cell signalling - exam Flashcards
cell signalling
- Part of a complex system of communication that governs basic cellular activities and coordinates cell actions
Receive signals -> process info -> action - This relay of information is known as signal transduction
cell signalling
involved diverse inputs and outputs
- Outputs feedback into the system so the messages are regulated
- Once cell has responded needs to be able to modulate the signal
cell signalling
involved diverse inputs and outputs
inputs
environmental stresses
signals from other cells
extracellular matrix
nutrient signals
internal states (homeostasis, cell cycle)
cell signalling
maintenance of tissue architecture
Cell signalling is essential for maintenance of tissue architecture
- Co-existence of many cell types
- Numbers and location of each are tightly regulation
- Achieved by exchange of signals
cell signalling
cell surface receptor proteins
- Membrane bound proteins
- How are messages received
types of signalling
paracrine
endocrine
juxtracrine (contact-dependent)
types of signalling
paracrine
- Cell secretes signalling molecules
- Acts ono cells in local vicinity (must express receptor that recognises ligand)
e.g. synaptic (paracrine) - Specialised type of paracrine signalling
- Ligand is released at remote location to the main body of the cell
types of signalling
endocrine
- Long distances
- Hormones from endocrine cells
- Some growth factors (e.g, HGF)
- Cytokines associated with systemic aspects of inflammation
types of signalling
juxtracrine
cells depend on physical contact
membrane bound signal molecules
cell signalling
cells depend on…
multiple extracellular signals
cell signalling
proteins involved in cell signalling
Message passed from protein to protein
reception:
- receptor protein
transduction:
- intracellular signalling proteins
response:
- effector proteins
ACTIVATION OF SIGNALLING MOLECULES
signalling nodes as switches
- Intracellular protein receives signal and switches from inactive to active state
- This is reversed to switch activity off and stop signal transduction
ACTIVATION OF SIGNALLING MOLECULES
modes of activation
- Binding/dissociation
- Post-translation modification
- Conformational change
- Localization
ACTIVATION OF SIGNALLING MOLECULES
phosphorylation
- The most common post-translational modification
o Conformational change by attracting +ve charged amino acids side chains
o Form a recognition site for other proteins (e.g. SH2 domain)
o Disrupt protein-protein interactions
ACTIVATION OF SIGNALLING MOLECULES
molecular switches: signalling by phosporylation
can be both activating and inactivating
e.g.
signal in -> protein kinase (ATP->ADP) -> ON
signal out -> protein phosphatase (P out) -> OFF
ACTIVATION OF SIGNALLING MOLECULES
molecular switches: signalling by GTP binding
- Proteins always inactive when bound to GDP
- Need an initiating event that will cause release of GDP so GTP can bind
- When bound to GTP always active
signal in -> GTP binding -> ON
signal out -> GTP hydrolysis -> OFF
ACTIVATION OF SIGNALLING MOLECULES
molecular switches: proteins
kinase
an enzyme that catalyses the transfer of phosphate groups from ATP to one or more serine, threonine or tyrosine residues on specific substrates
ACTIVATION OF SIGNALLING MOLECULES
molecular switches: proteins
phosphate
an enzyme that removes a phosphate from its substrate
ACTIVATION OF SIGNALLING MOLECULES
molecular switches: proteins
GAP
binds to activate GTPase (GPCRs or small GTPases), stimulating its GTPase activity, causing the enzyme to hydrolyse its bound GTP to GDP. Results in termination of the signalling event
o GTP Long intrinsic hydrolysis rate
o Irrespective of other proteins
o GAP increases this rate
ACTIVATION OF SIGNALLING MOLECULES
molecular switches: proteins
GEF
: binds to activated GTPase, causing it to release its bound GDP; thereby allowing GTP to bind
CELL SURFACE RECEPTORS
Classes of cell-surface receptors:
- Ion channel coupled receptors
- G protein coupled receptors
- Enzyme coupled receptors
CELL SURFACE RECEPTORS
ion channel coupled receptors
- Subunits of proteins that form a pore in the membrane
- Rapid signalling
o Electrically excitable cells
o E.g. nerve and muscle - Mediated by neurotransmitters that alter the permeability of membrane
- Alter electrochemical gradient across membrane
CELL SURFACE RECEPTORS: GPCRs
