Receptors Flashcards
Natural contributors to different receptor effect
- location of receptor expression
- different combination of receptors
- different signal transductions
- pathway branching and cross talk = co-ordination of signals from incoming ligands
pathway branching
primary signal transduction molecule can activate two different downstream molecules
pathway crosstalk
downstream signalling molecules from different receptors activate a common downstream target
GPCR signal transduction
In the resting state G protein exists as a trimeric unit composed of Ga, Gb, and Gy with GDP bound to Ga.
Ligand binding causes a conformational change in the receptor (outward movement of TM6 & TM2)
G protein binds to activated receptor in space created through movement
Conformation change in G protein causing dissociation of GDP
GTP binding causes dissociation of Gb/y heterodimer and Ga
Ga acts on adenylate cyclase, Gby acts on other targets (eg: ion channels)
GTP hydrolysis, return to resting state
downstream amplification (GPCRs)
single ligand binding to receptor can activate multiple adenylate cyclase enzymes, causing massive increase of cAMP.
cAMP can activate PKA causing a phosphorylation cascade which further amplifies.
Requirements for drug screening
- Cell line with appropriate receptor
(can be grown at high number) - assay with high sensitivity and accuracy (minimises false positives/negatives)
- appropriate negative/positive controls
- sustained response
(transient responses difficult to screen) - low cost and low volume
- amendable to automation
- high tolerance to solvents
efficacy
the ability of a ligand to bind to a receptor and exert a response, Emax = maximum possible response at full receptor occupancy
(if drug produces less than 100% response, is a partial agonist)
potency
a measure of how much a drug is required in order to produce a particular effect. This is defined in a concentration response curve by the EC50 – the concentration of drug required to produce 50% of maximal response.
(If a drug has high potency only a small amount is required to induce a response.)
affinity
a measure of a drugs ability to bind to its specific receptor
- defined by Kd: the concentration required to occupy 50% of receptors
- Bmax: maximum receptor number
types of in vitro cell models
cell lines/primary cultures
cell extracts
purified proteins
advantages of in vitro cell models
- reduce use of animals
- study the human target (instead of animal)
- can collect multiple data points due to ease of cell proliferation
- can be automates
- should produce readily reproducible results
disadvantages of in vitro cell models
- removed from true biological complexity
- cell phenotypes can drift or be contaminated
how to chose a cell for transfection
- robust growth, stability, readily transfectable
- low background activity (should not respond to drugs in absence of specific transfected target)
commonly used cell lines for transfection
HEK: human embryonic kidney
CHO: Chinese hamster ovary
COS-7: green monkey?
How to transfect a cell
- gene for receptor of interest is inserted into an expression plasmid which acts to transfect cell
- selection pressure (antibiotic) applied until all un-transfected cells die (transfected cells express antibiotic immunity)
- further isolation to obtain cell line with highest expression of receptor of interest
- maintain cells in antibiotic to maintain expression
expression plasmid
manmade circle of DNA containing
- MCS: multiple cloning site allows insertion of gene of interest
- Promoter: enables gene expression to be turned on (constitutive/inducible) once inside cell
- selection: antibiotic resistant genes
transfection methods
physical treatments: electroporation, microinjection, nanoparticles
chemical treatments: lipid-mediated DNA transfection via liposome vectors, biological particles (viruses)
stable transfection
long term expression of a transgene by integrating foreign DNA into host genome
WE WANT STABLE!! Maintain by continuously applying antibiotics
transient transfection
gene expressed off plasmid, expression of transfected gene lost with cell division
cell line isolation methods
clonal isolation: use limited dilution to isolate a single cell with desired expression, and grow.
expression sorting: use flow cytometry to select out high expressing cells
both approaches require antibody/tag that does not alter function
importance of further cell isolation following antibiotic application
cells can exist in heterogeneous expression of desired protein, and the foreign protein may slow down growth leading to low expressing cells taking over!
