Intracellular Signalling Flashcards
give a simplified overview of signalling?
a signal molecule will bind to a receptor and this will modulate a target protein
what is involved in intracellular signalling cascades?
- use proteins interaction motives, kinases, phosphatases, GTPases
- some can act in several different cascades
- network rather than linear
- allows control, diversification and cross talk
why are cell surface receptors important?
they initiate signalling cascades
- they receive and transfuse signals across the membrane
what are some examples of cell surface receptors?
G protein linked receptors
enzyme linked receptors
what do cell surface receptors do?
- cause a kinase cascade for example (from a G protein)
- signal onto second messengers (from enzyme)
what is a GPCR?
- around 500 in humans
- have very diverse types of ligands
- 50% of all drugs acto on GPCRs
- affect every level of human physiology
- receive a large range of signals
what ligands bind GPCRs?
amine hormones, peptides, small proteins, light, sugars and their derivatives
what is the structure of a GPCR?
- conformational flexibility of beta adreneric receptor changes upon ligand binding
- ligand binding, closes space at the extracellular side between TMs 3, 5 and 6
- they are forced apart at the cytosolic side
- induces a confomational change in the 5/6 loop coupling signal transduction
- increased probability of something
how many families of trimeric G proteins are there?
3: I, II and III
- families are determined by amino acid sequences relatedness of the alpha subunits
- grouped due to their downstream targets or a specific GPCR type they are activated by
what are the functions of G proteins?
- trimer G proteins bind GTP (hydrolyse) and then become activated
- they have 3 subunits (alpha, beta and gamma)
- the main one is the alpha subunit it binds GTP and is involved in signalling
what does the GPCR do?
- receptor determines how a signal is interpreted
- GPCR activates a G alpha protein
- discovered using a chimeric GPCR
describe the experiment involving GPCR
- adrenaline activates the alpa2 and beta2 adrenergic receptor
- these actiavtes differe Galpha subunits
- alpha2 activates G alpha i
- beta2 activates a G alpha s
- one inhibits a second messenger
- other stimulates the same second messenger
- same signal can exert different effects in specific cells by signalling through a different GPCR
how can you see these effects in GPCR in an experiment?
cut and past different parts of the receptors to see the effects
- find its transmembrane helix 5,6
- a loop between the two and a C terminus which are important
what happens once a Galpha protein is targeted?
- can be stimulatory or inhibitory
- both can couple to the downstream molecule adenylyl cyclase (a transmembrane protein)
- enzymes which generate cAMP
- when it couples GTP is hydrolysed and turn to GDP
- some GPCRs activate adneylyl cyclase in adipose tissue whereas others inhibit it
can G proteins hydrolyse GTP?
they cant on their own they need other proteins
describe what happens when a G protein binds to GPCR
- activates and binds GTP
- GPCR is GEF (guanine nucleotide exchange factor)
- adenylyl cyclase: GTP activating proteins it activates
- AC = GAP
what is the G protein cycle?
- G proteins bind tightly to nucleotide co-factor
- need GEF to release the GDP
- GTP spontaneuosly binds, due to higher concentration in the cell
- G proteins are GTPases - GTP hydrolysing enzymes - with extremely low activity so they need help from a GTPase activating protein (GAP)
what can ADP ribosyltion occur?
it can permanently activate signalling
- permenently activated signalling has serious pathological conequences
how does the cholera toxin affect GPCR signalling?
- colonises intestine
- changes G alpha unit
- catalyses covalent modification
- adds ADP ribose from NAD+ to an arginine residue at the GTPase active site
- prevents GTP hydrolysis by G alpha s and stimulatory G protein is permenantly activated
how does the Pertussis toxin affect GPCR signalling?
- catalyses ADP ribosyltion at cystein residue of the inhibitory G alpha i
- incapable of exchanging GDP for GTP
- blocks inhibitory pathway
how are secondary messengers involved in signalling?
