Week 3 Flashcards
What is the difference between G protein subunits and subtypes?
G proteins consist of three subunits which are alpha, beta and gamma.
G proteins have four subtypes which are Gs, Gi, Gq and G12/13.
- G-alphas: Many amine receptors (e.g. beta-adrenergic, histamine H2, dopamine D1). Stimulates adenylyl cyclase, causing increased cAMP formation.
- G-alphai: Muscarinic M2, M4, alpha-2-adrenergic, 5-HT!, D2 cannabinoid CB1 and CB2, opioid mu, delta, kappa receptors. G-alphai inhibits adenylyl cyclase, decreasing cAMP formation.
- G-alphaq: M1, M3 and M5, alpha1-adrenergic, angiotensin AT1, peptide and prostanoid receptors. Activates phospholipase C, increasing production of inositol triphosphate (IP3) and diacyglycerol (DAG). Stimulates Rho-GEF.
- G-alpha12/13: Thrombin PAR1, lysophosphatidic acid LPA4, angiotensin AT1, P2Y6 receptors. Stimulates Rho-GEF.
What are the differences between G-alpha and G-beta gamma subunits?
G-alpha
- G-alpha consists of a Ras-like GTPase domain and an alpha-helical domain. GDP/GTP sits between the two domains.
- Chaperone proteins may help G-alpha fold and move to the ER surface
- When G-alpha assemblies with G-beta gamma and is modified with fatty acids
- Fatty acids on G-alpha anchor it within inner cell membranes
- G-alpha is synthesised by transcription of encoding genes into mRNA, translating to proteins and undergoing cleavage.
G-beta-gamma
-G-beta folds into a beta-propeller with 7 blades. It’s N terminus forms tight alpha-helical coiled-coil dimers with G-gamma.
- Chaperone protein binds to nascent G-beta to promote G-beta beta-propeller folding
- Phosducin like protein binds and phosphorylates G-beta to make a G-beta gamma complex
- G-beta gamma complex moves to ER surface where G-gamma is modified with a fatty acid molecule
- Fatty acids within G-gamma anchor it within inner cell membranes
- G-beta and G-gamma are separately synthesised by transcription of encoding genes into mRNA, translating to proteins and undergoing cleavage
How do G proteins cycle between activate and inactivate states?
- In the inactive state, 3 G protein subunits are associated together and G-alpha binds to GDP
- Agonist-receptor binding causes receptor conformational change which encourages the receptor to bind to the G protein complex
- The receptor-G-protein coupling promotes the exchange of G-alpha(GDP) for G-alpha(GTP)
- G-alpha(GTP) dissociates from the receptor and G-beta gamma
- It then interacts with the target proteins and causes generation of second messengers
- The cycle is complete by the hydrolysis of GTP to GDP by the intrinsic G-alpha-GTPase which causes the re-association of G-alpha(GDP) with G-beta gamma
- Results in deactivation and return to the inactive state
Helper Proteins regulate the G protein activation process. These include:
- GEFs (guanine nucleotide-exchange factors) accelerate the release of bound GDP from G-alpha
- GAPs (GTPase-activating proteins) stimulate GTP hydrolysis to inactive G-proteins
- GDIs (guanine nucleotide-dissociation inhibitors) prevent GDP released from G-alpha subunit to maintain G-protein in an off-state
What are the characteristics of heterotrimeric G proteins?
Heterotrimeric G proteins are large, membrane-bound proteins that are made of alpha, beta and gamma subunits. There are four families according to alpha subunit Gs, Gi, Gq and G12/13.
What are the characteristics of monomeric G proteins?
Homotrimeric G proteins are small, not membrane associated proteins that are homologous to the alpha subunit. They are also known as GTPase or the Ras superfamily. Consists of Rab (vesicle trafficking), Arf (vesicle budding), Ran (nuclear transport), Rho (signalling), and Ras (signalling).
What are the structural features of G proteins and how are they pharmacologically targeted by toxins and small molecules?
CTX (cholera toxin) induces ADP-ribosylation of an Arg residue on G-alphas-protein and inhibits its GTPase activity, resulting in peristent G-alpha activation.
