Pathways Flashcards
steroid hormones
- Diffuse directly into cell
- Bind specific cytosolic receptors, causing release of inhibitory protein from DNA binding domain
- Translocate to nucleus , directly regulate gene transcription
ion channel linked receptors
- Ligand-gating – eg w/ neurotransmitters
- Conf change opens channel
- Eg nicotinic receptors, ACh and Na+
- Curare: blocks ACh receptor (antagonist)
- Nicotine: stimulates (agonist)
cAMP pathway
- Synthesised from ATP by adenylate cyclase, broken down by phosphodiesterase to 5’ AMP.
- Signal binds 7 membrane pass receptor, causing a conformational change
- Cytoplasmic domain 3 activates G protein (Gs)
- Gs activates adenylate cyclase
- Adenylate cyclase synthesises cAMP
- cAMP causes a response via protein kinase
- eg opening of Na+ channels and depolarisation
- G proteins = anchored to plasma membrane, bind GTP.
phosphoinositide pathway
o Signal binds 7 transmem pass receptor, causing a conformational change
o Cyto domain 3 activates Gq
o Gq activates PLC
o Phospholipase C enzyme hydrolyses PIP2 to IP3 and DAG, 2nd messengers
o DAG stimulates protein kinase C
o IP3 acts to release Ca2+ from the ER
o Ca2+ causes further Ca2+ release
o Ca2+ acts through protein kinase/ specific proteins (calmodulin, troponin c) to cause muscle contraction
o Or act directly on ion channels
tyrosine kinase linked receptors PDGF
platelet-derived growth factor:
1) PDGF => dimerization of 2 PDGF receptors
2) 2 tyrosine kinase receptors phosphorylate each other and then act as docking sites to bind specific amplifiers
3) PLC = amplifier (…)
4) Also attract proteins that regulate the GTP-binding ras. Ras = inactive when bound to GTP but activated by SOS, which exchanges GDP for GTP. Ras can also inactivate itself with a GTPase activating protein, GAP
Ras = commonly mutated in cancer via loss of GTPase activity
5) Active rasGTP starts a protein phosphorylation cascade, MAPK is phosphorylated
6) MAPKP moves into the nucleus and phosphorylates TFs
7) Active TFs initiate transcription of genes which control cell proliferation.
Ras
Ras = monomeric G protein and molecular switch. GAPs keep in default off state by promoting GTPase activity. GEFs promote GTP binding and on state.
Dictyostelium cAMP
- Founder cell emits cAMP and neighbouring cells emit own cAMP as signal relay. waves of cAMP from aggregation centre
- Cells move towards by chemotaxis
- cAMP binds to GPCR
- Activation of adenylate cyclase dependent cAMP pathway, causes signal relay as cAMP produced and secreted
- Activate PLC dependent IP3 pathway, triggers local cytoskeleton rearrangements via Ca2+, for chemotaxis.
- Movement = via directed extension of pseudopodia
- -ve feedback: when cAMP builds up in cell, sensing of cAMP inhibited by receptor phosphorylation. PDE breaks down cAMP so return to resting state
- +ve feedback: binding of cAMP secrete more cAMP
- Causes oscillations in [cAMP]
Vibrio fischeri quorum sensing
Vibrio fischeri – want to reach certain population size before starting to emit light, this depends on expression of lux A and B which encode luciferase enzyme, and is regulated by LuxI and R.
• OHHL = LuxI product and diffuses out of cell – so external concentration depends on bacterial cell density
• When OHHL conc reaches the critical for LuxR binding, stimulating gene expression
• luxI is induced more OHHL synthesis, +ve feedback
• luxA and B are induced, causing bioluminescence.
bacterial chemotaxis - general
- Bacteria can move due to rotation of flagella, motor = driven by pmf
- Biased random walk chemotaxis
- Runs: swim in straight line, anticlockwise rotation
- Tumbles: clockwise – then start new run in random direction
- Detect changes in concs of attractants etc. temporally rather than spatially (too small for this)
bacterial chemotaxis - proteins
- Mem-bound signal transducers, respond to specific chemoattractants, = MCPs, methyl accepting chemotaxis proteins
- Cytoplasmic signal transd: CheA=histidine kinase, CheY=response regulator, CheW=adapter, CheZ=CheY phosphatase
- Flagellar switch: FliM
- Adaptation: methylation levels of MCPs regulated by CheR (methyltransferase) and CheB (response regulator/ methylesterase)
bacterial chemotaxis - no attractant
- CheW adaptor couples CheA to MCP receptor
- CheA autophosphorylates
- Pi transfer to CheY (response reg)
- CheY-Pi interacts with FliM so flagella switch to clockwise tumble state, change direction
bacterial chemotaxis - attractant
- Attractant binds receptor, causes low rate of CheA phosph
- Unphosph CheA means no CheY phosph
- Also CheY dephosph by CheZ, phosphatase
- No int w/ FliM so anticlockwise flagella rotation and longer swims
ABA
o ABA = produced by plant roots in times of drought stress
o ABA binds intracellular PYL receptor
o ABA-receptor complex inhibits 2C protein phosphatase, allowing OST1 kinase to be active
o OST1 activates Ca2+ channel, allowing Ca2+ entry
o ABA-receptor complex also activates PLC/ IP3 pathway
o IP3 opens Ca2+, also have +ve feedback w/ Ca2+ induced Ca2+ release
o Elevated [Ca2+]: inhibit inward K+ channels and open outward Cl- channels
o Membrane depolarisation activates outward K+ channels, K+ leaves cell, water follows so lose turgor pressure and stomata close
Light responses: phytochrome and photomorphogenesis
Pr is inactive but can be converted to Pfr when absorbs red light. Pfr is active and can translocate to nucleus.