- cAMP activates protein kinase
- small molecules are produce or released upon activation
- can activate downstream targets
- amplify a signalling cascade
- localized and constant destruction ensures a localized target response
what are the targets of cAMP?
pKA
how does cAMP interact with pKA?
- cAMP binding releases pKAs catalytic subunit and allows it to phosphorylate targets
describe pKA
3 isoforms of the catalytic subunit
4 isoforms of the regulatory subunit
how is glycogen metabolism controlled by enzymes?
- glycogen phosphorylase, cleaves off glucose units - glycogen breakdown
- glycogen synthase adds glucose units, glycogen synthesis (uses UDP glucosen lose energy)
what are the effects of cAMP on glycogen dehydration?
- activate GPCR with adrenaline
- cascade to activate kinase A protein through cAMP
- phosphorylates GPK which phosphorylates GP and breaks down glycogen
- at the same time pKA inhibits glycogen synthesis
- simultaneous activation of synthesis and inhibition of degradation
- tightened control
what is an enzyme linked receptor?
on the binding of a ligand they activate some enzymatic activity on the cytosolic side
how do enzyme linked receptors work?
- induce receptor dimerization
- activates enzymatic activity
- on cystolic side
how can receptor dimerization be induced?
- mutual trans-phosphorylation of the two subunits
- recruitment of a catalytic subunit from the cytosol (becomes activated)
what happens as a result of enzyme linked receptor becoming activated?
active enzyme may phosphorylate and activate targets - initiates a signalling cascade
what is RTK signalling?
receptor tyrosine kinase signalling - involves lots of domains
- it is a dimer needs dimerization
how does RTK signalling work?
- it phosphorylates itself (2 halves trans phosphorylates)
- fully activates receptor
- recruits and activates proteins
- activated receptor binds a multidocking proteins (IRS-1) and phosphorylates it
- proteins recruited by their ability to bind phosphorylated tyrosine
- their own tyrosines get phosphorylated as well
what roles does IRS-1 play in RTK signalling?
- gets phosphorylated by RTK
- provides further docking sites
- recruits multiple factors would individually be inactiv
what are the 2 types of modules capably of binding phosphorylate tyrosine?
PTB (phosphotyrosine binding domain)
SH2 domain
what is PTB?
- only has the property to bind the phosotyrosine
- PTB domains are similar to each other
- there are many
- evolved from a common ancestor that was able to bind phosotyrosine
- good feature
- needs specificity
how does specificity work in PTB?
domains are specific to one phosphotyrosine
- have amino acid residues in the vicinity
- important for binding
how can phosphotyrosine binding switch signalling on or off?
it phosphorylates or dephosphorylates
how does the insulin receptor work?
- PTB of IRS1 binds specifically to Tyrp of the insulin receptor and IL4R
- binding specificity - amino acid before the Tyr
- Alanine (in IL4R) is best
what are SH2 domains?
- amino acids around pTyr provide a specificty signature
- SHC needs a Leu or a Val at the third position of pTyr
- amino acids after the pTyr at a specific distance
what is phosphoinositide kinase mediated signalling?
- one signalling molecule recruited by IRS1 is phosphoinositol 3 kinase (PI3K)
- p85 subunit of PI3K contains an SH2 domain that binds phosphorylated IRS1
what is the function of phospholipases?
PLC can transduce both GPCR and RTK initiated signal
what is PLC activated by?
a G alpha protein or an activated RTK
how does an activated RTK activated a PLC?
- the RTK will dimerize
- activated kinase domain
- recruited PLC or γ(SHC 2 domain
- promotes enzymatic activity of PLC gamma
- its an enzyme that amplifies the signals
- generates second messengers from an important signalling lipid
- cross talks between GPCRs and RTKs
what is the activity of PLC?
- activated by GPCR or RTK
- cleave a specific [PI(4,5)P2]
- PLC doesnt have direct access to the [PI(4,5)P2]
- when recruiteded in the same vicinty PLC can more easily find its target
what is - [PI(4,5)P2]?
