03-02-22 - Signal Transduction Flashcards
Learning outcomes
- Examine the role of signal transduction and why it is needed.
- Outline trimeric G-proteins, structure and function
- Identify the role of the phospholipase C/Ca2+/protein kinase C pathway in the cell in context with G-protein signalling
- Identify the role of the adenylate cyclase/cAMP/protein kinase A pathway in the cell in context with G-protein signalling
- List and relate the other major intracellular signalling pathways
How do most things that affect cell activity interact with the cell?
What are 2 ways extracellular signalling molecules can lead to cellular effect?
- Most things that affect cell activity or function do not enter the cell
- They act on membrane-bound receptors that control signalling proteins via the production of second messengers which mediate cell activity
• Extracellular signalling molecules can either:
1) Alter protein function, which leads to altered cytoplasmic machinery and altered cell behaviour
• This is fast (
What do many signalling proteins act as?
What are 2 common ways of activating/de activating signalling proteins?
What are the 2 main types of kinases?
- Many signalling proteins act as molecular switches
- 2 common ways of activating/de activating signalling proteins:
1) Phosphorylation
• A signal comes in, resulting in a protein kinase phosphorylating the signal protein using ATP
• When the signal leaves, the signalling molecule is dephosphorylated by protein phosphatase
2) GDP and GTP binding
• When the signal comes in, the signalling molecule releases GDP and binds GDP to become active
• When the signal leaves, GTP hydrolysis takes place, which dephosphorylated the GTP back to GDP, leaving the signalling protein inactive
• The two main types of kinases:
1) Tyrosine kinases
2) Serine/threonine kinase
• Two main types of GTP-binding proteins:
1) Trimeric G proteins
2) Monomeric GTPases
Describe the structure of a G Protein Couple receptor (GPCR)
- GPRC consist of 7 transmembrane helices
- This is known as a β2 adrenergic receptor (7TM receptor
- The amino termini is on the extracellular surface
- The carboxyl termini and loop is on the cytoplasmic surface and interacts with intracellular machinery
- This receptor interacts with a heterotrimeric G protein signal transducing complex on the intracellular side, which consists of alpha, beta, and gamma subunits
- These G-proteins can bind GTP (active) or GDP inactive
Describe the steps involved in the action of GPRCs
• steps involved in the action of GPRCs
1) Binding of hormone induces a conformational shape change in the β2 Adrenergic receptor
2) The activated receptor binds to the Gα subunit of the G protein
3) This binding induces a conformational shape change in the G-protein
4) The bound GDP dissociated and is replaced by GTP
5) The Gα subunit dissociated from Gβγ
6) The hormone dissociates from the β2 Adrenergic receptor while the Gα subunit binds to the effector, activating it (effector can be an amplifier like adenylate cyclase, which generates a large amount of secondary messenger)
7) The hydrolysis of GTP to GDP causes Gα to dissociate from the effector and reassociate with Gβγ
What 3 things does the GPRC system need to function
• 3 things does the GPRC system need to function:
1) A receptor
2) A transducer (G-protein)
3) An amplifier (adenylate cyclase) that generates a large amount of secondary messenger
What are the 3 main types of Gα subunit?
What do they each stimulate?
• 3 main types of Gα subunit:
1) Gαq (or Gq)
• Stimulates phospholipase C (enzymes that produce secondary messengers)
2) Gs
• Stimulates adenylate cyclase, increases cyclic adenosine monophosphate (Camp)
3) Gi
• Inhibits adenylate cyclase, decreases Camp
Describe the 5 steps in the inositol phospholipid signalling pathway
1) A signalling molecule binds to a G-protein-linked receptor, which activates a G-protein α subunit
2) This subunit then activates phospholipase C, which then cleaves PIP2, so that the inositol group and 3 phosphate becomes cleaved, forming IP3 and diacylglycerol
3) IP3 binds to calcium receptors on the ER, causing it to open, allowing calcium to move into the cell cytosol
4) Protein Kinase C (PKC) can then bind to calcium and diacylglycerol and become active
5) PKC can then phosphorylate proteins in the cell and alter their function
What are the cytosolic calcium levels at rest and while calcium channels are activated?
What does cellular response depend upon?
What are cytosolic calcium levels?
What are on and off pathways?
What are examples of each?
- Resting calcium concentration – 100nM
- Activated calcium concentration – 0.5-1 µM
- Cellular response depends upon duration of signal
- E.g exocytosis is fast signalling (µs) and only requires a short elevation in calcium, while processes like fertilization are long signalling (hr) and require a larger elevation in calcium
- Cytosolic calcium levels are dynamic?
• On pathways are ways in which calcium can enter the cytosol, either from an organelle or the extracellular environment
• Examples:
1) Calcium can enter from intracellular stores or from outside of the cell via calcium channels (may be receptor or voltage operated
2) Buffer calcium binding proteins can control the amount of calcium available to machinery
• Off pathways are how calcium gets pumped back out of the cell or into organelles. Usually pumps/transporters
1) SERCA ATPase that transports cytosolic calcium back into the SR
2) Calcium channels
3) Buffer proteins
How is fertilization of an egg by sperm triggered by calcium channels?
- When sperm penetrate an egg, they release protein PLC-Zeta, which triggers the opening of surface calcium channels
- Calcium wave triggers the start of embryonic development and prevents other sperm from entering the cell
How many proteins kinase C are there?
How are most isoforms present?
What does a rise in cytosolic calcium levels cause?
What 2 things can it be activated by?
What can it then phosphorylate?
- PKC is a large family of 12 different isoforms
- Most isoforms are present as catalytically inactive, soluble proteins in the cytoplasm
- Rise in cytosolic calcium levels cause PFC to bind to the cytosolic leaflet of the plasma membrane
- It can then be activated by membrane associated DAG or Ca2+
- PKC can then phosphorylate a wide variety of substrates on serine and threonine residues
Where does PKC have substrates?
