Intracellular Signalling Flashcards
Define intercellular and intracellular signalling
Intercellular signalling signalling from one cell to another (extracellular signals) Intracellular signalling –signalling sent within the cell to stimulate a change in an activity of another or the same part of the cell
Why do we need transmembrane receptors?
Proteins, peptides, charged molecules can not cross the lipid bilayer.
the signal needs to reach the inside of the cells
Transmembrane proteins span the membrane and transmit the signal into the cell
how do ligands work to activate a receptor?
- Ligands may interact directly with the receptors or by binding to co-receptors or accessory molecules on the cell surface.
- Receptor activation causes a conformational change in the tertiary or quaternary structure that allows initiation of signalling
Different Signal transduction molecules
Hydrophobic proteins - membrane-associated
Hydrophilic proteins - in the cytosol
Second messengers - e.g. cAMP
Ions - e.g. Ca2+
Signal amplification explain:
The ligand binds to the receptor leading to the secondary messenger release
secondary messengers are a lot higher in concentration and cause an amplified reaction in the cell
divergence in pathway allows multiple effects from one stimulus
How are signalling molecules controlled?
By post translational modification, e.g phosphorylation
By regulating whether a G protein has bound GDP or GTP
By provision of activators such as Ca2+ and cAMP
Explain protein phosphorylation
Kinases phosphorylate proteins and phosphatases remove proteins.
This can either switch on or off the signal
Which amino acids are phosphorylated?
serine, threonine and tyrosine
Two types of kinases and example
Serine/Threonine kinases:
- Ca2+/calmodulin-dependent protein kinases or CaM kinases
- Protein Kinase A (PKA)
- Protein Kinase C (PKC)
- Mitogen-activated protein kinases (MAPK)
Tyrosine kinases
- Non receptor tyrosine kinases e.g. Src family kinases - Knowledge of these kinases has been fundamental to our understanding of cancer
- Receptor tyrosine kinases (RTKs) – e.g. Epidermal growth factor receptor (EGFR)
Explain GTP binding proteins
Many GTP binding proteins are individual proteins (small GTPases)
Also used by heterotrimeric G proteins
Hydrolyse GTP-GDP by their intrinsic GTPase activity
Act as a molecular switch
Signalling must be regulated
Cancer - Changes in cell proliferation in response to growth factors or in response to signalling errors e.g. Ras mutations (aa12 Glycine-Valine)
What do provisional activators of proteins do and 2 examples?
Calcium and cAMP
Binding changes the conformation of target proteins which changes their activity
What are the three categories of ion channels?
Ion channels
G proteins
Enzymes
Ion channels features
ØTransport Na+, K+, Ca2+, Cl- Ions along an electrochemical gradient
ØSpecificity of the channel is defined by the amino acids lining the channel
ØChannels are formed of protein subunits
ØFast regulated opening/closing mechanism (mSec)
ØActivated by either a change in membrane voltage or by a ligand
Voltage gated ion channel structural features?
a-subunit contains the pore which has 4 homologous domains, each with 6 transmembrane regions
Β subunits traffic the channel and regulate its kinetic properties
How do voltage gated Na+ channel work?
Opens in response to a change in voltage
Region 4 has amino acids with positive R-groups which sense the voltage across the membrane causing movement of region 4 opening the channel
What are ligand gated ion channels and how do they work?
Transmembrane proteins consisting of a receptor part and a channel which traverses the membrane
Open in response to binding of a ligand
Receptors are often classified based on which agonists they bind
Nicotinic acetylcholine receptor
- Receptor has 5 subunits (2α, β, γ and δ)
- Region 2 of each subunit forms the channel
- 2 acetylcholine molecules bind the α subunits causing movement of the M2 helices opening the channel
Name a few events regulated by Ca2+
Secretion, transcription factor activities, Skeletal muscle contraction
Many effects of Ca2+ are mediated through:
CAM kinases calmodulin-dependent protein kinases
Calcineurin Ca2+-dependent protein phosphatase
What is the conc of Ca2+ in the cytoplasm and how is it maintained?
Low concentration of 100nM and maintained by ATP dependent pumps
How many TM domains does a GPCR have?
What are the subunits of a G-protein?
typically 7 TM domains - NOT a pore
G-protein consists of three subunits: a, b and g
GPCR activation explained
- When a ligand binds to receptor the affinity for G-protein increases. The receptor and the G-protein may sometimes already be in a complex at the membrane.
- Receptor activation changes the conformation of the internal portion of the receptor releasing GDP
- Action of the receptor causes GDP attached to a subunit to be replaced by GTP
- G-protein splits up in bg complex an α-subunit
- Both complexes then initiate further cell signalling
- GTP is then hydrolyzed to GDP and bg recombines with a ready to associate with another GPCR
Explain the role of cAMP and Adenylate cyclase
G-protein activates adenylate cyclase which produces cAMP from ATP in the cytosol
cAMP is a second messenger and works on PKA to activate it.
PKA phosphorylates many different proteins leading to increased transcription
Phospholipase C activation and pathway
activated via Gaq and M1 ACh receptors
How do GPCRs activate ion channels (muscarinic M2 receptor)
Acetylcholine binds GPCR, activated bg unit binds to potassium channel causing it to open
Potassium ions flow out, cells become hyperpolarized, heart slows down
G proteins in Cholera
Cholera toxin
- Inhibits the GTPase activity of the subunit Gαs (stimulates adenylyl cyclase)
- Prolonged signal causes water and Cl- to move out of the cells lining the intestine
- Results in diarrhoea, dehydration and death
G proteins in Whooping cough
- Bordetella pertussis releases an active adenylyl cyclase domain
- Pertussis toxin renders Gαi inactive (Gαi inhibit adenylyl cyclase)
- Modifies Gαi preventing association with GPCRs
- Prolonged signal that stimulates coughing
GPCR signalling pathways
Receptor linked to enzymes (Receptor tyrosine kinases)
- Ligand binding activates enzyme activity within the cytoplasmic domain
- The response usually requires receptor dimerisation
- Tyrosine residues in the intracellular domains are auto-phosphorylated in response to the signal
