G-proteins Flashcards
What are G-proteins?
They are GTPases (GTP hydrolysing enzymes)
- Input from cell surface receptors
- Guanine nucleotide exchange factors (GEFs) add a Pi onto GDP (they use GTP hydrolysis to supply this)
- The now GTP G-protein can initiate signalling responses
- GTPase-activating factors hydrolyse GTP into GDP
There are 2 types of these:
Heterotrimeric (alpha/beta/gamma) - Gs, Gi, Gq, G12
Small GTP-binding proteins - Ras, Rho, Rab, Ran, Arf
They function monomerically without other subunits
What are some signalling responses of g-proteins?
Cyclic AMP signalling InsP3/DAG signalling MAP kinase signalling Ptdlns 3-kinase signalling PLD signalling Redox signalling Cytoskeletal remodelling Ion channel modulation
Describe G-protein coupled receptors (GPCRs)?
Membrane proteins - 7 hydrophobic transmembrane domains
GPCR interacts with a heterotrimeric G-protein (abg) - GTPase associated
Once the subunits are dissociated they regulate signalling pathways
Cells express several varieties of each GPCR
GPCR-specific pathways can trigger different responses
Cytosolic calcium rise is an important feature of GPCR activation
Signal (agonist) -> GPCR -> G-protein -> effector
What is involved in the evolution of GPCRs?
Highly diverse
Used for many biological responses
Found in yeast to man
Conserved mechanism during evolution
Describe GPCR activation?
Inactive GPCR binds to Gabg-GDP
Binding of ligand to GPCR triggers exchange of GDP for GTP on the Ga subunit (within Gabg)
Activated Gabg-GTP complex dissociates from GPCR
Cytosolic activated Gabg-GTP complex further dissociates into free a-GTP and bg subunits
a-GTP subunit can activate different target proteins
bg subunit can activate different target proteins
What is the heterotrimeric G-protein cycle?
G-protein cycling - moves between GTP (On) and GDP (Off) states
Receptors promote activation by allowing GDP dissociation and GTP association
Spontaneous exchange of GTP/GDP on Ga is very slow
RGS proteins and phospholipase C-βs are GAPs for G proteins
Ga and Gbg can independently target different pathways and cause different physiological responses
What are some downstream responses for each subunit?
Ga responses - InsP3, DAG, cyclic AMP, cyclic GMP, cytoskeleton
Gbg - InsP3, DAG, cyclic AMP, cytoskeleton, PIP3
Inhibitors or activators of GPCR signalling pathways have therapeutic use
What are some similarities between GPCR and RTK signalling?
Arrestins act as molecular scaffolds that recruit MAPK enzymes
Activation of the MAPK pathway - but using differential activators
Differential cellular responses depending on cell and tissue type
Describe molecular scaffolds?
Membrane-based scaffolds increase the local concentration of signalling proteins and insulate signalling proteins
Therefore increasing the signalling efficiency
Scaffolds localize signalling pathways to sites of action
What pathways can GPCRs activate?
GPCR can activate lipid signalling pathways and MAPK pathways
Lipid signalling pathway: It recruits phospholipase C beta1 This converts PIP2 into DAG and IP3 DAG leads to activation of protein kinase C - used in phosphorylation IP3 increases Ca2+ concentration
How does the receptor work?
They work in response to concentration levels
Constant and repeated GPCR activation leads to desensitisation and activation of a different signalling response over time e.g. cAMP production, PLC activation, etc.
In the adrenergic GPCR response to agonist concentration:
At low agonist concentration - only activate adenylyl cyclase
At high concentrations - interact with he Src protein, activating the MAPK
Shows different pathway activation depending on the amount of ligand
Describe ligand diversity in cellular responses?
Differential responses can be evoked by the GPCR ligand or agonist
Types of ligands: biogenic amines, amino acids/ions, lipids, peptides/proteins and others
Types of alpha units and their downstream signal activation:
Ai - adenylyl cyclases, inhibition of cAMP production, ion channels, phosphodiesterases and phospholipases
Aq - PLC-b, DAG, Ca2+ and PKC
As - Adenylyl cyclases and increase in cAMP concentration
A12 - RhoGEFs and Rho
Biological responses - proliferation, differentiation, development, cell survival, angiogenesis, hypertrophy, cancer etc…
Describe the life cycle of GPCRs?
