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Flashcards in GPCRs Deck (73)
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How many GPCRs are currently known

over 800


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.


How many GRK isoforms are there and which are expressed ubiquitously?

7 and 2/3 are expressed ubiquitously.


What is the structure of GRK2/3

These GRKs have a pleckstrin homology domain built into their structure which binds B/Y subunits. So when a receptor becomes active and has caused the G protein to be active/ B/Y subunits to come apart. If they remain present long enough they will bind GRK2/3


Which GRKs are restricted to the visual pathway and phosphorylate rhodopsin

GRK 1 and 7


How many B arrestin isoforms are there and which is the most common

2 different beta arrestin isoforms and the most common is B arrestin 2.


How is B arrestin recruitment and activation by GPCRs monitored

FRET probes - Track the recruitment of the beta arrestin/GRK to the receptor by live cell fluorescent imaging. To quantify this interaction you can do FRET pair analysis in the presence of an agonist.


What is tolerance

The process whereby an increased dose of drug is required for equivalent effect


Which arrestin is responsible for opioid receptor tolerance

arrestin 2


What is the effect of beta arrestin 2 knockout mice

Analgesia is reduced


What is DAMGO

A modified version of the endogenous enkephalins, which is incapable of being degraded.


What is the result of treating opioid cells with a large amount of DAMGO

Internalisation of the receptors, at a much higher level than when morphine was used to activate the receptor.


What type of agonist is morphine to its receptor

It is a partial agonist, which means the structure of the receptor that morphine stabilizes is different to that of DAMGO. It means that it remains stuck on the cell surface in an inactive form, acculming a group of desensitised receptors leading to desensitisation.


What is the result of morphine binding of its receptor as a partial agonist on GRK recruitment

It recruits GRK6 which has an impact on which carboxy terminus residues are phosphorylated which will recruit PKC - This is not the case for other enkephalins


What is the result of the L851 SNP receptor mutant with morphine induced internalisation

The mutant receptor is more likely to be internalised and recycled more efficiently so will not be desensitised to morphine - individuals could be resilient to development of morphine tolerance.


Sites for phosphorylation in the carboxy terminus of the receptor by GRKs are dependant on what?

The agonist and will have an impact on what subsequent proteins are able to associate to the receptor to control its function/signalling.


What are class A GPCRs in the context of desensitisation

GPCRs that undergo rapid loss of the B arrestins and become dephosphorylated and get sent for degradation or recycling.


What are class B receptors in the context of desensitisation

Don't have a rapid pathway and exist for much longer on the endosomes. Angiotensin receptor - B arrestins remain for a long time. Theory that when receptors remain in this endosome they may continue to signal by recruiting additional proteins.


What are biased ligands

Receptors may signal completely independently of G proteins


What role does B arrestin have in G protein independent signalling

Not only do they provide a scaffold for endocytotic machinery but also as a scaffold and adaptor protein to extent the signalling capacity of the receptors


What binding domains are found on B arrestin for independent signalling

JNK3, MAPKs, SH3 and SH1 domains, IP6


How do agonists induce two temporally distinct phases of ERK activation

1st phase, transient, Gs and PKA dependent
2nd phase, delayed onset but prolonged


What was the result of using a TYY mutation on B2 adrenoreceptors

Abolishes G protein activation but leaves arrestin-dependent signalling intact


What effect did the TYY mutation have on ERK signalling

ERK activation is enhanced in the mutant receptor - B arrestins can signal completely independently of the G protein


Can the same receptor show different bias to G protein bias and arrestin bias signalling

Yes, depends on the ligand that binds to it


Why is bias signalling important in Opioid receptors in analgesia

The signalling pathway for analgesia is biased to G protein signalling and the regulation of ion channels etc. The beta arrestin pathway is responsible for the opioid effects on respiration, as well as nausea and constipation. So an opiate which is biased towards the g protein signalling pathway only would avoid problems with max efficacy.


