G Proteins Flashcards
What are the two types of G proteins?
How is Ras activated briefly?
Where is Ras? What happens on activation?
How is Raf in its inactive form?
What makes up Raf?
How does Ras activate Raf? 2 steps
SHtG & Small monomeric
Through EGFR signalling and GRB2-Sos complex
Membrane-bound - binds to complexes & travel through cytosol
Bidentately bound to 14-3-3 at its termini at pSer residues
RBD (ras binding domain) and Raf kinase
- Ras-GTP binds to RBD of Raf & causes dissociation of 14-3-3 from raf
- Raf is then phosphorylated at its activation loop for active kinase
How does active Raf continue down the MAPK signalling pathway to activate MAPK? 3 steps
How does the active MAPK regulate the transcription of c-Fos? 3 steps
What is the overall result of this?
- Raf kinase phosphorylates MEK kinase at its activation loop
- MEK double phosphorylates MAPK at its activation loop
- Two doubly-phosphorylated MAPK associate to form its active homodimer
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- Active homodimer MAPK phosphorylates Rsk (ribosomal s6 kinase)
- MAPK & Rsk accumulate in the nucleus & go on to phosphorylate TCF & SRF response factors
- Phosphorylated TCF & SRF form a complex & binds to the SRE (steroid response element) upstream of the c-Fos gene
Expression of c-Fos involved in cell proliferation & differentiation
What type of proteins does the Ras superfamily include?
What 6 proteins are in the Ras-like sub family of small G proteins?
What is the accessory protein that ACTIVATES Ras and how?
What is the accessory protein that INACTIVATES Ras & how?
Briefly describe the basic structure of Ras?
Small monomeric G proteins, large ones, HtG
Ras
Rho
Ran
Rab
Arf
Kir/Rem/Rad
GEF - binds & induces conformational change
GAPs (GTPase activating proteins) which cleaves gamma phosphate of GTP to induce conformational change
Mixed alpha-beta structure with a guanine-binding pocket at 1 side of the beta sheet and 5 loops around the binding pocket
What are the 3 functions of the G1 loop in Ras?
What kind of mutation is G12V?
What kind of mutation is S17N?
- Binds to alpha & beta phosphates of GDP/GTP to position them
- Contains consensus motifs/fingerprints that characterises GTP binding sites
- S17 coordinates Mg2+
Activating mutation of Ras - becomes oncogene
Dominant negative mutant - decreases affinity for GTP but not GDP
What are the 2 functions of the G2 loop in Ras?
What other name does the G2 loop have?
What are the 2 functions of the G3 loop in Ras?
What is its other name?
What is the role of the G4 & G5 loop?
- Connects Mg2+ binding sites to gamma phosphate
- T35 of backbone makes bonds to gamma phosphate
Switch I
- Contains consensus sequence that characterising GTP binding sites
- G60 of amide backbone makes bonds to gamma phosphate
Switch II
Recognise the guanine nucleotide
What bonds does Mg2+ form when GDP is present? (hint: it’s 6)
What changes when GTP is present?
How does this change account for the conformational change of Switch I and Switch II?
What is the mechanism by which they change conformation?
How would you describe Switch II in this state?
What happens if GTP hydrolysis were to occur by intrinsic GTPases?
How would you describe the conservation of this mechanism?
4 with water
1 with the hydroxyl of S17 in the G1 loop
1 with the ß phosphate of GDP
Only 2 bonds with water
1 with the ß-phosphate of GTP
1 with OH of T35 in Switch I
1 with the gamma phosphate of GTP
Allows for T35 and G60 of the amide backbone to form hydrogen bonds with the gamma phosphate of GTP - causing the loops to move TOWARDS the gamma phosphate/nucleotide-binding site
High energy state
Loaded spring
Switch I & II springs back & Ras is in its inactive state
Loaded spring mechanism & switch are highly conserved while the amino acids can be different in different proteins
How would you describe the affinity for alpha-GTP and alpha-GDP to the beta-gamma complex in both states?
What are the 3 domains of the alpha subunit from heterotrimeric G proteins?
What is the cap made up of?
Where is it inserted into the Ras-like domain?
How is the cap removed?
What are the steps for alpha subunit activation following the removal of the cap? 3 steps
How does Switch III move?
Where is Switch III in small monomeric G proteins?
Alpha-GTP = low affinity for them
Alpha-GDP = high affinity for them
- Ras-like domain (GTP/GDP binding)
- Cap domain
- Two linkers between the two domains
Made up of 1 long alpha helix & 4 smaller helices packed against it by hydrophobic interactions
Before Switch I & after alpha helix 1
Major conformational change by the 7TM or GPCRs
- GTP can now bind at the Ras-like domain
- T35 & G60 of the amide backbone coordinates with Mg2+ and as such form hydrogen bonds with the gamma phosphate
- Induces conformational change of Switch I, Switch II and Switch III that puts the alpha subunit in the “strained” active conformation
Mechanical propagation of Switch I and Switch II due to their hydrogen bond formation with T35 & G60
Between ß4 and a3
What is the problem with GEF when G-protein is in its empty state?