considerations with over-expression model
transfected cells express the receptor at a greater level than normal conditions
= increased chance of finding an initial hit, but may amplify low affinity interactions
detection of phosphorylation signalling event
use phosphorylation specific antibodies
detection of changed protein location signalling event
microscopy, BRET approaches
detection of altered gene transcription signalling event
western blotting/microscopy to quantify levels of protein
detection of environmental modulation signalling event
electrophysiology
cAMP
- activates protein kinase A
- hydrolysed by phosphodiesterase
phosphodiesterase inhibitors
methylxanthines: caffeine, theophylline, IMBX
often included in cAMP assays to enhance cAMP detection
forskolin
agonist at adenylate cyclase. used to increase the basal cAMP levels to allow detection of decreased cAMP levels (Gai)
considerations in signalling assays
- sensitivity
- dynamic range
- requirement for transfection
- kinetic changes or accumulation
- time
- cost (if expensive but single use can be justified over cheaper, less effective assays)
accumulation assays
good for drug screening but not indicate actual time course of signalling
dynamic range
the range of values that can accurately measured by the assay
- want to use concentrations that fall within the linear part of the standard curve
- always do a standard curve so you can understand where the data falls on it
scintillation cAMP assay
- incorporate 3H-adenine (radioactive) into ATP, ADP, cAMP by overnight incubation
- stimulate/inhibit cAMP production with drug
- lyse cells and use sequential chromatography to separate 3H-cAMP from other tritium labelled molecules
- detection via scintillation counting
pros/cons of scintillation counting assay
Pros:
- direct measure of cAMP
- very sensitive
- unlimited dynamic range
Cons:
- time consuming & low throughput
- typically used as an accumulation assay in presence of PDE inhibitor (pick a time point to measure)
- limited kinetic range, as each time point is a different cell preparation
competition cAMP assay
cAMP produced by cell competes for antibody binding against a (radioactive/fluorescent/luminescent) labelled cAMP. The more cAMP bound, the less signal detected; labelled cAMP interacts with donor on antibody to produce fluorescence.
- stimulate cells with drug
- incubate for set time point
- lyse cells
- incubate with donor (antibody) and acceptor (labelled-cAMP)
- detect signal
pros/cons of competitive cAMP assay
Pros:
- high throughput & automation/miniaturisation possible
- doesn’t require cell transfection, but cells must still be lysed
Cons:
- limited dynamic range (lysate must be diluted to ensure cAMP levels fall in range of assay)
- accumulation assay; set time points
- reagents $$$
- susceptible to interference from media (can be overcome)
biosensor cAMP assays
resonant energy transfer between two proteins in close proximity
BRET cAMP assay
cAMP bound to EPAC = RLuc (bioluminescent donor) activated by luciferase. No activation of YFP (effector)
cAMP not bound = Rluc donates light to YFP, which emits bioluminescent signal
- detected on plate reader > cell population based > high throughput
- enzyme based, requires substrate (coelenterizine) in excess, as depletion could interfere with results
FRET cAMP assay
donor is fluorescent and activated by light, transfers energy to acceptor when in close proximity.
Eg: antibody = donor, d2-cAMP = acceptor.
increase signal when less endogenous cAMP.
Eg: CFP = donor, YFP = acceptor
- microscope detection allows analysis of single cells
- activated by laser, thus can cause photobleaching/laser damage over time
- NO HIGH THROUGHPUT
EPAC
endogenous cAMP-binding protein.
Used in BRET; when cAMP not bound RLuc & EYFP held in close proximity = signal. When cAMP bound they are distanced = no signal
pros/cons of biosensor cAMP assays
Pros:
- kinetic assay; can measure response across time
- generally good sensitivity/dynamic range
- high throughput/miniaturisation (BRET)
- cost effective
Cons
- population based response (BRET)
- requires transfection of living cells, may over exaggerate response + limits type of cell can be used
issues with cAMP as a measure in assays
cAMP is a highly activated pathway
- can detect weak signals
BUT
- limits ability to detect partial agonism
Good for initial drug screening, but need additional screening to characterise ligand effect
Other uses for BRET/FRET
- dimerisation
- movement of proteins (can label protein and receptor, thus can observe when in close proximity)
Migraine
Sensitisation to CGRP, which is abundant in pain-modulating nerves including trigeminal
CB1
a cannabinoid receptor with widespread distribution throughout the brain, that mediates the psychoactive effects of cannabis
orthosteric binding assays
radio ligand binding assays
+ saturation binding assay (determine affinity of radio-labelled compounds)
+ competitive binding assay (determine affinity and selectivity of endogenous ligands)
considerations when choosing radio-ligand
- high affinity (low Kd) for receptor of interest
- specific for receptor of interest
- labelled with 125I/3H
- high specific activity (amount of radioactivity/molecule) to increase signal:noise ratio
125I vs 3H
Addition of (125)I can alter the structure and therefore function of molecules, so is often used less despite it’s increased sensitivity.
