L7: Intracellular Mechanisms of GPCR signalling Flashcards
What are the 4 main families of GPCRs according to phylogenic analysis?
- Glutamate,
- Rhodopsin,
- Frizzled,
- Secretin
What are the Membrane Resident Proteins in GPCRs?
G protein, middle messenger between receptor and effector
What are effector systems in GPCRs signalling?
Second messengers
What is the function of G-proteins in GPCRs signalling?
- To recognise activated GPCRs
- pass on the message to effector systems
Which part of GPCRs is coupled to G-proteins during signalling?
Long 3rd cytoplasmic loop couples to G-protein
What do G-proteins interact with?
G-proteins interact with Guanine nucleotides (GTP; GDP)
How many subunits G-proteins have and where are they located in the cell?
All 3 subunits bound of G-proteins are bound to the cell membrane
Does single pool of G-proteins interact with only one receptor?
Single pool of G-proteins can interact with several different receptors
In which subunit of G-proteins is GTPase domain?
GTPase is in alpha-subunit
What is the function of GTPase domain of G-proteins?
- Hydrolyses GTP
- provides binding surface for G-beta-gamma GPCRs and effector proteins
Shortly describe beta-gamma subunit of G-proteins
Tight functional unit, only dissociated by denaturation
What is ‘collision coupling’ regarding GPCRs?
The collision coupling model describes interactions between receptors and G-proteins as first requiring the molecules to find each other by diffusion.
Describe G-proteins in resting state.
G-protein trimer unattached, GDP occupying alpha-subunit
Describe the changes in GPCRs and G-proteins followed by occupied receptor
1) conformational change in cytoplasmic domain; high affinity for alpha-beta-gamma
2) GDP dissociates from alpha-subunit of G-proteins
3) GDP replaced by GTP
4) G-protein trimer dissociates
5) ‘active’ forms of the G-protein diffuse in the membrane
6) associate with enzymes / ion channels; activates targets
What is the function of beta-gamma subunit of G-proteins?
Beta-gamma subunit of G-proteins is considered a chaperone, controls effect but higher concentrations required.
As for Gi/o, also:
- activates potassium channels
- inhibits VGCCs (voltage-gated calcium channels)
- activates GPCR kinases (GRKs)
- activates MAP kinase cascade
What happens to G-proteins upon signal termination of GPCRs?
- attachment of a subunit to target increases its GTPase activity
- hydrolysis of GTP to GDP
- alpha-subunit moves back
- returns to homeostasis
How does a single pool of G-proteins allow each receptor to produce a distinct cellular response?
It’s not a single entity, molecular variation within subunits, >20 subtypes, 4 main classes: Gs, Gi/o, G12/13, Gq
What is the function of Gs class of G-proteins?
Adenylate Cyclase, stimulation, Increasing cAMP
What is the function of Gi/o class of G-proteins?
Adenylate Cyclase, inhibition, decreasing cAMP
What is the mechanism of Adenylate Cyclase?
- adenylate cyclase converts ATP to cAMP
- cAMP then converts PKi to PKa
- PKa phosphorylates the protein
CHECK L7, slide 18 for diagramm
How can levels of cAMP be reduced?
cAMP can be converted to 5’-AMP by PDEs (hydrolysis reaction), which reduces cAMP levels meaning that PKi conversion to PKa is obstructed, thus no protein phosphorylation.
Explain the mechanism of Adenylate Cyclase in smooth muscle by the action of isoprenaline
FOR DIAGRAMM check L7, slide 20
1) beta-adrenergic receptor is activated by isoprenaline
2) G-alpha-s dissociates and attaches to adenylate cyclase
3) adenylate cyclase converts ATP to cAMP
4) cAMP works on PKi conversion to PKa
5) PKa phosphorylates MLCK
6) MLCK-P becomes inactive, thus prevents muscle contraction
7) result - relaxed muscles
Explain the mechanism of Adenylate Cyclase in smooth muscle by the action of clonidine
FOR DIAGRAMM check L7, slide 21
1) alpha-2-adrenergic receptor is activated by clonidine
2) G-alpha-i dissociates and attaches to adenylate cyclase
3) adenylate cyclase is inhibited
4) no cAMP to work on PKi conversion to PKa
5) MLCK does not get phosphorylated, thus it’s active
6) result - muscle contraction
What is the function of Phosphodiesterase (PDEs) Inhibitors? Give example of inhibiting PDEs
Inhibit cAMP by hydrolysis to 5’-AMP, for example PDE4 produced by inflammatory cells inhibits cAMP, causing contractions. They can be inhibited, rolipram is used during asthma, it inhibits PDE4 to reduce secretion