What was the traditional method of screening for GPCR drugs

Analysis of second messengers. Therefore only drugs affecting G protein mediated signalling to second messenger producing effectors will be discovered


How did screening methods change after the discovery of beta arrestin dependent signalling

New screens for B arrestin signalling were introduced (Fluorescent gene reporter) which may identify a novel pathway selective drugs with valuable therapeutic properties


Howis the Gi/o pathway analgesic

It inhibits adenylate cyclase which prevents cAMP production - cAMP would normally lead to PKA activation which phosphorylates nociceptors.
It inhibits the activation of voltage dependent calcium channels reducing neurotransmitter release.
Also directly hyperpolarizes neurons by activating the G-protein gated potassium inwardly rectifying channels (GIRKs) resulting in reduced neuron excitability.


What is PZM21

A potent Gi activator with exceptional selectivity for mew opioid receptor signalling with minimal B arrestin 2 recruitment, so is an effective component of analgesia without the side effects of morphine.


Why was rhodopsin easier to crystallize

The ligand is permanently associated with the receptor, so it doesn't have the same dynamic properties that other GPCRs have


Why did it take 15 years to crystalize and solve the structure of a GPCR

Expressed at low levels so hard to purify.
Integral membrane proteins don't fold properly outside the membrane
Due to their dynamic structure they don't crystallize easily - proteins that constantly change their shapes do not form crystals.


What modifications were made to the B adrenoceptor in order to stabilize the GPCR and make it easier to crystallise.

Truncated the C terminus to remove the phosphorylation and G protein binding sites for the receptor. Also they replace the third intracellular loop with a protein whos structure was already known and whos size from the structure was predicted to be about right as a replacement for the third intracellular loop, maintaining the distance between the transmembrane domains.


How did they show the hybrid receptor still maintained a ligand binding site representative of the original receptor

Ligand binding experiments - using a series of drugs which were well known to bind the B adrenoceptor (salbutamol)


how did they make sure that the hybrid receptor still allowed the transmembrane domains to move relative to each other as they would be expected after ligand binding.

Fluorescent ligands were used. Fluorescence of the ligand will change when the environment it finds itself in changes. i.e. The transmembrane domains are moving. Domains 5 and 6 move relative to each other to change the fluorescence.


What do inverse agonists do to the receptor upon binding

Stabilise an inactive configuration of the receptor. Prevents the dynamic structure of the receptor so it doesn't randomly become activated.


How do B1 and B2 receptors differ in their constitutive activity and what is this due to in terms of their structure

Whilst they have a very similar overall architecture, extracellular loops and ligand binding regions differ. B2 has a loop connecting transmembrane domains with greater flexibility to its B1 counterpart, so more likely to adopt an active conformation.


How does the GABA receptor activate a G protein

GABA B1 and GABA B2 Dimerise for the formation of a functional GABA B receptor - Carboxy terminus contains coil-coil interactions


What receptors exist as dimers due to disulphide bonding on the N terminal

mGluRs and Calcium receptors


Which receptors dimerise and have interactions between TM regions

D2 receptors as well as Opioid receptors.


Can mew opioid receptors dimerise with non mew opioid receptors?

Yes, dimers with delta and kappa receptors with distinct interactions. Useful as ligands can be designed with increased specificity to areas where different dimers are expressed.


What are DREADDs

Designer receptors exclusively activated by designer drug - artificial GPCRs with no natural ligand to activate them.


What is the first DREADD produced

Muscarinic receptor 3 coupled to Gq signalling - PKC - TM3 and TM5 mutations introduced into the receptor created a receptor that would be activated by CNO uniquely (no longer binds Ach), without a change in G protein coupling


How can DREADDs be utilised?

Some with altered pharmacology
Intracellular domains can be changed as to change the bias of its signalling to either G proteins or B arrestins
Intracellular domains can also control location of where the receptor will be located within a cell.


How have DREADDs been used in transgenic mice

Express a DREADD in the hypothalamus to alter its feeding habits. Manipulation of behaviour by controlling expression of the receptor.


How are DREADDs used to treat diabetes

Transgenic mice expressing a DREADD at the beta cells of the pancreas. Gq DREADD used - Insulin secretion is dependent on cAMP this activates a potassium channel through the regulation of ATP leading to depolarisation and calcium signalling. Or by raising calcium to directly trigger insulin containing vesicle exocytosis. After feeding the mice CNO the mice increased insulin release and improved blood glucose levels.