(3)H/tritium on the other hand is less sensitive, but is easily substituted in place of normal hydrogens
receptor source for radioassay
- transfected cell line: enriched cell membrane > whole cell prep (HEK/CHO)
- homogenised tissue sample from rodent
competitive radio-ligand binding
Essentially a measure of how much a known agonist/antagonist is displaced by a novel compound. More displacement = higher affinity of novel c.
- radioligand applied at conc below/close to Kd (to stay in sensitive portion of binding curve; effective visualisation)
- add increasing [unknown] until equilibrium
- incubate with receptor preparation
- @ equilibrium separate bound ligand from free ligand (filtration & wash/centrifuge & wash/wash) FAST AND COLD to prevent dissociation
- measure via scintillation counting
- data analysis; remove non-specific binding window
scintillation counting
sample is trapped in a filter, and the liquid scintillant/melted solid scintillant is applied, releasing fluor molecules in response to radioactive emission to allow quantification
non-specific binding window
The portion under the bottom plateau of the curve, due to non-specific binding, which occurs due to:
- Distribution of ligand into lipid components of the preparation.
- Free ligand which is not separated from bound ligand during the separation phase of the experiment
- Increases linearly with radio-ligand concentration, and are non-saturable
The specific binding window can be defined with a high concentration of a confirmed orthosteric ligand
- Remove from analysis graph
IC50
a proxy for affinity; measures relative displacement of radio-ligand = relative affinity
convert to KI
KI
concentration of competing drug that binds to 50% of receptors at equilibrium in absence of radio ligand
KI equation
KI = IC50/(1+ [L]/KL
KL = Kd(radio-ligand - ligand)
[L] = concentration of radio ligand
Ki equation, when radio-assay carried out at Kd of radio-ligand
Ki = IC50/2
effect of allosteric modulators on affinity of radio-ligand
negative affinity modulators result in less radio-ligand binding at normal Kd, thus can resemble competitive binding
- specific binding curve will never reach 100% displacement = 0% radio-ligand bound
Cholera
toxin from V.cholera that does ADP-ribosylation to stabilise the active conformation of Gas
polymorphisms
common variants in amino acid sequence found in >1% of individuals of a given population
germline/hereditary mutations
occur in reproductive cells, thus are passed to offspring where they are present in all cells
somatic/acquired mutations
mutations that are not present at birth, acquired over lifetime
silent mutations
result in an sequence change that results in coding of same AA as original
nonsense mutations
mutations that result in a stop codon, therefore early termination of AA translation
conservative missense mutations
mutation that results in translation of an AA similar to orginal
non-conservative missense mutations
mutation results in AA that is very different than the original AA
Questions to ask when considering effect of receptor mutation
- does the mutation affect cell surface expression?
- does the mutation affect coupling to intracellular signalling/regulatory proteins
- does the mutation affect endogenous ligand interaction?
- how does the mutation affect drug activity at the receptor?
