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