GPCRs Flashcards
What are GPCRs?
G-protein coupled receptors. 7TMD which anchor receptors in PM with an EC amino terminus and an IC carboxy terminus. Associate with G proteins, which recognise the fold of the C terminus and match to appropriate G protein groove. Made up of Ga, B and y (trimeric) Ga subunit is needed for downstream signalling inside the cell, binds to a guanine nucleotide.
800 known receptors which can be subclassified according to their structure and how they bind their endogenous agonist.
Ligands include hormones, peptides, neurotransmitters and lipids.
What are the characteristics of each GPCR family?
Family 1a: Receptors for catecholamines, opiates. B-adrenoreceptor best studied. Ligand-binding site is buried within TMD, agonist must be small.
Family 1b: Receptors for small peptides, cytokines. Ligand-binding site incorporates N terminus and some residues that are poking out from TMD.
Family 1c: Receptor for larger peptides like LH, TSH, FSH. More complex structure. Receptors have longer amino terminus and Ligand-binding site is further up in N terminus.
Family 2: Receptors contain very little sequence homology to family 1. Ligand-binding site is within the extended NH2 terminus.
Family 3: Receptors such as metabotrophic receptors glutamate and GABA (GABAb exists as a dimer), Ca receptor. Ligand-binding site found within clam shell structure whereby when the ligand binds it snaps shut.
Family 4: Contains odourant receptors, hundreds of examples.
Family 5: Receptors involved in developmental such as Smo and Frizzled. Not yet common drug targets.
How does GPCR become active?
When the receptor is occupied by an agonist, G protein becomes active. Conformational change in a binding site, giving higher affinity for GTP. GDP replaced by GTP on a subunit. By dissociates and a+GTP can go and activate downstream targets.
Eventually hydrolysis occurs, GDP replaces GTP. Ga is then highly attracted to By, receptor becomes inactive.
What are the different types of Ga subunit?
Usually classified according to the a subunit.
- Ga s: stimulation of adenylyl cyclase. Linked to B-adrenoreceptors in the heart, lungs and smooth muscle
- Ga olf: regulates a variety of different effectors including src tyrosine kinases
- Ga o/i: inhibition of adenylyl cyclase
- Ga t: acts in visual transduction, increases cGMP
- Ga q: regulates activity of phospholipase Cb to generate IP3
- Gby: activate a family of potassium channels which leads to hyperpolarisation of neurons and inhibition of neurotransmission. Can also inhibit voltage-gated Ca channels in neurons and endocrine. Opoid receptors uses both function depending on location.
What methods can you use to visualise GPCR signalling?
The a subunit is loaded with radioactive GTPyS. The final phosphate group is connected to a sulfate which is non-hydrolysing and permanently bound, so it cannot be broken down to GDP. Purify and quantify a subunits by measuring radioactivity.
Cholera toxin causes ADP ribosylation step, adds a sugar group to Ga s subunit which irreversibly stimulates cAMP production which you can measure.
Pertussis toxin irreversibly inactivates Gai, causing ribosylation as well. Stops the exchange of GDP to GTP.
How can you measure GPCR by analysing downstream products?
Measure 2nd messenger production (cAMP and Ca). You can buy cheap fluorescent small molecules that can detect Ca. Initially you would only be able to detect Gq in this way, but drug companies have modified receptors so that Ca is their output.
Measure Ca using FLIPR assay whereby cells containing receptor of interest are loaded with calcium indicator dye.
Fluorescent biomarkers - fluorescent engineered proteins that bind to second messengers, measure fluorescence.
What are the two hypothesises about G protein interactions?
- The G protein is precouplled to the receptor
- The receptor and the G protein only come together when the receptor is activated ‘Collision theory’
How was FRET analysis used to determine whether the G protein’s interaction with the receptor?
1) FRET donor put on the G protein, FRET receiver put on the receptor
2) Check that the proteins are expressed and trafficking. Light absorbed by fluorescence and fluorescence emits a different wavelength
3) Both proteins found at the plasma membrane. Look at the fluorescence transfer between the two molecules.
4) In the presence of the agoinst, the amount of fluorescence emitted is decreased, but fluorescence of GPCR increases due to absorbance, increasing FRET. Shows that the protein and receptor are only getting close enough in the presence of the agonist. So not preoccupied.
How can B2-adrenoreceptors have short and long term effects on the heart?
A G protein can switch its coupling because of second messenger activity. B2 adrenoreceptors bind to Ga s to increase cAMP but can also cause long term effect in the heart.
