GPCRs Flashcards
What is the structure of a GPCR
7 transmembrane proteins in a single chain.3 intra and 3 extra cellular loopsN-terminus extracellularC-terminus intracellular
Name the 5 families of GPCRs
SecretinAdhesionGlutamateFrizzled/Taste2Rhodopsin
What is the special feature of the Adhesion family of GPCRs
Large N-terminus which includes similar regions to adhesion molecules
What are the special features of the Glutamate family of GPCRs
Mainly neuronalSmaller extracellular regionCan be ligand gated ion channels
What are the special features of the Frizzled/Taste2 family of GPCRs
Generally involved in controlling development and cell growth
What are the special features of the Rhodopsin family of GPCRs
Biggest family of GPCRs. Include amine receptors, light receptors, olfactory receptors etc.
How was the structure/function relationship of GPCRs determined?
They created chimeras. Fused two different receptors and measured response
What region of a GPCR determines function
Function is usually controlled by intracellular loops.Specifically intracellular loop 3 and carboxyl (C)-terminus.
How does a GPCR signal?
Receptor activation causes the alpha subunit of the G-protein to exchange a GDP for a GTP. The GTP bound alpha subunit is able to activate an effector molecule
How does a GPCR signal switch off
Each G-protein has an intrinsic GTPase. It hydrolyses the GTP to give GDP (inactive)
What are the 4 stages of the G-protein cycle
Basal stateReceptor activationEffector modulationGTP hydrolysis
What happens in the basal state of the G-protein cycle
Nothing. Alpha bound to GDP
What happens in the receptor activation state of the G-protein cycle
Agonist activates receptorG-protein coupled and GDP exchanged for GTPG-protein subunits dissociated
What happens in the effector modulation state of the G-protein cycle
Effector is bound to the G alpha subunit
What happens in the GTP hydrolysis state of the G-protein cycle
GTP bound to alpha subunit dissociates.Alpha G protein bound to GDPSubunits re-associate
What is the role of the alpha subunit of a G-protein
Bound to GDP/GTPActivates effector molecule
What is the role of the beta subunit of a G-protein
Propeller structure that creates stability
What makes up a G-protein
Alpha subunitBeta subunitGamma subunit
What is the effector pair of a Gs protein
Adenylate cyclase
What is the effector pair of a G (alpha i) protein
Adenylate cyclase
What is the effector pair of a G (alpha o) protein
K+ and Ca2+ channels
What is the effector pair of a G (alpha t) protein
cGMP phosphodiesterase
What is the effector pair of a G (alphah gust) protein
cGMP phosphodiesterase and possibly adenylate cyclase
What is the effector pair of a G (q / 11) protein
Phospholipase C - beta
What is the effector pair of a G (12 / 13) protein
Rho AMisc small proteins
What are the main downstream signalling molecules associated with GPCRs
Adenylyl cyclasecAMPProtein Kinase A (PKA)PhosphodiesteraseRGS (regulator of G-protein signalling)
Name a common downstream signalling pathway
Adenylyl (adenylate) cyclase activation -> increased cAMP levels -> Increased PKA levels -> lots of intracellular pathways
How does cAMP activate protein kinase A
PKA is made up of four subunits - two catalytic subunits, and two regulatory subunits.cAMP binds to and mediates the dissociation of the regulatory subunits, enabling PKA to function.
What is Forskolin and why is it used?
It is a drug that increases the effect of GPCRs coupled to a Gs protein. Usually used in labs to show effects of adenylyl cyclase as it bypasses the need for GPCR activation
Give an example of a downstream event triggered by PKA activation
Calcium-handling proteins activated, increases force of contraction in the heart
Which G-proteins change intracellular levels of cAMP
Gs - increasesGi - decreases
Define phospholipase
An enzyme that hydrolyses phospholipids (e.g. cAMP) into fatty acids and other lipophilic substances
What are the three groups of Protein Kinase C
Calcium-dependent, DAG-activated (cPKC; conventional)Calcium-independent, DAG-activated (nPKC; novel)Calcium-independent, DAG-non-responsive (aPKC; atypical)
What does adenylyl cyclase do
Synthesises cAMP from ATP.
How is adenylate cyclase regulated by GPCRs
Gs alpha protein stimulates AC effector. (e.g. beta-adrenoceptor)Gi alpha protein inhibits AC effector (e.g. alpha2-adrenoceptor; and CB1 cannabinoid receptor)
What is the structure of adenylate cyclase (AC)
Two transmembrane domains; C terminus is bound to the N terminus of the other
How many isoforms of Adenylate Cyclase (AC) have been identified
9
How does adenylate cyclase act as a catalyst
The two cytoplasmic C-domains in the second transmembrane domain interact.