Where can certain isoforms of PKC translocate?
What does this effect?
How can PKC indirectly alter gene expression?
- PKC has substrates in the cytoplasm, and some isoforms can translocate to the nucleus to phosphorylate nuclear proteins
- This allows PKC to work in a transient, or more permanent way (by mediating gene transcription)
- PKC can also function indirectly to alter gene expression by phosphorylating kinases:
1) MAP Kinase
• When phosphorylated, MAPK kinase translocates to the nucleus where it can phosphorylate protein ELK-1
• This forms a complex with SRF, which binds to SRE
• This triggers transcription upstream to create Mrna and proteins
2) IkB
• When phosphorylate, the NFkB subunit translocates to the nucleus, where it binds to binding sites upstream from genes where it can switch on transcription
What does binding of adrenalin to the B2 adrenergic receptor mediate?
What 3 cellular effects does this have?
What is PKA? What does it consist of?
How does Camp activate PKA?
What is CAMP degraded by?
- Binding of adrenalin to the B2 adrenergic receptor mediates the bodies stress of fear (fight or flight)
- Cellular effects:
1) Release of glucose anfd fatty acids from liver/fat cells for rapid source of energy
2) Increased contraction of cardiac muscle to give the body more oxygen
3) Increases intracellular concentration of camp (cyclic AMP), which is synthesised from ATP by the enzyme adenylate cyclase
- PKA is a camp-dependent protein kinase made up of 2 regulatory subunits and 2 catalytic subunits
- When camp is released, it binds to the regulatory subunits of PKA, causing the catalytic subunits to be released
- These can then go and phosphorylate a range of different targets in a cell to help invoke cellular effect
- Camp is degraded by the enzyme camp phosphodiesterase
What does signal transduction result in?
- Signal transduction results in an amplification of a signal
- A small change at the top can have a large change at the bottom
Where does the catalytic subunit of PKA phosphorylate substrates?
What 3 places does it have substrates?
What can PKA activate the transcription of genes for?
What does CREB do? When is it active?
- The catalytic subunit of PKA can phosphorylate substrates on the serine or threonine residues
- It has substrates in the:
1) Membrane
2) Cytoplasm
3) Nucleus
- In the nucleus, PKA can activate the transcription of genes contain Camp response elements (CREs) in their promoter
- CREB (Camp response element binding protein) binds to this sequence and activates transcription of downstream genes
- When CREB is unphosphorylated, it is inactive, only when it is phosphorylated does CREB activate transcription
What is cholera toxin?
When does it become active?
What does it do after this?
What does the overstimulation of camp production cause?
What acts in a reverse manner to cholera toxin?
- Cholera toxin is an oligomeric complex
- It becomes active after cleavage, and enters the intestinal epithelial cells to stimulate Gαs, and stimulates Camp production
- This overstimulation of camp production results in a release of water and ions, including Na+, K+, Cl- and HCO3- into the lumen of the small intestine
- This leads to rapid fluid loss and dehydration
- Pertussis toxin acts in reverse manner, inhibiting GαI (inhibitor) to increase camp production in lung epithelia
What are enzyme-linked receptor tyrosine kinases?
When are RTKs used?
Describe the steps involved in the function of RTKs
- Enzyme-linked RTKs are receptors that have enzyme activity of their own
- Insulin-like growth factors activate RTKs to control cell proliferation
• Function of RTKS:
1) Signal molecule in the form of a dimer bind to the RTK (which is also a dimer – 2 different receptor molecules forming a complex)
2) This catalyses the phosphorylation of the receptor itself on the intracellular side (phosphorylated tyrosine groups)
3) Different proteins inside the cell recognise the phosphate groups and bind to interact with the receptor, switching the proteins on
What is Ras?
When is it active?
How many Ras are there?
What 4 cellular processes does Ras regulate?
Describe the 3 steps in Ras activation?
- Ras is a small GTPase
- It is active when it binds GTP, and inactive when it binds GDP
- Ras superfamily has over 100 members
• Ras regulates:
1) Proliferation
2) Cytoskeletal dynamics
3) Membrane trafficking/vesicular transport
• Ras activation:
1) When the RTK becomes activates, the adaptor proteins binds to one of its phosphates
2) This acts an adaptor for Ras activating protein
3) When RASAP becomes active, it then activates RAS, which binds GTP and becomes activate
Describe the steps in Ras activating MAPK (mitogen-activated protein kinase) pathway.
What are the 2 different types of substrates of MAPK?
• Ras activating MAPK (mitogen-activated protein kinase) pathway:
1) Active Ras protein activates MAP 3 Kinase
2) MAP 3 Kinase activates MAP 2 Kinase
3) MAP 2 Kinase activates MAP Kinase (MAPK)
• MAPK substrates:
1) Substrates that cause changes in protein activity
• Protein-X
• Protein-Y
2) Substrates that cause changes in gene expression
• Gene regulatory protein A
• Gene regulatory protein B
What is the EGFR?
Describe the steps in the EGFR pathway.
What are mutations in proteins in this pathway associated with?
What do these mutations cause?
- EGFR is the epidermal growth factor receptor, which is an important RTK in cancer
- Steps in the EGFR pathway:
1) EGFR is activated by TGFα (Transforming growth factor alpha)
2) The receptor activates Ras via Grb2/SOS proteins
3) Ras activates Raf kinase, which stimulates gene transcription via other kinases
- Mutations in EGFR, Ras and Raf are associated with tumorigenesis
- These mutations cause overexpression and/or hyperactivation of the respective proteins
Signalling pathways interconnected
