)

Used in various biological responses

Conserved mechanism

Question 5

GPCRs binding heterotrimeric G protein (alpha-beta-gamma)

Answer 5

Activates downstream signalling proteins and enzymes

• different signalling proteins for different responses
• calcium release and cytosolic flux key feature

Question 6

Mechanisms of activating heterotrimeric G protein

Answer 6

Ligand binding causes structural rearrangement of cytoplasmic parts, opening a binding site of G-alpha subunit so GTP can bind

• GTP binding activates G-alpha-beta-gamma

Active alpha subunit (alpha-GTP complex) dissociates and acts as signalling molecule

Question 7

The Heterotrimeric G-protein cycling

Answer 7

1) resting complex = GDP bound
2) ligand binding triggers GDP dissociation and GTP binding on alpha subunit
3) Active form (GTP bound): alpha subunit dissociates to trigger signalling cascade; beta-gamma subunit complex dissociates
4) Upon alpha-GTP hydrolysis, the alpha-GDP re-associates with the beta-gamma subunit (mediated by RGS proteins) and return to G-protein at membrane

Question 8

G-alpha signalling pathway examples

Answer 8

1) stimulating PLC enzyme: triggers PIP2 hydrolysis, DAG release - important activator of Protein Kinase C
2) Stimulating Adenylyl cyclase to convert ATP into cAMP
3) Activating MAPK pathway and ERK phosphorylation

Question 9

Molecular Scaffold increasing efficiency

Answer 9

Complex molecular scaffolds enhance signalling efficiency and spatial organisation of pathways

• Increase local concentration of certain signalling proteins
• localise signalling pathways to sites of activation

Question 10

B2 adrenergic receptors (ligand concentration dependent)

Answer 10

Receptors respond differently to different agonist levels

• Low agonist concentration = one pathway, high = another

Question 11

GPCR ligands

Answer 11

Biogenic amines, amino acids, ions, lipids, peptides…

• different ligand = different signalling pathways

Question 12

Monomeric GTPase subfamily

monomeric nature, functioning

Answer 12

Exist as monomers as there are no other molecules to form complexes with.

Similar function to heterotrimeric G proteins:

• also prenylated (lipid attachment on C/N termini) allowing for membrane attachement
• have functioning alpha subunit
• rely on same ON/OFF GTP cycle (GEF activation; GAP deactivation; GDIs slowing cycle)

Question 13

Effects of Ras mutation on GTP cycle

Answer 13

Oncogenic mutation in Ras causes permanent GTP-bound (active) state

• Blocks GTP hydrolysis
• Constant ERK activation = proliferation (& cancer)

Question 14

Ras-coupled growth factors

Answer 14

• EGFR
• Nerve growth factor receptor (TrxA)
• MAPK (big drug target as related to cell proliferation)

Also activates Raf (Ras-GTP recruits a Raf/HSP heterodimer

Question 15

Ras activating Rac and Rab

Answer 15

Rab: Regulates actin dynamics (polymerisation/depolymerisation)
- important in moving cytoskeleton (e.g. phagocytosis)

Rab: regulate vesicle docking/fusion
- interact with SNAREs to alter their fidelity

Question 16

Rho-like GTPase

Answer 16

Regulate intracellular signalling, cell shape and migration
- important in pathogenic infection: often subverted during infection due to pathogen’s own proteins

Normally regulated by Adhesion-linked TKs (Focal Adhesion Kinase, FAK)
- Active FAK stimulates Rho-like GTPases (exchanging GDP for GTP)