- head groups is a sugar like molecule
- many hydroxyls
- one hydroxyl used to bind to the phosphate
- other hydroxyls used as signalling platforms (depending on which is phosphorylated you get different signalling outcomes)
what is the structure of - [PI(4,5)P2]?
contains a phosphate in the 4th and 5th position
what happens when - [PI(4,5)P2] is PLC activated?
- hydrolyses and cleaves between the glycerol and phosphate group that links the ring to the phospholipid backbone into DAG and IP3
what is inositol 1,4,5 trisphosphate?
releases Ca2+ form the endoplasmic reitculum (ER)
what is DAG?
activates protein kinase C remains membrane bound
what is IP3?
- diffuses to the ER
- activates a channel to release Ca2+ ions
- Ca2+ release is transient
- increase Ca2+ concentration locally around the channel
- released from the ER into the cytosol
- IP3 degradation is by phosphates removing 4’ or 5’ or a kinase adding 3’
can Ca2+ levels in the cytosol be generated chemically?
no
why is Ca2+ important?
they are important second messengers
describe Ca2+ in the cytosol?
- low concentration in the cytosol
- Ca2+ pumps it out of the cytosol to organelles or to the outside
- Ca2+ channels in the plasma membrane and the ER when activated provide a local increase in cytosolic Ca2+ through IP3 gated channels from the ER
- ER Ca2+ pumps constantly removes excess cytosolic Ca2+ into the ER
- doesnt take long to get a large increase in calcium concentration
where is Ca2+ signalling found?
Muscle contraction
neurotransmitter release
activation of calmodulin
what is the activation of calmodulin?
- important calcium binding protein
- 2 different structures: one calcium free and one calcium bound
- conformational cahnge
- when it changes calcium binds to a kinase helix
- activates the kinase and goes on to phosphorylate its targets
what is the target of calcium?
protein kinase C (PKC)
what is the conventional isoform of PKC?
- alpha, beta, gamma
- has a calcium binding domain
- needs a second messenger to be activated and has a DAG binding domain
what is the novel isoform of PKC?
- need DAG only (activated)
what is the atypical isoform of PKC?
- activated by ceramide
what can PKC affect?
adhesion, proliferation, differentiation, migration, apoptosis, autoimmunity, IgG switch
give an overview of SH2
- binds pTyr generated by receptor tyrosine kinase
- once phosphorylated SH2 can bind
- binding/not binding determine by phosphorylation state
- specificity is determined by amino acids nearby
what does insulin isgnalling do?
regulates metabolism
what kind of receptor is the insulin receptor?
An RTK
how does the insulin receptor function?
- insulin (the ligand) binds and there is dimerization
- activation of tyrosine kinase activity
- phosphorylates the receptor
- phosphorylates molecules that bind to the receptor via phosotyrosine binding
- recruits proteins (IRS-1 which is a scaffolding protein)
- recruits several different signalling proteins (makes sure they are next to each other)
what does IRS-1 do?
provides docking sites for further signalling proteins and provides a further site for regulation
how does phosphoinositide 3 kinase transmits insulin signalling?
- recruited by IRS-1
- the p85 subunit of PI3K contains and SH2 domain that binds phosphorylated IRS-1
- important component of membranes
- PI45 is a substrate for the kinase
- creates binding sites for yet other domains
- needs to recruit PI3 kinase to the membrane
how does the local genertion of PI(3,4)p2 activates protein kinase B(PKB)?
- recruits 2 proteins: PKB and PDK1
- PDK1 phosphorylates PKB
- PKB needs to be recruited and the phosphorylated
what is PDK1?
phosphorylates PKB and contains a PH domain
what is PKB?
- needs to be recruited and then phosphorylated
- in the cytosol (folded in on itself)
- has a PH domain
- binds and unfolds
- PKB will be next to the PDK1
- full activation of PKB
what are some examples of PKB targets?
- e.g. IRS-1, GSK3, GS, PDE3B, mTOR
- effects glycogen synthesis, protein synthesis, cAMP
what is insulin?
reduces blood glucose levels so therefore the body wants to store glucose as glycogen
- mostly opposes adrenaline
what is PD£?