mutagenesis
a molecular pharmacology technique whereby cell lines expressing wild type/mutated receptor sequences are used to
quantify receptor expression
quantify ligand binding
= determine effect of mutation
loss of function mutations
- genomic alterations
- misfolding of proteins
- reduced protein production
- abnormal post-translational modification
- expression of inactive mutant receptors
- disrupted ligand/tethered agonist binding
- blocked/biased signalling
leydig cell hypoplasia
caused by A593P and S616Y mutations
= retention within cell, and reduced signalling capacities
gain of function mutations
- increased transcription
- increased transport to cell surface
- increased membrane expression
- expression of constitutively active mutant receptors
- increase in agonist potency
- promiscuous agonist recognition
- decreased arrestin signalling
- increased recycling, decreased degradation
glycoprotein hormone receptor GOF mutant
Receptor can now be weakly activated by hCG, which can cause spontaneous ovarian hyper-stimulation due to high hCG levels in pregnancy
treatment for LOF mutations
- activate adenylate cyclase (forskolin)
- activation of co-expressed receptors with same signalling pathway
- alternate agonist/ allosteric agonist
- PDE inhibitors to increase PKA activation
- stop codon suppression
- misfolding prevention through chaperones
- gene replacement
treatment for GOF mutations
- inactivating antibodies
- receptor specific inverse agonists
- RNA interference
- CRISPR-CAS9
pharmacoperones
pharmacological chaperones that are specific receptor ligands that help receptor folding and assist in cell surface expression
EGF receptor
a RTK for epidermal growth factor. GoF mutations result in excessive cell growth… cancer
GNAS mutation
Mutated in ~5% of cancerous cells, causes expression of a constitutively active Gas protein
life cycle of a receptor
- translation
- folding and PTMs in ER
- trafficking to cell surface via golgi
- signalling
- desensitisation; internalisation
- vesicle forms early endosome
- splits into either lysosome (degradation) or endosome (recycling)
splice variants
variations in splicing resulting in different expression of exons that alter the protein composition/structure causing functional changes
accessory proteins
interact with receptors to create a novel complex with altered functional capacity
RAMPs
receptor activity modifying proteins
act as chaperones for GPCRs, influence transport to cell membrane from golgi and internalisation/recycling
types of post-translational modification
- disulphide bond formation
- glycosylation
- palmitoylation/lipidation
- phosphorylation
- ubiquitination
phosphorylation
the addition of a phosphate to tyrosine (Y) serine (S) or threonine (T) to increase/decrease protein activity
variation from phosphorylation
An individual receptor can be phosphorylated differently according to:
- The ligand that activates the receptor
- Where it is expressed
- Which proteins are available to phosphorylate it
The specific phosphorylation pattern (barcode) is cell/tissue specific and affects subsequent protein interactions with the receptor
GPCR desensitisation
PKA/PKC phosphorylate active & inactive GPCRs, reducing capacity for interaction with G proteins thus reducing signalling
GRKs
GPCR kinases, exist in 7 isoforms which are present in varying concentration across tissue types
phosphorylate GPCRs to reduce interaction w/ G proteins (desensitisation) and increases affinity of receptor for arrestin (promotes internalisation)
arrestins
exists in 4 isoforms: 1&4 (vision) and 2&3 (b-arrestin1 & b-arrestin2) which mediate receptor internalisation by acting as a scaffold for other protein interactions
b-arrestin
- desensitisation (block G protein binding)
- signalling via kinases (MAPK, PI3K, AKT), transcriptional control, and transactivation
- trafficking via (clathrin-mediated) internalisation or translocation via interactions with ERK/APs
PDZ/non-PDZ effect on GPCR signalling
binds to C terminus of GPCRs to increase/decrease signalling & influence rate of internalisation.
PDZ is a AA sequence associated with anchoring proteins to the cytoskeleton of membrane.