So B-adrenoreceptor using Gs to stimulate increased cAMP, leading to activation of PKA through phosphorylation (regulated by cAMP) which can also cause coupling of receptor from Gs to Gi. The By that is released works on Src kinase which leads to MAPK activation.
Therefore:
Gs: PKA leads to short term effects
Gi: MAPK pathway leads to derogatory long-term effects on the heart
How are signalling pathways regulated?
Signalling pathway activated by a receptor is context specific, giving greater control over the physiological outcome.
Regulated by scaffold proteins, adaptor proteins, lipids (association with rafts/lipid microdomains), endocytosis, splicing, post-trans modifications.
Regulation through interacting proteins: RAMPS (proteins in PM, can alter pharmacology of G proteins), GSKs, B-arrestins, RGS (regulators of G-protein signalling).
How can the receptor itself regulate signalling?
Two ways:
- Homologous: the receptor acts back on itself to inhibit
- Heterologous: 1 receptor activated affects another receptor
Both regulate desensitisation of the receptors, restricting signalling.
How can GRKs and B-arrestin regulate a receptor [homologous]?
Agonist occupies the receptor for a long time/frequently repetitively.
This causes it to recruit a G-protein receptor kinase, a serine/threonine kinase which phosphorylates the receptor.
Phosphorylation affects C terminus so G protein cannot associate with the receptor even if the agonist is present.
Phosphorylation creates a binding site for B-arrestin, a scaffold protein which attracts machinery involved in endocytosis.
Receptor becomes internalised.
C terminus can become dephosphorylated, agonist release from receptor.
THEN
Endosome can fuse with a lysosome for degradation OR
Receptor is recycled back to the membrane
What are the different types of GRKs?
Different isoforms of GRKs are found in different locations and are regulated differently. Grk2 and 3 are most common and contains a PH domain to bind By subunits. Grk4 has a very restrictive pattern of expression. GRK5 and 6 are found associated with the PM, not well-understood and required for ERK activation.
How can you visualise the interactions of GRK and B-arrestin with the receptor?
You can track the recruitment of GRK or B-arrestin to the receptor by doing live cell fluorescence imagine using fluorescent tags on each. According to the intensity of FRET, a colour look up table is used to assign an artificial colour system. Red is high intensity and Blue is low intensity.
Interaction requires continued presence of agonist.
What is morphine tolerance?
After repeated doses of morphine, higher doses are needed to achieve the same pain relief. Continual use of opiates reduces the receptor reserve meaning higher doses give more unpleasant side-effects. Dose-response curve moves to the right.
How were mice used to investigate whether B-arrestin is required for opiate tolerance?
Knockout of B-arrestin2 in mouse. Give mouse repeated doses of opoids at high frequency or continual low dose. In WT mouse, analgesia is dramatically reduced over 5 days. In B-arrestin KO, analgesia is maintained over time but by day 9 the dose-response curve for WT has moved to the right.
Conclusion: Desensitization pathway involves B-arrestin.
How is DAMCO used to look at how morphine tolerance occurs?
DAMGO is a modified version of natural enkephalin peptide that is destruction-resistant and can be used for experiments, has high u-opoid receptor specificity. DAMGO causes considerable internalisation, but by adding morphine, internalisation of receptors is reduced. This is because morphine is like a partial agonist so when it is desensitised it doesn’t undergo proper internalisation, it simply remains on the membrane in an inactive state. Because morphine is not internalised it cannot be resensitized like DAMGO, meaning that it can cause a high tolerance to develop.
How can genetics predispose you to addiction?
Natural SNP found in opiate receptors in people predisposed to alcoholism. When WT and mutant receptor are compared, it was found that the SNP had an impact on the ability of morphine to cause receptor internalisation. In mutant there is increased internalisation via a mechanism distinct from that seen with DAMGO in WT receptor. This could mean that some individuals could be resilient to development of morphine tolerance.
How can GPCRs be classified according to fate after internalisation?
We can classify receptors into groups depending on what happens after internalisation.
- Class A: Majority. Very rapidly become recycled back to the membrane.
- Class B: Remain for a long time on endosome, often still able to signal. Important therapeutic pathway.
What do B-arrestins do?
-Provide a scaffold for endocytosis to facilitate receptor internalisation
-Can be an adaptor for a E3 ubiquitin ligase to promote ubiquitination in calcium channels and growth factors
-Provide a scaffold for adaptor proteins which can amplify signalling. Their binding domains are diverse.
Src, MAPKs, MAPKKKs, JNK, p38, PI2 kinase Akt can all bind which can regulate diverse functions. Can lead to regulation of transcription to produce long-term changes in cell function.