- Made on the RER and cotranslated into the ER
- Then sent to the Golgi, before reaching the cell surface
- It binds ligand and undergo different activation states: phosphorylation, trafficking to endosomes, degradation in the lysosome and recycling back to the cell surface
What are some g-proteins within the monomeric GTPase subfamily?
Ras family Sar1/Arf family Ran family Rab family Rho/Rac/Cdc42 family
Describe monomeric G-proteins?
The small monomeric G-proteins are 20-25 kDa (200-250 residues) in size
The Ga subunits are 35-40 kDa (350-400 residues) in size
Small monomeric GTP-binding proteins are:
active when bound to GTP
inactive when bound to GDP
Both monomeric and heteromeric (a/b/g) G-proteins are prenylated
Meaning they have lipid tails attached to their N- or C-termini that enables membrane attachment
Describe G-protein cycling factors?
GDP/GTP exchange catalysts known as GEFs (guanine nucleotide exchange factors) promote activation
GAPs accelerate hydrolysis and deactivation
GDP dissociation inhibitors (GDIs) slow spontaneous nucleotide exchange
Describe Ras?
Ras has ~200 residue small GTPase conserved from yeast to man
Regulates many aspects of signal transduction pathways
Links cell surface receptor activation to MAPK pathways
It switches between GDP and GTP bound states - which are conformationally different
How is Ras linked to proto-oncogenes?
Ras mutations affect cell proliferation, death and/or differentiation
Due to a lack of a negative feedback loop
Many human cancers have Ras mutations
Oncogenic mutations in Ras cause a permanent GTP-bound state (on)
How is Ras and RTK signalling linked?
It is coupled to growth factor and RTK signalling
Growth factor activation of RTKs can lead to downstream Ras activation
Ras activation causes downstream MAPK activation
Ras activation triggers proliferation in many cell types
What RTKs can regulate Ras activity?
Epidermal growth factor receptor (EGFR) is coupled to Ras - they are functionally linked
EGF can activate intracellular signalling pathways leading to the same outcome, potentially one utilising Ras - activating the MAPK pathway
Nerve growth factor receptor (TrkA) is also coupled to Ras
Describe Raf?
This is a serine/threonine protein kinase
It contains 3 regions:
Protein kinase domain - catalytic (at C-terminus)
Hinge region - highly flexible
Autoinhibitory domain block - Ras binding domain and C1 domain (at N-terminus)
How is Raf activated?
Ras activation leads to a GTP-bound state
Ras-GTP recruits a Raf/HSP/14-3-3 heterodimer complex
Phosphorylation of Raf by PKC and c-Src unfolds and activates Raf
Describe Rac activation?
The Rac/Rho/Cdc42 small monomeric G-proteins regulate actin dynamics
Ras signalling regulates the GTP/GDP status of the Rac/Rho/Cdc42 family
Thus Ras activation triggers altered actin polymerisation and membrane dynamics = changes in cell shape and migration
Each G-protein regulates different aspects of actin-linked dynamics
Describe Rab?
Rabs are Ras-like GTPases - they are membrane associated and often interacting with vesicles
15-20 Rabs in yeast and 40-60 Rabs in mammals
Rabs regulate membrane traffic between intracellular compartments
Rabs act as switches that regulate transport vesicle docking and fusion
Describe the function of Rabs?
The Rab GTP/GDP-bound states regulate vesicle dynamics - as they are conformationally different
Different proteins can bind to each state
GEFs or GAPs regulate Rab activity
Rabs usually act alongside but separately to SNAREs
Describe the function of Rho-like GTPases?
Regulate intracellular signalling, cell shape, migration (changes in actin remodelling)
Important in pathogenic infection
Subversion of GTPase function by bacterial proteins
How do Rho-like GTPases function during pathogen infection?
Pathogenic bacteria secrete proteins into the cell
Rho-like GTPases are used to regulate actin dynamics correctly
Actin dynamics are manipulated by the bacteria to allows it bacteria to insert itself into the infected cell and to get out of the cell after multiplication/infection
What is the role of focal adhesion kinase (FAK)?
FAK activation stimulates GDP/GTP exchange (GEF) on Rho-like GTPase
Rho and FAK regulate cell shape and migration
Describe the similarity of GTPase activation and cycling?
The GDP and GTP-bound of states of different G-proteins are similar
Specific motifs in exposed/hidden sequences can bind proteins that have regulatory functions e.g. Raf, etc.