endosomal signalling
endosomes can be degraded via lysosome, recycled via golgi, or enact cellular responses on gene expression, cytoskeleton dynamic, mitogenic signalling
sources of GPCR signalling bias
- ligand bias
- biased signalling
- location bias
subdomains
- protein conformation
- receptor dimerisation
- signalling kinetics
- protein isoforms
- other GPCR effectors (eg: GRKs, AKAPs, JAK kinase)
GPCR structure
- 7 transmembrane helical domains
- 3 extracellular loops
- 3 intracellular loops
- extracellular N terminus
- intracellular C terminus
GPCR ‘motifs’
highly conserved sequences of amino acids that play roles in G protein binding pocket and shape folding
CWcP, D(E)RY, NPxxY
class A ligands
small molecules (amines, light or odorants, peptides, purines, and lipids) that bind with equal depth and orientation in a pocket of the upper transmembrane bundle of class A GPCRs
- similar position of binding AAs
class B ligands
peptides of 20-50 AAs that bind to the both the large extracellular domain and the transmembrane bundle of class B GPCRs
RAMPs x ClassB GPCRs
RAMPS can completely alter class B binding by providing extra ligand contact points
- CGRP, and adrenomedullin receptors form from different RAMPS acting on CLR
GPCR Class A drugs
often derived from structure of endogenous ligand, with variations that allow changed receptor response
eg: b1/b2 adrenoreceptor response
GPCR Class B Drugs
Challenging- peptides are difficult to successfully deliver as drugs as large, polar molecules are poorly absorbed and peptides generally have a short half-life
** RAMPS can change receptor function
Success in using natural peptide (+ slight modifications to increase stability)
eg: GLP-1
some small molecule antagonists
eg: CGRP receptor antagonist
Amylin
a class B GPCR ligand that suppresses food intake
Class C GPCRs
require dimerisation to form the orthosteric binding site. Ligands (amines, glutamate, and calcium ions) binds venus fly trap (extracellular) portion
- contains a cysteine rich domain (CRD)
influence on receptor conformation
Every single molecular interaction
- top down = extracellular (ligands, ion concentration)
- bottom up = intracellular factors
G proteins
guanine nucleotide binding proteins composed of a heterotrimeric structure
- exist as Gas, Gai, Gaq, Ga12
GPCR conformation change
TM5 extends by 2 helical turns
TM6 moves outwards by 14A
= G protein pocket
GPCR - Gas protein interaction
a5 helix of Gas docks in cavity formed by TM5 & TM6 and interacts with DRY and NPxxY causing disruption of H1-H5 interaction, promoting GDP dissociation
GPCR - arrestin interaction
arrestin interacts with TM6 via a ‘finger loop’ (which is very similar to the interaction between a5 helix of G proteins and TM6)
arrestin anchors to membrane at C-edge loop
how are conformational changes identified?
structural biology (X-ray crystal structure/cyro electron microscopy)
molecular dynamic simulations
biochemical and biophysical methods
- site directed mutagenesis
- conformational biosensors
- hydrogen-dueterium exchange mass spectroscopy
- cross linking
multiple conformation model
Receptors exist in different conformational landscapes; binding of other proteins (G proteins & agonist) decreases the energy required to shift into an activated state
two state model
states that there are two potential receptor conformations: active/inactive, and the overall receptor response is governed by the ratio of receptor conformations
multistate model
suggests that there are multiple active conformations of the receptor which may favour in downstream signalling
- states may be independent states with specific downstream actions OR intermediate states with different downstream action
rhodopsin
GPCR containing covalently bound 11-cis-retinal, which when exposed to light is isomerised causing receptor activation
- has no basal activity
b2 adrenergic receptor
when inactive contains proline kinks within TM5/6/7 and a distance of 28A between TM6 & 2.
Ligand binding causes disruption of proline kinks and increased distance between TM6/2 to 42A.
Unique conformation model of partial agonists
inactive states,
active states;
one of which has different downstream activity and preferentially favours partial agonist
other has full downstream activity and is favoured by full agonist
A(2A) receptor
different equilibrium model of partial agonism
partial agonists stabilise active and non active state, preventing equilibrium from reaching 100%
(b2-adrenergic receptor)
models of biased ligand action
- induce different conformations within receptor, resulting in different downstream recruitment
- induce a conformation of the receptor that changes conformation of scaffolding proteins such as arrestin, which in turn activate other downstream pathways (eg:MAPK)
(arrestins can bind loosely or tightly) - induce distinct conformational rearrangements within G proteins that results in different rate of GTP association/hydrolysis
= faster GTP action = more G protein and downstream signalling/unit time
allosteric modulators - conformational change
Majority of molecules bind at the orthosteric site, which is heavily conserved thus limits potential variation in conformational changes
Allosteric ligands may bind anywhere on a receptor, resulting in a plethora of potential conformational changes that can impact receptor activity
Cmpd-15 (NAM)
binds to the intracellular region of the b2-adrenergic receptor, and results in decreased G protein & arrestin interactions
Likely due to steric blocking of the intracellular binding site
LY211 (muscarinic M2 allosteric modulator)
binds to the extracellular region, and increases the affinity of ligands, by locking the ligand in to prevent dissociation
