Week 6 (signalling & intracellular messengers) Flashcards
Describe G protein coupled receptors
- largest class of human cell surface receptors
- involves in smell, taste, vision and hormone detection
- bind a huge array of molecules
- characterised by 7 transmembrane domains
GPCR activation
- ligand binds to an extracellular site
- changes to the conformation of the receptor
- this recruits the G protein
- at this point the G protein is inactive (GDP bound)
- binding to the receptor induces GDP release; GDP is replaced by GTP
- GTP bound G alplha separates from the GCPR and G beta gamma
- Ga goes on to activate signalling cascades
- An active GPCR can activate many G proteins.
Explain the properties of G proteins
- G proteins act as molecular switches.
- In the inactive state they are bound to GDP.
- Activation of the G protein results in release of GDP.
- This GDP is then replaced by GTP producing an active G protein.
G-proteins can activate Adenylyl-cylase: what is the function of adenylyl cylase?
- Adenylyl-cyclase is a transmembrane protein
- Adenylyl-cyclase can form cyclic AMP (cAMP) from ATP
- cAMP is a potent SECOND MESSENGER
What are second messengers?
A diverse family of low molecular weight compounds such as cyclic AMP and calcium ions, which transmit the biological signals initiated by receptor-ligand binding at the cell surface to intracellular targets such as gene expression.
Give some examples of second messengers
Second messengers – alter metabolism or affect effector enzymes that modulate target proteins and eventually cell behaviour. Include: cAMP Ca2+ IP3 DAG
What are the G protein families?
4 families based on sequence homology.
Gs – stimulates adenylate cyclase.
Gi/o – inhibits adenylate cyclase.
Gq/11 – stimulates PLCB (phospholipase C beta).
G12/13 – activates RhoGEFs.
What are the cellular effects of cAMP?
- Activation of cAMP-dependent protein kinase A (PKA)
- Protein kinase A can phosphorylate multiple targets
What are some cellular effects of Ca2+?
- Activates calmodulin (calcium binding proteins)
- Activates together with Calmodulin Ca2+/calmodulin dependent kinases (CaM-kinases)
- Modulates the activity of loads of other proteins
How can G-proteins control channel opening?
Indirectly (via cAMP, IP3 and Ca2+ or phosphorylation by PKA, PKC, CaM-kinase)
Directly by binding to the channels and modulating their activity
what are Downstream events of gated
channel signalling?
opening of channels leads to ion fluxes across the membrane
as a result the membrane potential is changed (hyperpolarisation or depolarisation)
intracellular ion concentrations rise
(Most importantly: Ca2+)
What is the function of receptor signalling cascades?
Can detect a single molecule of ligand e.g. a photon.
Transduce this signal across the membrane.
Rapidly amplify this signal.
Activate a cascade of proteins that bring about appropriate cell behaviours.
What are the 3 subunits of G proteins
Alpha (Bound to GDP)
Beta
Gamma
What can Gq proteins do?
They can activate phospholipids C (PLC beta)
What are the the 4 subunits of protein kinase A?
It is a tetromer:
2 regulatory subunits
2 catalytic subunits
How many units of cAMP can bind to protein kinase A?
4
What happens after cAMP bind to protein kinase A?
Regulatory subunits dissociate from catalytic subunits which become active
What is the function of phospholipase C?
It cleaves (hydrolyses) phospholipids
Describe the prices by which Gq proteins activate phosholipase C
- signal molecule binds to GPCR
- Gq protein recruited
- Gq protein exchanges GDP for GTP and becomes active
- the alpha subunit activates phosphlipase C
- phosplipase C cleaves the inner leaflet of the membrane (phosphoinositde 4,5- bisphosphate )
- phospholipase cleaves part of that molecule leaving part of it (diacylglycerol) attached to the inner leaflet of the membrane
- produces IP3
What are the consequences of phospholipase C activation?
- IP3 is the ligand for gated Ca2+ channels
- binding causes the release of Ca2+ (second messenger)
What two secondary messengers activate protein kinase C?
Diacylglycerol and Ca2+
Explain the receptor signalling cascades involved in vision:
- one rhodopsin molecule absorbs one photon
- G protein molecules are activated
- cGMP phosphodiesterase molecules are activated
- cGMP are hydrolysed
- Na+ channels close
- Na+ prevented from entering the membrane
- hyperpolarisation occurs
G proteins as molecular switchses: what acts as the GEFs (Guanine nuclrotide exchange factors)?
GPCRs
How can protein kinase A modulate transcription?
-Protein kinase A can modulate transcription (gene expression) by phosphorylating transcription factors
How many subunits does protein kinase A have?
4 subunits
2 Regulatory +2 catalytic
How many cAMP molecules can bind to cAMP dependant protein kinase A?
4
What happens when 4 cAMP molecules bind to the 2 Regulatory subunits of cAMP dependent protrin kinase A?
- The catalytic subunits will dissociate from the regulatory subunit.
- Catalytic subunits are not active so can phosphorylate target proteins with serine or threonine residues
What happens when 4 cAMP molecules bind to the 2 Regulatory subunits of cAMP dependent protrin kinase A?
- The catalytic subunits will dissociate from the regulatory subunit.
- Catalytic subunits are now active so can phosphorylate target proteins with serine or threonine residues
Why is cAMP short lived?
The enzyme cyclic AMP phosphodieterase converts cAMP to (5’) AMP
What happens when phospholipase C is activated?
It cleaves a component of the inner leaflet- phosphoinositide 4,5 bisphosphate
- diacyl glycerol stays attached to the inner leaflet
- inositol 1,4,5 triphosphate (IP3) is released
What is the function of diacylglycerol?
Activates protein kinase C
What is the function of IP3?
Releases Ca 2+ from ER
What 2 second messengers activate protein kinase C?
Diacylglycerol
Ca2+
What is a calmodulin?
A calcium binding protein
What are CaM-kinases?
Calmodulin dependent kinases
What activates phosphodiesterase?
A G protein