ion channel coupled receptors
example: : Acetylcholine receptors
- Acetylcholine causes a conformational change
- Influx of Na+
- Membrane depolarisation causes muscle contraction
CELL SURFACE RECEPTORS
G-protein coupled receptors (GPCRs)
- ligand binds to receptor
- GPCR then activates an associated G protein
- A subunit of this G protein then dissociates
- Affects other intracellular signalling/target proteins directly
CELL SURFACE RECEPTORS
enzyme coupled receptors
- Ligand binding outside and catalytic domain inside
o Most commonly catalytic domain on the intracellular of protein - Some have intrinsic enzymatic activity
- Some rely on associated enzymes
CELL SURFACE RECEPTORS
activity cycle of GPCRs
structure
- GPD had 3 different subunits
o Alpha, beta, gamma - Basal
o Galpha bound to GDP + in complex with Gbetagamma - Ligand binding
o Conformational changes induce GEF activity - Conformational changes in Galpha lead to dissociation with GPCR and Gbetagamma
- Beta and gamma subunit can also initiate cell signalling pathways
CELL SURFACE RECEPTORS
activity cycle of GPCRs
once conformational changes have happen
can then bind to target protein
activation o a target protein by the activated alpa subunit
hydrolysis o GTP by the subunit inactivates this subunit and causes it be dissociate from the target protein (get downstream signalling from the activated protein)
Inactive alpha subunit reassembles with By complex to reform inactive g protein
CELL SURFACE RECEPTORS
activity cycle of GPCRs
- GEF activity
Activated receptor has the GEF activity
(GAP switches OFF cycle)
- GEF: Guanine nucleotide exchange factor
- GAP: GTPase activating protein
- Betagamma can also signal
CELL SURFACE RECEPTORS: GPCRs
Many G proteins activate membrane bound enzymes that produce…
small messenger molecules
secondary messenger molecules diffuse to act on intracellular signaling molecules
- Can rapidly diffuse
- Mechanism of activation
o This is a generic example
o Lot of G proteins
CELL SURFACE RECEPTORS: GPCRs
GPCRs and cAMP
- cAMP: second messenger
o causes activation further downstream - associated with GPCRs
o heterotrimeric G protein (3 subunits) - cAMP binds to regulatory subunit
o this releases catalytic subunit
action
CELL SURFACE RECEPTORS: GPCRs
cAMP synthesis
precursor –adenylyl cyclase–> signalling mediator (3-5-cyclic AMP) –cAMP posphodiesterase–> inactive product (5’-AMP)
Rapid increase in cAMP in response to signal
Target tissue: Heart
Hormone: Adrenaline
Major response: Increase in HR and force of contraction
Target tissue: Kidney
Hormone: Vasopressin
Major response: Water resorption
Target tissue: Muscle
Hormone: Adrenaline
Major response: Glycogen breakdown
Target tissue: Ovary
Hormone: Luteinising hormone
Major response: Progesterone secretion
Activation of PKA
cAMP binds regulatory subunits
end up with:
2 active subunits
2 regulatory subunits (bound to cAMP)
How a rise in intracellular cAMP concentration can alter gene transcription
- ligand binding (e.g. adrenaline)
- activation of GPCR
- activation of adenylyl cyclase
- ATP to cAMP
- Activation of PKA
- Translocation of PKA to the nucleus
- Activation of CREB transcription factor
- Target genes: PER1 and PER2 – circadian clock; c-Fos – cell proliferation
o Linked to cancer
CELL SURFACE RECEPTORS – enzyme-coupled receptors
- Ligand binding outside and catalytic domain inside
- Some have intrinsic enzymatic activity
- Some rely on associated enzymes
CELL SURFACE RECEPTORS – enzyme-coupled receptors
receptor tyrosine kinases
- Different extracellular domains depend on which ligand they bind to
CELL SURFACE RECEPTORS – enzyme-coupled receptors
EGF receptor tyrosine kinase
activation involves
-> binding of EG to EGF binding site
-> Tyr (on cytosolic tail) phosphorylated
CELL SURFACE RECEPTORS – enzyme-coupled receptors
Receptor tyrosine kinase (RTK) autophosphorylation
- Intrinsic kinase activity of receptor responsible for intramolecular phosphorylation
- Recruitment of specific ‘effector proteins
CELL SURFACE RECEPTORS – enzyme-coupled receptors
RTK-induced phosphorylation of scaffold proteins
- Scaffold protein – multiple protein-binding sites
o Concentrate groups of proteins
see image in notes for best understanding
CELL SURFACE RECEPTORS – enzyme-coupled receptors
Protein interaction domains
- Conserved structural units of a protein (35-150 a.a.)
