M&R S7 - Signal Transduction in Biological Membranes Flashcards
What is signal transduction?
A process by which an extracellular signal (ligand binding to a receptor) can bring about an internal response in the cell
What are G-proteins?
A receptor superfamily that act by altering the activity of effectors (E.g. Ion channels, enzymes)
This is done through the activation of one or more types of Guanine nucleotide binding proteins (G-proteins)
What are some of the cellular functions controlled by G-protein receptors?
Muscle contraction
Light, smell and taste perception
Metabolic processes
Secretion
Describe the structure of G-proteins
Heterotrimeric (3 distinct subunits)
Alpha, beta and gamma subunits bind tightly to one another and function as a single unit
Alph subunit has a guanine nucleotide binding site which binds to GTP and slowly hydrolyses it
In what form is G-protein found in basal conditions? (receptor inactivated)
Where is it found?
In heterotrimeric form
Bound to GDP
Found at the inner face of the plasma membrane
What happens to G-proteins when G-protein receptors are activated?
Activated receptors have a high affinity for GDP bound G-proteins and they will bind
A protein-protein interaction occurs leading to the release of GDP and the G-proteins subsequently binding to GTP
Once GTP bound, receptor affinity falls and GTP-alpha and beta-gamma are released separately to interact with effectors
By what mechinism is the interaction between effector and G-protein terminated?
Terminated by the intrinsic GTPase activity of the Alpha subunit
Once GTP is hydrolysed the affinity of the alpha subunit for the Beta-gamma subunit is increased and they will reform the heterotrimer to await reactivation
Why can G-proteins be considered as on/off switches?
What else can they be considered as?
GDP to GTP exchange and GTP hydrolysis can be considered the on/off switches
Can also be considered as timers
The length of time taken for GTP hydrolysis governs length of effector activation
G-protein activation can have one of two effects on the effector, what are these effects?
Activation
Inhibition
Give some examples of different G proteins
Gs
Gi
Gq
Gt
What G protein is activated when noradrenaline binds to Beta-adrenoceptors and what is the effect?
Gs (Alpha s-GTP)
Stimulates adenylyl cyclase
How might adenylyl cyclase activity be inhibited?
Noradrenaline binds to Alpha2 adrenoceptors
OR
Ach binds to M2 cholinoceptors
The G-protein Gi is activated
Gi inhibits adenylyl cyclase
What does adenylyl cyclase do?
Creates cAMP
What two G-proteins bind to an effector other than adenylyl cyclase?
Gq
Gt
What is the action of Gq?
Stimulates Phospholipase C to cleave Posphatidylinositol-4,5-bisphosphonate (PIP2)
This results in Inositol-1,4,5-triphosphate (IP3) and Diacylglycerol (DAG) being produced
Where is rhodopsin found and what does it do?
In the eye, retinal photoreceptive cells (rods and cones)
It’s a G-protein receptor that activates Gt
This in turn activates a phosphodiesterase enzyme that hydrolyses cGMP to 5’-GMP
For each G protein give:
- An example receptor
- It’s effector and action on that effector
- An example physiological response to the effector in this case
Gs:
- B-adrenoceptor
- Stimulates adenylyl cyclase
- Stimulates glycogenolysis, lipolysis
Gq:
- M3 muscarinic
- Stimulates phospholipase C
- Smooth muscle contraction
Gi:
- M2 muscarinic
- Inhibits adenylyl cyclase and stimulates K+ channels
- Slowing of cardiac pacemaker cells
Gt:
- Rhodopsin
- Stimulates cGMP phosphodiesterase
- Visual excitation
Give a list of Adrenergic and muscarinic receptors and their G proteins along with that G protein’s action on its effector
Adrenergic:
A1 - Gq - Stimulate phospholipase C
A2 - Gi - Inhibit adenylyl cyclase
B1+B2 - Gs - Stimulates adenylyl cyclase
Muscarinic:
M1+M3 - Gq - Stimulates Phospholipase C
M2 - Gi - Inhibits adenylyl cyclase
How many G-protein subunits are coded for in the human genome?
How many possible combinations of G proteins are there?
20 G-alpha
5 G-beta
12+ G-gamma
Over 1000 G-alpha-beta-gamma combinations
How many G-protein receptor types are there?
How specific is their action?
At least 800
Can interact with different G-alpha subtypes to activate/inhibit 10 or more effectors (enzymes/ion channels)
Why can an extracellular signal binding to a GPCR bring about specific cellular responses?
A specific GPCR will activate a single or small sub-population of G-proteins and effectors
Due to the large numbers of different receptor types and G-protein subtypes this will bring about a very specific response
Describe the action of Cholera toxin on the body from a biochemical standpoint
CTx will ADP-ribosylate the s-alpha subunit of Gs
This eliminates the GTPase activity of Gs-alpha and it becomes irreversible activated
Describe the action of Pertussis toxin on the body from a biochemical standpoint
PTx will ADP-ribosylate the i-alpha subunit of Gi
This interferes with the GDP/GTP exchange on Gi-alpha and it becomes irreversibly inactivated
What two conditions can be caused by loss of function mutations to GPCRs?
Name the GPCR that mutates in each case
Retinitis pigmentosa:
- Loss of function mutation to rhodopsin
Nephrogenic diabetes insipidus:
- Loss of function mutation to V2 vassopressin receptor
What condition can be caused by gain of function mutation to a GPCR?
What is the GPCR in question?
What is meant by ‘gain of function’ in this case?
Familial male precocious puberty:
- Gain of function mutation to the luteinising hormone receptor
The receptor is active without a ligand
How does adenylyl cyclase activity bring about increased/decreased cellular activity?