Where is the AC1 receptor located
Brain retina, adrenal medulla
Where is the AC3 receptor located
Olfactory neurones, brain, retina, heart, lung
Where is the AC8 receptor located
Brain
How do the AC1, AC3 and AC8 receptors work
Activated by calcium/calmodulinInhibited by G[alphai] subunits (AC1 and AC3)Inhibited by G[betagamma] subunits (AC1)
Where is the AC2 receptor located
Brain, olfactory tissue, lung
Where is the AC4 receptor located
Widespread
Where is the AC7 receptor located
Widespread
How do the AC2, AC4 and AC7 receptors work
Activated by Protein Kinase C (PKC)Unaffected by calcium/calmodulin/G[alphai] (unlike AC1,3,8)Stimulated by G[betagamma] subunits
Where are the AC5 and AC6 receptors located
Heart, Brain, Kidney
How do the AC5 and AC6 receptors work
Inhibited by free calcium; unaffected by Calcium/calmodulinInhibited by protein kinase A (PKA) phosphorylationunaffected by G[betagamme] subunits
Where is the AC9 receptor located
Testis/widespread
How does the AC9 receptor work
Inhibited by:Calcium/calcineurinG[alphai] subunitsProtein Kinase C (PKC) phosphorylationUnaffected by G[betagamma] subunits
What is the specific activity of Protein Kinase A
Acts as a serine/threonine kinase. Transfers terminal phosphate on ATP onto target. Involves esterification between phosphate group and serine/threonine.
What is the consensus sequence recognised by Protein Kinase A
NH2 —- Arg-Arg-X-Ser/Thr-X
What does CREB stand for
cAMP response element binding protein
How does Protein Kinase A activation affect the heart? how?
Phosphorylates several calcium handling proteins. Increases force of contractions
What is CREB
A transcription factor that regulates gene expression
How does protein kinase A affect CREB
IndirectlyModifies other proteins that are able to promote/inhibit transcription
What genes are targeted by Protein Kinase A activity
Phosphoenolpyruvate carboxykinase (PEPCK) (rate limiting in glyconeogenesis)AC8, c-fos, glutamate receptor, tyrosine hydroxylase (important in learning and memory)
What does PIP2 stand for
phosphatidylinositol-4,5-bisphosphate
How is PIP2 affected by GPCR receptor activated
GPCR q effector is phospholipase C. Phospholipase C cleaves PIP2 (yielding DAG and IP3)
What does DAG stand for
Diacyl Glycerol
What does IP3 stand for
Inositol-1,4,5-triphosphate
How are DAG and IP3 formed
Through PIP2 cleavage by phospholipase C
What does DAG do
Activates protein kinase C which then moves from the cytosol to the plasma membrane. PKC then phosphorylates other proteins.
What does IP3 do
Stimulates calcium release from the ER by activating calcium-release channels in the ER membrane.
Give some examples of protein kinase C inhibitors
Non-selective protein kinase inhibitors (H7, staurosporine etc)Selective inhibitors (calphostin C etc)
What modifications can happen to IP3
Phosphorylated (to IP4/5/6)Sequentially dephosphorylated - eventually yields inositol (can be used to synthesise phosphatidylinositol PI)
What signalling role does IP4 have
Can stimulate Calcium entry (activation of plasma membrane calcium channels maybe?)
What does GAP stand for
GTPase Activating Proteins
What does a GAP do, how
Promotes GTP hydrolysisInserts an amino acid which helps to stabilise the transition state.
What does RGS stand for
Regulator of G-protein signalling
What do RGS proteins do
Negative regulators of G protein signalling. Stimulate GTP hydrolysis. Are a type of GAPAccelerate the rate of GTP hydrolysis
What does GEF stand for
Guanine Nucleotide Exchange Factors
What do GEFs do
Promote GDP/GTP exchange
What can act as a GEF
An activated GPCRAGS (activator of G-protein signalling)
How many families of RGSs are there
2Modulators and integrators
How does RGS4 work
GPCR modulatorReversibly associates with membranes via N terminus amphipathic alpha-helix.Targets specific GPCRsCan bind to PIP3, which blocks the RGS. (stimulated by G[beta/gamma] subunits)(inhibited by calcium/calmodulin)
What is the molecular basis of calcium oscillations (RGS4/PIP3)
GPCR activates; intracellular Ca incRGS4 forms complex which shuts off Ca mobilisation (Ca dec)PIP3 binds to RGS4; inhibits; allows Ca mobilisation (Ca inc)Ca/Cam complex forms. Inhibits PIP3. RGS4 continues. (Ca dec).Lowered Ca; CaM deactivated. PIP3 able to rebind to RGS4. PROCESS CONTINUES
What does PDE stand for
phosphodiesterase
What are phosphodiesterases
Diverse family of enzymes.Degrade phosphodiester bond in cyclic nucleotices (cAMP, cGMP etc).