o Some of these amino acids have conserved regions that form domains
o Evolutionarily conserved
o Important role in forming protein-protein complexes - Essential in the formation of multi-protein complexes linking cell surface receptors to intracellular signalling pathways
- Recruit and confine
- Different type of domains
CELL SURFACE RECEPTORS – enzyme-coupled receptors
Protein interaction domains
what domains?
enzymes: BtK
adaptors: Grb2
docking protein: Shc
transcription factors: Stat
cytoskeletal proteins: Tensin
inhibitory factors: Sap
part of the domains
o SH2 and PTB – pY
o SH3 – proline-rich
o PH – lipid
CELL SURFACE RECEPTORS – enzyme-coupled receptors
SH2 domain binds…
phosphotyrosine
- Not just phosphotyrosine recognised
- The flanking amino acids and recognised as well
two binding sites
CELL SURFACE RECEPTORS – enzyme-coupled receptors
Binding of one interaction domain can provide further targets for a catalytic domain
- IRS1 PTB recognises pY-insulin receptor
- IRS1 phosphorylated by receptor ]leads to recruitment of specific signalling molecules
- Docking: Generally means they don’t have any enzymatic activity.
- Bridging
CELL SURFACE RECEPTORS – enzyme-coupled receptors
A specific signalling complex formed using…
modular interaction domains
CELL SURFACE RECEPTORS – enzyme-coupled receptors
Most RTKs activate..
Ras
- RAS: Type of G protein, Small
o Ras proteins tethered to membrane
o Bound to GDP inactive bound to GTP activate
o Intrinsic hydrolysis activity means GTP can be hydrolysed back to GDP - Interaction of Grb2 and Sos brings Sos into close proximity with Ras
CELL SURFACE RECEPTORS – enzyme-coupled receptors
MAP kinase cascade
- 3 different kinases
inputs –> MAPKKK –> MAPKK –> MAPK –> outputs
CELL SURFACE RECEPTORS – enzyme-coupled receptors
Ras activates the MAP kinase cascade
- RAS will hold RAF and stabilise it
o This is complex - RAF is phosphorated because of the interaction between RAS and RAF (this is not RAS phosphorlysing RAF)
CELL SURFACE RECEPTORS – enzyme-coupled receptors
Localisation of signalling molecules
- ERK can bind proteins in the cytosol and the nucleus
- Some of the nuclear proteins are transcription factors
- E.g. Myc – drives the cell through the cell cycle (cell proliferation)
PATHWAY REGULATION
Signalling contains activating and inhibitory steps
- Balance of activating and inhibitory steps
PATHWAY REGULATION
Different pathways have…
different rates of response
pathways involving gene expression are usually slow
PATHWAY REGULATION
Cellular regulation integrates…
cell signalling and gene expression networks
PATHWAY REGULATION
Cellular regulation integrates signalling and gene expression networks
cell signalling
o Enables transmission from outside of cell to nucleus
o Fast ON and OFF (seconds to minutes)
o Transient changes (minutes to hours)
o Spatial/directional responses and organization
o Energetically cheap (no protein synthesis)
PATHWAY REGULATION
Cellular regulation integrates signalling and gene expression networks
gene expression
o Slow ON and OFF (minutes to hours)
o Stable changes (hours to years)
o Limited spatial responses
o Energetically costly (transcription and translation)
PATHWAY REGULATION
signal integration
- Complexities of signalling
o Not one pathway one interaction
o There are multiple and overlap
