Physiology and Pharmacology: GPCRs Flashcards
Main difference between GPCRs and RTKs
GPCRs have no intrinsic enzyme activity
General structure of GPCR
Extracellular N-Terminus
7 alpha-helices spanning membrane
Intracellular C-Terminus
Describe active and inactive states of Heterotrimeric Gproteins
- how do they self regulate?
- Which subunit most important here?
GDP bound state - inactive
GTP bound state - active
- activation causes dissociation of alpha monomer and beta-gamma dimer
Gproteins have their own GTPase activity
- > converts GTP to GDP
- > ‘turns off’ Gprotein
Alpha subunit most important here
- contains GTP binding domain
- has intrinsic GTPase activity
What is a GEF? What is its function? How is it related to GPCRs?
GEF = Guanine nuclotide exchange factor
GTP displacement of GDP (i.e Gprotein activation) is stimulated by GEFs
Activated GPCRs have regions which undergo shape change and can act as GEFs
Very general overview of GPCR signalling pathway
- > GPCR
- > Gprotein
- > Effector protein (enzyme)
- > Secondary messenger
- > Target protein
- > Response
3 MAIN Gprotein subtypes and their typical activation pathways
G-alpha-s -> activated Adenylyl Cyclase
G-alpha-i -> inhibits Adenylyl Cyclase
G-alpha-q -> activates Phospholipase C (IP3/DAG signalling)
Describe G-alpha-s pathway
GPCR activated
- conformational change to enable GEF activity
- GEF catalyses GTP -> GDP exchange
- Newly bound GTP activates Gs-protein, alpha subunit dissociates from beta-gamma
- Gs-alpha stimulates adenylyl cyclase activity
- Stimulates production of cAMP through ATP conversion
Describe effects and mechanism of cholera toxin
Inhibits G-alpha-s GTPase activity
- > GTP-alpha subunit remains active, cannot switch off
- > Excessive stimulation of AC
- > Build up of cAMP conc.
Describe G-alpha-i pathway
Very similar to G-alpha-s
GPCR activated
- conformational change to enable GEF activity
- GEF catalyses GTP -> GDP exchange
- Newly bound GTP activates Gi-protein, alpha subunit dissociates from beta-gamma
- G-alpha-i binds to and inhibits AC activity
Describe effects of Pertussis toxin
Inhibits G-alpha-i
-> reverses AC inhibition
Describe G-alpha-q pathway
GPCR activated
- conformational change to enable GEF activity
- GEF catalyses GTP -> GDP exchange
- Newly bound GTP activates Gq-protein, alpha subunit dissociates from beta-gamma
- G-alpha-q binds to and stimulates Phospholipase C (PLC)
- PCL acts on PIP2 membrane lipid, cleaving it and releasing IP3
- remaining membrane lipid is DAG
- IP3 secondary messenger binds to ligand gated calcium channels on ER resulting in flux of calcium to cell
- Calcium goes on to activate downstream proteins e.g. Protein kinase C, calmodulin
- DAG can also activate Protein kinase C
- Phosphorylation of MLCK
Example of each type of GPCR
- name of receptor and ligand
G-alpha-s -> Beta-adrenoceptor
- ligand = adrenaline/noradrenaline
G-alpha-i -> alpha2-adrenoceptor
- ligand = adrenaline/noradrenaline
G-alpha-q -> alpha1-adrenoceptor
- ligand - adrenaline/noradrenaline
Example of effector, mechanism and response stimulated by G-betagamma subunit
GIRK channel in heart - Gprotein coupled Inwardly Rectifying Potassium Channel
Regulated by Muscarinic M2 receptor
- G-betagamma binds to and opens GIRK
- Increases K+ membrane permeability
- Hyperpolarises membrane
- Reduces heart muscle contraction
- Reduces heart rate
How is GPCR signalling terminated?
- Upon activation, GEF site is revealed, but also several phosphorylation sites
- Gprotein-coupled receptor kinase (GRK) phosphorylate these sites -> has 2 effects
- prevents intracellular domain from interacting any further with gproteins
- shape change via phosphorylation creates a binding site for proteins including Beta-arrestin
- Once assembled, plasma membrane invaginates, brings receptor into endosome
- receptor is then either degraded in lysosome, or recycled back to plasma membrane
- Desensitisation of tissue as a result