- G-alphas is activated by CTX which blocks GTPase activity, thus preventing inactivation
PTX (pertussis toxin) catalyses the ADP-ribosylation of the Gi/Go interfering GDP exchange for GTP
- G-alphai/o is blocked by PTX which prevents dissociation of G-alpha beta gamma complex
Small Molecule Inhibitors
- FR is a naturally occurring cyclic depsipeptide that is originally isolated from the Ardisia crenata plant
YM is a synthetic compound
- Both are highly selective inhibitors for Gq signalling
- Ripasudil and netarsudil inhibit Rho-kinase and are used to treat glaucoma and ocular hypertension as they increase trabecular meshwork outflow to reduce intraocular pressure
- betaARK-ct, gallein and Mu9 interfere with GRK2 binding to the G-beta gamma dimer and causes inhibition of the recruitment of GRK2 to the membranes and reduces receptor phosphorylation and internalisation (used in heart failure which is caused by upregulation of GRK2 in the damaged heart)
What are the main transductions pathways used by GPCRs?
Gs-alpha -> Adenylate cyclase -> cAMP -> Protein Kinase A -> Increased protein phosphorylation
Gq-alpha -> phospholipase C -> DAG + IP3 -> Protein Kinase C + Ca2+ release
-> Increased protein phosphorylation and activated Ca2+ binding proteins
Gi-alpha -> Adenylate cyclase -> cAMP -> Protein Kinase A -> Decreased protein phosphorylation
G12/13-alpha -> RhoGEF -> RhoA -> Increased protein phosphorylation
What are the major enzyme families responsible for second messenger generation?
Adenylyl Cyclase Family
- AC1-9 are membrane bound
- AC10 soluble, only has catalytic domains
- AC1-8 respond to forskolin
- different locations within the body for each member
- all have two cystolic domains C1 and C2 subdivided into catalytic (C1a and C2a) and regulatory (C1b, C2b)
Phospholipase C-beta Family
- PLCbeta hydrolysis of PIP2 leads to DAG and IP3 production
- 4 isoforms
- share the same domain structure
- different ratios of membrane to cytosol localisation
- X-Y linker as gatekeeper for active site
- highly conserved amphipathic helical domain
What are the two major signal transduction pathways used by G-alphai coupled receptors?
Adenylyl Cyclase Pathway
- through this pathway Gi-alpha decreases cAMP
- Gi-alpha binds to the C1a and favours the closed state
- only a subset of ACs bind to Gi-alpha subunits
GIRK Pathway
- through this pathway Gi-alpha activates GIRK channels
- GIRK channels are G protein-gated inwardly rectifying potassium channels expressed throughout the body
What is the difference between kinase cascades and second messenger transduction pathways?
Kinase Cascade Pathway
- protein kinases are part of a phosphorylation cascade that transduces a signal
- critical component of most signal transduction pathway
- Protein Kinase A and C, Rho Kinase, and ERK1/2 (directly or indirectly in all 4 pathways)
- a kinase catalyses the transfer of a phosphate group from ATP to a target protein
- a kinase cascade is a series of reactions in which a signal passes to downstream proteins by sequential protein phosphorylation and activation of the cascade components
Second Messenger Transduction Pathway
- a second messenger is a small, nonprotein molecule or ion that rapidly diffuses and relays a signal throughout a cell
- signal transduction is the mechanism by which cell surface receptors receive information from extracellular signals such as hormones and neurotransmitters, and amplify this information through the actions of second messengers
What are some examples of signal transduction components as drug targets?
Phospholipase C-beta
- inhibitors that prevent movement of X-Y linker or that occupy the site
- small molecules that prevent close interactions of N terminus
- protein-protein interaction inhibitors that occupy space so Gq can’t bind
- Inhibitors that prevent action of CTD with plasma membrane (difficult because long stretch)
- Activators that force allosteric changes that Gq does to cause activation (agonist)
Adenylyl Cyclase
- Analogs for Forkslin binding site
- Protein-protein inhibition of Gs-alpha binding
- Inhibitor to block Gi-alpha binding site
- Target catalytic site
GIRK Channels
- Channel blocker
- Protein-protein inhibitor to stop interaction site to stop activation
- Small molecule that interacts with residues lining the pore - doesn’t block but stop activation
- Small molecule that acts as a terminal wedge and inhibits interaction between subunits