What shifts the equilibrium between the bound states?
How is this a cyclic process?
What does increasing the activity/amount of GEF do to the equilibrium?
What does increasing the activity of GAP do to the equilibrium?
Why would you use a G-protein analogue?
How else can you create the same effect?
Can either bind GTP or GDP
GTP or GDP concentration
Hydrolysis of GTP by GAP is irreversible
Shifts it towards either the on state (G-GTP)
Shifts it towards off-state (G-GDP)
To trap the G-protein in its active form/GTP-bound form
Use a cell permeable GTP analogue & soak cells in these
What are the 4 important features of the 7 transmembrane receptors?
What are the 3 elements crucial to the activity of 7TMR?
What does the less conservation of amino acids between classes indicate?
What 2 ways is the mechanism for G-protein activation impacted by?
- 7 transmembrane alpha helices that span the membrane
- N terminal extracellular tail & C-terminal intracellular tail
- 3 extracellular loops (E1-3) including glycosylation sites
- ## 3 intracellular loops (C1-3) including palmitoylation sites
- C3 intracellular loop (between E & F)
- F-helix
- C-terminus
Very similar transduction mechanism for downstream signalling with G proteins but G-protein coupling varies
- How deep ligand buried in 7TM receptor
- Extent & nature of interactions between ligand & E loop
How does retinal bind to the rhodopsin 7TM receptor? 2 steps
How does this activate HtG proteins?
What does this say for the essential movement for activating HtG proteins?
- Photoactivation/isomerisation with light, causes the ligand retinal to be converted from the cis to the trans config
- Trans retinal buries deeply into the core of the rhodopsin GPCR barrel to associate compactly with alpha helices via hydrophobic interactions
Helix F moves relative to Helix C causing displacement of the C3 loop
Twisting of the 7TM helix barrel
How do biogenic amides bind to activate G proteins in their 7TMR?
How do peptide hormones activate G proteins from their 7TMR?
How do receptor modified ligands/thrombin activate their G protein?
How do receptors for large ligands bind and activate their G proteins?
Penetrate 1/3rd deep
Interactions in core & extracellular loops
Cleaving the N-terminus revealing a tethered ligand
Receptors have globular N-terminus that binds to the ligand & this interacts with the core to change receptor conformation
Describe the structure of the beta subunit of HtG
Describe the structure of the gamma subunit
What happens to the alpha subunit when GDP is exchanged for GTP?
What happens when GTP is exchanged for GDP?
What are the GEFs for monomeric G proteins and HtGs?
7-bladed propeller that forms a barrel & packs against the alpha subunit
Wraps around the beta subunit in complex
Switch II changes to temporarily remove interaction site for beta
Alpha subunit site is recreated to interact with the beta-gamma complex
Monomeric = Sos
HtG = 7TM receptors
How does Sos stabilise Ras for GDP to GTP exchange? 3 steps
Briefly describe what Sos does to Ras for its activation?
What are the positions of Switch I and Switch II in Ras and how does this further support its activation?
What do localising proteins do generally?
What do Rho/Rho-GDI localising proteins do?
What do effectors do?
- Sos inserts its H-helix into the structure to displace the Switch I region
- This causes the nucleotide-binding site to open
- Glutamate and lysine side chains of H-helix destabilises the environment around Mg2+ and the GDP-binding site
Stabilises Ras in an open conformation such GTP binding is more favourable
Further apart than if GDP was bound so that H-helix can be inserted
Interact with G proteins in different states
Bind to G-GDP & pull them off membranes to keep a pool of G proteins in the cytosol
Interact with active G proteins
What are the heterotrimeric G protein GAPs?
How?
What is the monomeric G protein GAP?
What can you add to G-GDP to mimic the state where the phosphate gamma has been cleaved but hasn’t left yet?
In this state, what state is the G protein in?
How are GAPs similar to GEFs on interaction with Ras?
Gaq & PLCß
PLCß can inactive Gaq by causing hydrolysis of GTP
p120
[AlF4]-
Inactive
Interacts with Switch I & II and the P-loop (around nucleotide binding pocket)
What are the 3 steps for p120-GAP’s binding to Ras?
What other residue is there for the arginine finger support?
What would happen without the arginine finger?
What does the mutation of Q61 induce?
What does the mutation G12V/T induce?
- L1 loop/reactive arginine finger of p120-GAP is inserted into the guanine nucleotide binding site of Ras-GTP
- Arginine finger neutralises the negative charge and stabilises the transition state
- Q61 of Switch II stabilises this transition state further by hydrogen bonding to the arginine finger
G12 of the P-loop
Negative charge would accumulate & hydrolysis would be prohibited
Ras activation & hence tumour formation as no GTP hydrolysis
Ras activation due to steric hinderance of a longer side chain interfering with the arginine finger & no hydrolysis