- Pfr (PHY) phosphorylates PIF3 (-ve regulator of photomorphogenesis), targeting it for degradation – allowing photomorphogenesis to proceed
- COP1 = negative regulator of photomorphogenesis, an E3 ubiquitin ligase which targets HY5 for degradation. HY5 = TF which activates genes needed for photomorphogenesis
- So when COP1 is in the nucleus it represses photomorphogenesis.
- COP1 accumulates in the nucleus in the dark and is exported in the light – allowing expression of light-activated genes eg those activated by HY5.
Gibberellic acid and regulation of growth
- Regulates growth via abundance of DELLA proteins, repressors of gene expression.
- GID1 = receptor which binds gibberellic acid and targets DELLAs for degradation by 26S proteasome – via ubiquitination by an E3 ligase
- DELLA degradation allows for gene expression
Convergence: of phytochrome and gibberellin signalling at PIFs to regulate growth
- Light: low GA, DELLAs accumulate, bind PIF3 (TF) and prevent growth
- Dark: high GA, DELLA degradation allows PIF3 to regulate genes that promote growth for photomorphogenesis
- DELLA degradation = relief of repression
- Light: Pfr phosphorylates PIF3 (negative regulator of photomorphogenesis) and targets for degradation, allowing HY5 to activate genes needed for photomorphogenesis
- Dark: COP1 E3 ligase targets HY5 for degradation so photomorphogenesis repressed
Negative regulation/ + relief of
hormones bind proteins associated with SCF complexes, which are part of E3 ubiquitin ligase complexes. Recruitment of negative regulators to SCF, ubiquitination of negative regulators by E3 ligase to target them for degradation by the 26S proteasome.
G proteins general
o Gs has 3 subunits, Ga, Gb and Gy. (heterotrimeric) Ga and Gy are associated with the plasma membrane.
o Unstimulated Gs = trimer with GDP bound to Ga so cannot interact with adenylate cyclase
o Binding of hormone to GPCR changes receptor conformation so dissociates from GPCR and GDP is replaced by GTP on Ga.
o This causes dissociation of Ga from Gb and Gy.
o Ga binds and activates adenylate cyclase
o Adenylate cyclase synthesises cAMP
o Ga has GTPase activity so hydrolyses GTP
o Ga then detaches from adenylate cyclase and reassociates with Gb and Gy in inactive form.
adrenaline
- Stimulates glycogen hydrolysis in muscle cells
- Low blood concentration, response is proportional to hormone concentration
- Adrenaline binds its receptor, a 7 transmembrane pass GPCR, causing a conformational change in the 3rd intracellular loop.
- The GPCR activates Gs (see below for G protein pathway)
- Ga activates adenylate cyclase, which synthesises cAMP
- cAMP acts as a second messenger, activating protein kinase A
- PKA activates phosphorylase kinase by phosphorylation
- Phosphorylase kinase then activates glycogen phosphorylase by phosphorylation.
- Glucose-1-phosphate is produced and can be used in glycolysis to generate ATP for muscle contraction.
Nervous pathway of phosphorylase kinase activation:
o at neuromuscular junctions, motor neurone endings release acetylcholine, which binds to nicotinic acetylcholine receptors on the muscle fibre membrane.
o This leads to an increase in Na+ permeability and depolarisation.
o Voltage-gated calcium channels in the sarcoplasmic reticulum membrane are activated, causing calcium efflux into the sarcoplasm.
o Ca2+ activates phosphorylase kinase and stimulates muscle contraction.
adrenaline pathway downregulation
- Hormone conc in blood falls due to metabolism
- GTP hydrolysed to GDP by Ga GTPase activity, Ga reassociates with rest of G protein, then inactive
- cAMP hydrolysed by PDE
- Phosphorylase kinase and glycogen phosphorylase dephosphorylated by phosphatases
- Reuptake of cytosolic Ca2+ into ER by SERCA reverses this mechanism of phosphorylase kinase activation
- So glycogen breakdown only continues with persistent signals.
adrenaline pathway amplification
– via cascade mechanism
- 10^6 fold (nM to mM)
- Each activated receptor can activate multiple G-proteins
- Adenylate cyclase, protein kinase and phosphorylase kinase all act catalytically so each activated enzyme produces many products.
caffeine
phosphodiesterase inhibitor, so increases cAMP levels – causing alertness, muscle tremors
cholera
produce cholera toxin in intestine. Toxin ADPribosylates Ga, inhibiting its GTPase activity – GTP is permanently bound to Ga so adenylate cyclase is permanently activated. [cAMP] increases, Cl- and water flow into the intestine lumen and cause dehydration.