e.g. in some cases two kinases need to be activated to phosphorylase target protein
PATHWAY REGULATION
GPCRs and RTKs activate…
multiple signalling pathways
PATHWAY REGULATION
desensitization
a process where repeated exposure to a stimulus reduces an individual’s emotional and/or behavioral response to it
receptor sequestration
receptor down regulation
receptor inactivation
inactivation of signalling protein
production of inhibitory protein
PATHWAY REGULATION
Receptor-mediated endocytosis + desensitiation
GPCR desensitisation via beta-arrestin
- G protein phosphorylates by kinase (GRK)
- Phosphorylation recruits beta-arrestin
- Further complication: we know we can get signalling from endosomes
o Internal signals
METHODOLOGY
methods to study cell signalling
- Phosphoantibodies
- Perturbation of cell signalling pathways
- Mutant proteins
METHODOLOGY
Phosphoantibodies
control cells (unstimulated) and stimulated cells
–> quench and lyse cells run extract on denaturing protein gel
–> transfer to membrane
–> probe with a phosphospecific antibody
–> immunoblot
done over time e.g. can see an increase (pathway becoming activated)
- These antibodies will only bind to phosphorylated version of the protein
o (also recognises the amino acids around the phosphorylation)
METHODOLOGY
Perturbation of cell signalling pathways
manipulating either the external environment or internal cellular components to observe how changes in these pathways affect cellular responses
control, knockout/knowdown, inhibitor
- Often inhibitor not specific
o So can be some off target effects
METHODOLOGY
mutant proteins
can disrupt or alter signaling pathways in specific ways, allowing researchers to understand the roles of different proteins and pathways in cellular processes
example: changes to ALANINE
-> seeing where specific Y areas bind
WHEN CELL SIGNALLING GOES WRONG
- Signal not sent at the right time
- Signal doesn’t reach target
- Target doesn’t respond
- Cells respond without a target
WHEN CELL SIGNALLING GOES WRONG
Aberrant cell communication ->
DISEASE
WHEN CELL SIGNALLING GOES WRONG
cancer
many pathways implicated in cancer
- GROWTH FACTORS
- ANTI-GROWTH FACTORS
- Similarities and differences between:
o Cells
o the same cells in different environments - Elk and Fos involved in transcription
WHEN CELL SIGNALLING GOES WRONG
cancer
REAL CELLS
cells expressing a mutant form of Ras
- blocks hydrolysis of GTP
- uncontrolled growth
WHEN CELL SIGNALLING GOES WRONG
cancer
growth factors and Ras
Ras inactive –> upstream simulatory signal and Ras activation triggered by GEF –> Ras active
–> blockage caused by oncogenic mutation –> GTP hydrolysis and Ras inactivation induced by GAP –> Ras inactive
WHEN CELL SIGNALLING GOES WRONG
cancer
evidence of Ras implication in cancer
tumour types and proportion o tumour carryibg a point-mutated Ras gene
pancreas: 90 K
thyroid (papillary): 60 (H, K, N)
SUMMARY
- Receptor kinases rely on…
on phosphorylation to transmit signals through the cytoplasm
o MAPK signalling
SUMMARY
- Cells receive many different signals that they…
transduce into cellular responses
SUMMARY
- Signalling proteins can be activated by a…
variety of mechanisms including de/phosorylation and conformational changes
SUMMARY
- G-protein signalling makes the use of…
cAMP and lipids as second messengers
SUMMARY
- A cell signalling pathway can result in…
an alteration in gene expression