Why are phosphodiesterases important
Can degrade cyclic nucleotides - Regulators of signal transduction mediated by these secondary messenger molecules.
How many member of the PDE family are there
11 families (PDE1-11)Over 50 isoforms known
What are the key structural features of the PDE family members
Conserved catalytic site (in the C-terminus)They have regulatory sites/interactions at the N terminus
How are the members of the PDE family involved in signalling
Each can be inhibited or stimulated by PKA, PKG, cGMP (and one by calcium/calmodulin)They each have varying affinity for degrading a specific type of cyclic nucleotide (cAMP, cGMP etc).
What is the molecular basis of Antiphasic calcium oscillations
cAMP increases = Calcium decrease (and vice versa)Activation of store-operated/voltage-gated calcium channels. Simulates Ca/CaM dependent PDE1 activation. Stimulates cAMP hydrolysis for duration of calcium increase. Anti-phasic because they do the opposite of each other. (PDE1 dependent)
What is the molecular basis of Phasic calcium oscillations
cAMP increase = calcium increaseCalcium increase stimulates adenylyl cyclase to increase cAMP production. Calcium decrease causes cAMP hydrolysis by PKA-dependent PDE4 activity. Phasic because they do the same thing as each other. (PDE4 dependent)
How can IP3 signals be terminated
Dephosphorylation to inositol (minus 3 phosphates)
What is G protein uncoupling
Occurs when phosphorylated (by specific kinases and G-protein coupled receptor kinases (GRKs))GRKs interact with agonist activated GPCRs. GRK phosphorylation allows beta-arrestins to bind.Beta-arrestins uncouple GPCRs from G-proteins
What do beta-arrestins do to GPCRs
Uncouple G-proteins, preventing activationCan target for internalisation in clathrin-coated vesicles.
What happens to a GPCR when bound in a clathrin-coated vesicle
Internalised.Then either degraded (by lysosomes); or dephosphorylated and recycled back to the surface (GRK activity reversed).
How does GPCR desensitisation occur
Constant GPCR stimulation leads to phosphorylation by GRK. This binds beta-arrestin which targets the GPCR for internalisation in a clathrin-coated vesicle. From here can be degraded (downregulation) or recycled.
What is cholera toxin
Protein toxin that acts intracellularly to enzymatically disrupt GTPase activity of G[Salpha] and G[t].
What is the structure of cholera toxin
Made up of A1, A2 and B subunits
How does cholera toxin work
B subunits bind to cell surface. A2 subunit is an ‘adaptor piece’ which enables the A1 subunit to enter the cellA1 subunit binds to ARF6 (ADP-ribosylation factor 6). Disrupts GTPase activity.
What is pertussis toxin
Protein toxin - responsible for whooping cough
What is the structure of pertussis toxin
Made up of A subunits (S1)and B subunits (S2-5)S2/S4 and S3/S4 are dimers linked together by S5.
How does pertussis toxin work
Released in inactive form. Binds to cell receptor and internalised. S1 subunit is pathogenic. Activated by many factors (including ATP and reducing agents).
What is the mechanism of cholera toxin
Catalyses the transfer of ADP-ribose from NAD+ to an arginine residue on the G[Salpha] GTPase active site. This prevents GTP hydrolysis.Therefore the stimulatory G-protein is permanently activated (because no GTPase to switch off)
What is the mechanism of pertussis toxin
Catalyses the transfer of ADP-ribose onto a cysteine residue of the inhibitor G[ialpha]. This makes the protein incapable of exchanging GDP for GTP. Therefore the inhibitory pathway is blocked (and stimulatory continues)
What are the cellular effects of cholera toxin
G[Salpha] permanently activatedMassive increase of cAMP. PKA activatedActivates CFTR-mediated chloride efflux in gut epitheliaRapid loss of fluid in gut ensues.
What are the cellular effects of pertussis toxin
G[ialpha] permanently inactivated. cAMP levels increase. Causes:Increased insulin secretion (and hypoglycemia)CoughReduced neutrophil/macrophage recruitment (immune)
Why are GTP analogues used
Different analogues can cause different effects (such as perma activation or inhibition).Allows to detect a GPCR, and identify the pathway
Give some examples of GTP analogues used
Gpp(NH)p - Guanosine-5’[betagamma-imido] triphosphate; leads to perma activationGDP/AIF4- (Aluminium fluoride). Activates G proteins. Commonly used.
