Flashcards in GPCRs Deck (73)
How many GPCRs are currently known
How are different subfamilies identified
by structural features that relate to their ability to bind their endogenous ligands and bring about subsequent signal.
What receptors are members of the family1a receptors
Retinal, catecholamines and opiate receptors
Where is the ligand binding site in family 1a receptors
Buried within the transmembrane domains creating a binding pocket
what requirements of agonists are there for binding of 1a receptors
Quite small to be able to reach down into the binding pocket
Where is the binding site for 1b receptors
encoumpasses part of the amino terminus as well as some residues that poke out from the transmembrane domain
What endogenous ligands bind to 1b receptors
Peptides, cytokines, IL-8, Thrombin - all larger molecules than 1a ligands
Where is the binding site for 1c receptors
Further out into the extracellular space with a much larger amino terminus - evolved to recognise much larger peptides e.g. LH, FSH, Glycoproteins
What characteristics do family 3 GPCRs have
small molecule receptors (Calcium, glutamate, GABA) Clam shell structure extracellularly (amino terminus) which contains the ligand binding site, when the ligand binds it snaps shuts and holds it allowing signalling to occur.
What receptors are part of the family 4 GPCRs
The odorant receptors
What receptors are part of the family 5 GPCRs
Smoothened and frizzled etc, developmental
What occurs to GPCR subunits after ligand binding
After binding the receptor undergoes a conformational change and attracts the G protein. The alpha subunit gets an increased affinity for GTP so bound GDP dissociates and GTP associates. The GTP-bound alpha subunit has a reduced affinity for the beta/gamma subunits and dissociates from them to give two functional subunits. Each functional subunit can independently control different effectors
How is GPCR signalling stopped
The alpha subunit has a built in timer mechanism to control its duration of signalling. Its enzymatic domain hydrolyses GTP, this hydrolysis increases the affinity of the alpha subunit for the beta/gamma subunits and reassociation occurs.
What is the effector of the Galpha s subunit
Increased adenylate cyclase
What are the effectors of Galpha olf
Activation of calcium channels and c-Src tyrosine kinases.
What is the effector of Galpha i/o
Inhibition of adenylate cyclase. Also activate Src tyrosine kinases.
What is the effector of Galpha q
Activation of Phospholipase C - subsequently IP3 and therefore a calcium signal
What are common tools for studing GPCR signalling
1. GTPgammaS binding - alpha subunit gets loaded with radioactive GTP and is non hydrolysable. This can be purified and quantified by measuring radioactivity. Also downstream targets are permanently on.
2. Cholera toxin - Irreversibly activates Galpha S. Causes an ADP ribosylation - adds sugar to G alpha S and can no longer hydrolyse GTP.
3. Pertussis toxin - irreversibly inactivates G alpha i/o - adding a sugar to alpha subunit which stops the exchange of GTP for GDP so can't be activated.
4. Measure second messengers (cAMP, Calcium) by biosensors (fluorescent engineered proteins) - measure fluorescence.
How has calcium signalling of Gq coupled receptors led to drug discovery
Genetically modify the carboxy terminus of the receptor of interest so it has a Gq domain - Drug screening uses FLIPR - plates cells expressing the Gq receptors and a fluorescent calcium dye. Computer analyses fluorescence in wells, Agonists or antagonists can be identified this way.
How was FRET analysis utilised to investigate Receptor and G protein interactions(where they pre coupled or not)
FRET donor put on the G protein. FRET acceptor put on the receptor. CFP on the BY subunit, YFP on the carboxy terminus. YFP emission went up whilst CFP emission went down (due to absorbance by the acceptor) Only when agonist was added showing that the receptor and g protein come together after ligand binding.
Is it a one to one relationship between G proteins and the GPCR?
No, multiple G proteins can couple to one receptor
Which G protein does the B2 adrenoceptor activate
Gs pathway via cAMP - but also the MAPK pathway via the Gi pathway over time.
How was the dual activation of Gs and Gi discovered in B2 adrenoreceptors
B2 was overexpressed in an APK and MAPK cell line
treated cells with PTX, which enters cells and adds a sugar to the alpha subunit, rendering them inactive.
This inhibits signalling from Badrenoreceptors which prevented signalling from the Gi pathway so it must be involved.
A second experiment used B-ARKct which sequesters the B/y subunit, inhibiting the coupling to MAPK
A Src inhibitor was also used which had the same effect
H-89, a PKA inhibitor was then used, this too abolished MAPK coupling
What is the importance of a PKA inhibitor preventing MAPK coupling
PKA is a effector of the Gs pathway, which when inactivated is capable of preventing the coupling of MAPK - of the Gi pathway - shows that GPCRs can regulate their own G-protein signalling.
How does PKA regulate its own G protein signalling
It phosphorylates the B2 adrenoreceptor which switches its G protein coupling. It then couples to Gi, and the B/Y subunit it releases works on a Src kinase which =leads to the activation of MAPK.
What is the result of continuous presence of agonist in B2 adrenoreceptors
Increased MAPK signalling - increased ERK which leads to transcriptional changes and heart disease.
What is homologous desensitisation
Where the receptor that's being activated by its agonist acts back on itself to inhibit its own function.
The agonist has to occupy the receptor for a long time/repetitively, when this happens it recruits a GRK (Gprotein receptor kinase) which phosphorylates the receptor. (serine and threonine kinases) This causes the coupling to change, the G protein can no longer bind to it due to the phosphorylation. Phosphorylated state creates a docking site for beta arrestin, which arrests signalling further, attracting endocytic machinery
What is heterologous desensitisation
One receptor being activated impacts on the signalling of another type of receptor.
What can occur to the endocytosed GPCR
It can be dephosphorylated so the agonist can come off the receptor in the endosomal compartment. Then the receptor can be targeted for degradation or recycled back to the plasma membrane and become available again to signal.