- inactivates PKA signalling
- inactivates cAMP
- degraded by phosphodiesterase (PDE)
- signalling through adenylyl cyclase is antagonised by the continuous breakdown of cAMP through PDEs
- ensures cAMP only acts when the cell is stimulated
how are PDE’s regulated?
by themselves, PKA, PKB, calmodulin
- allows a further layer of regulation
what is mTOR?
- a kinase
- central regulator of cell growth
- TOR: target of rapamycin
what is mTOR influenced by?
- nutrients, insulin signalling
- protein synthesis, self-eating, respiration
what does mTOR do?
- regulates cellular metabolism
- modulates how energy is used
- physiologically (influences how we age)
- oxidative stress if we take in too much
- pathological = cancer
what is rapamycin?
- inhibiting TOR, strong influence on growth
- used as a drug for: immunosuppression, cancer, longetivity, psychiatric conditions
what can be the negatives of mTOR?
- overworking mitochondria produces reactive oxygen this can damage cells
- mTOR activity has an effect on how quickly our cells age due to damage
- highlevels = obesity
- restrict calroic intake, to a degree, live longer
- medication that does the same thing as limiting calorie intake that inhibits mTOR
how does insulin affect glycogen synthesis?
- insulin and adrenaline come together at the same target, insulin receptors take charge
- IRS-1
- p85 and p110 (PI3 kinase)
- activates PDK1
- inhibits PGSK3 (activates glycogen synthase)
how does adrenaline affect glycogen synthesis?
- switches off insulin signalling
- short term signalling effects
how do the intracellular signals cross talk?
- PKA, PKC, CAM kinase etc all have common targets
- can be phosphorylated by more than one of these kinase
- can phosphorylate and regulate members of other pathways
- PLC is a common component of GPCR and RTK signalling
how does PKB inactivate IRS-1?
- negative feedback
- inhibits cAMP and affects glycogen synthase
why is compartmentalisation important?
- allows streamlining of cross talk
- recruiting activated signal transducers and targets to the same place (endosomes) where signalling can be channelled
what do EEA1 and APPL do?
they are scaffolds that bind to endosomal PIPs
what is endosomal recruitment?
endosomal recruitment of activated Akt promotes cell survival rather than growth by presenting Akt with a different substrate
what is endosomal activation?
Akt by the MAPK pathway leads to overgrowth (hypertrophy)
describe receptor down regulation by sequestration
- release of the ligand is not promoted in the endosome
- receptors are delivered to the multivescular body (MVB)
- MVBs have internal vesicles (formed from invaginations of their own limiting membrane)
- once in the internal vesicle of MVB the receptors active cytosolic portion is sequestered from the cytosol portion is sequestered from the cytosol
- the physical separation stops signalling
- receptors in MVBs can be degraded or recycled
what is ubiquitin?
- surface receptors are often ubiquintinylated following prolonged stimulation
- monoubiquitinylation of receptors induces their endocytosis
- endosome often provides a second signalling patteren
- sequestered into internal vesicle of a compartment called the MVB
Name a key signalling protein that ensures efficient cross talk between the insulin and adrenaline signalling pathways
PDE
list four ways in which a cell surface receptors downstream signalling can be modulated
- Extracellular ligand binding (followed by dimerization and/or conformational change in the TM region)
- Internalization (following ubiquitinylation)
- Phosphorylation
- Binding of an intracellular accessory protein (e.g. arrestin, scaffolding protein)
What is the role of the phosphotyrosine residue for signal transduction via the PTB and SH2 domains?
It is an on/off switch for signalling
What are the three key features that allow Ca2+ to be used as a second messenger?
- Cytosolic concentration of Ca2+ is 1000-fold lower than in organelles or outside of the cell
- Ca2+ specific channels respond to signalling molecules (e.g. IP3)
- Ca2+ binding can cause large conformational changes in the targets of Ca2+ signalling