Hydrolyses ATP to generate cAMP which interacts with cAMP-dependent protein kinase (PKA)
PKA phosphorylates a variety of proteins within the cell to affect activity
How is adenylyl cyclase activity controlled?
Gi binding inhibits
Gs binding stimulates
Give some examples of the effects of adenylyl cyclase activation
Increased glycogenolysis and gluconeogenesis in the liver
Increased lipolysis in adipose tissue
Relaxation of some smooth muscle types
Positive ionotrophic and chronotrophic effects on the heart
Describe how Phospholipase C activation can affect cellular activity
Cleaves Posphatidylinositol-4,5-bisphosphonate (PIP2)
This results in Inositol-1,4,5-triphosphate (IP3) and Diacylglycerol (DAG) being produced
IP3 interacts with specific intracellular receptors (IP3 receptors) on the ER to allow Ca2+ to leave the ER lumen
DAG interacts with protein kinase Cs
Give a list of GPCRs that can activate Phospholipase C and their ligands
What G protein is responsible for Phospholipase C activation?
M1+3 muscarinic (Ach)
H1 receptors (Histamine)
5-HT2 receptors (5-HT aka. serotonin)
What physiological effects can Phospholipase C activation lead to?
Vascular, GI tract and airway smooth muscle contraction
Platelet aggregation
Mast cell degranulation (whatever that means)
What is the physiological effect of increased or decreased rhodopsin activity and what controls the level of rhodopsin activation?
Activated by a photon
In the dark, there is less cGMP breakdown and levels remain sufficient to open a secondary messenger controlled ion channel to allow Na+ and Ca2+ into the cell cytoplasm
On exposure to light there is more cGMP breakdown leading to decreased cGMP and the closure of the ion channel and membrane hyperpolarisation, thus altering output to the CNS
What are the secondary messengers that exert their effect via protein kinases?
For each, name the protein kinase they activate
cAMP:
- cAMP-dependent protein kinase (PKA)
cGMP:
- cGMP-dependent protein kinase (PKG)
DAG:
- Protein kinase C (PKC)
Ca2+:
- Ca2+/calmodulin-dependent protein kinase (CaM-kinase)
How do protein kinases activated by secondary messengers change cellular activity?
Cause phosphorylation of a distinct family of target proteins
These proteins may be enzymes, ion channels, transporters, structural proteins etc.
Their activities may be increased or decreased or unaltered by this covalent modification
Protein kinases activated by secondary messengers commonly phosphorylate what amino acid residues in the target proteins?
Serine or threonine
Give an example of regulation of Chronotropy in the heart by GPCRs
Ach release from parasympathetic nerves will bind to M2 muscarinic GPCRs on sinoatrial node cells
This results in the activation of Gi which increases the open probability of K+ channels
Increased membrane permeability to K+ causes hyperpolarisation resulting in a negative chronotropic effect
Give an example of ionotropic regulation in the heart by GPCRs
Sympathetic innervation (leading to release of noradrenaline) of the cardiac ventricles or circulation adrenaline will lead to activation of beta-adrenoceptors (mostly B1)
This results in activation of Gs which in turn activates adenylyl cyclase and leads to intracellular increase in cAMP levels
cAMP binds to PKA and causes phosphorylation of voltage operated Ca2+ channels (VOCCs)
which increased the open probability the VOCCs
The increase in intracellular Ca2+ brings about positive ionotropic effect (increase in force of contraction)
Explain how arteriolar vasoconstriction is brought about by GPCR activation
Sympathetic release of noradrenaline activates A1-adrenoceptors in the arterial smooth muscle
This activates Gq which stimulates Phospholipase C activity
Phospholipase C produces IP3 which releases Ca2+ from the ER and initiates contractile response
Give an example of how GPCR activation can lead to modulation of neurotransmitter release
Pre-synaptic u-opioid receptors can be stimulated by opioids (endogenous or analgesics)
This activates Gi and the beta-gamma subunit released from Gi interacts with Voltage operated Ca2+ channels (VOCCs) to reduce Ca2+ entry into the cell
This reduces neurotransmitter release
Why must GPCR - G-protein - Effector system allow for amplification?
A small signal (activation of a few GPCRs) requires amplification to generate a large intracellular response
By what mechanisms is the GPCR - G-protein - Effector system amplified?
Activated GPCR can activate more than one G-protein
Activated G-protein subunits (either alpha- GTP or beta-gamma subunits) can activate multiple effector molecules
Enzyme effectors can create/breakdown 100 - 1000s of secondary messengers
Ion channel effectors allows 100 - 1000s of ions to cross the plasma membrane
Secondary messengers often activate enzymes which can convert 100 - 1000s of molecules or initiate enzyme cascades
Over what time scale do activation/deactivation of signalling pathways in a cell occur?
Rapid, often a few seconds
What aspects of the GPCR signalling pathway facilitate deactivation?
Productive interaction of GPCR and G-protein weakens the GPCR-agonist binding, agonist dissociation more likely to occur
While activated the GPCR is vulnerable to protein kinases that phosphorylate the receptor and prevent it activating further G-proteins (receptor desensitisation observed in most GPCRs)
Active lifetime of a GTP-alpha G-protein subunit may be limited by cellular factors which stimulate the GTPase activity of the alpha subunit
Cells contain high levels of enzymes which metabolise secondary messengers
Enzymes/protein kinases activated by secondary messengers have their activities opposed or reversed
Give two examples of how a cell might metabolise secondary messengers
cAMP metabolised to 5’-AMP by phosphodiesterases
IP3 metabolised to inactive IP2 (inositol-1,4-bisphosphate) by 5-phosphatase activity
Give an example of how a cell might oppose the effects of secondary messengers
Target protein phosphorylation by protein kinases reversed by active cellular protein phosphatase activity
