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Flashcards in Cell signalling Deck (69):

What is the purpose of the plasma membrane?

- Acts as a barrier between intra- and extracellular environments.
- Controls movement of substances into an out of cells.


What are the ways that substances may be transported across a plasma membrane?

1. Simple diffusion: For lipid soluble substances and small molecules such as O2 and CO2.
2. Channels: Allows passive diffusion of specific substances through selective pores across the PM. Gated and can be open or closed.
3. Carriers: Transporter proteins that rely on conformational changes in order to transport substances across PM. Can be passive or active.
4. Vesicles: Bulk transport of substances into and out of cell by exocytosis/endocytosis.


What are the ways by which information can be passed between cells?

- Hormones (endocrine)
- Neurones (neurocrine)
- Autocrine
- Paracrine


What is the principle of cell signalling?

- Passing information across plasma membranes.
- Information need to be transported out of the plasma membrane of cells at origin.
- Information needs to be transported into the plasma membrane of target cells.


What are the different ways in which information is transported out of cells?

- Simple diffusion: Lipophilic messengers are able to diffuse across the plasma membrane.
- Carriers/channels: Transports messengers out of origin cell.
- Vesicles: Messengers are packed into vesicles and are secreted out of origin cell by exocytosis. This only happens in eukaryotes.


What are the different ways in which information is transported into cells?

- Simple diffusion: Lipophilic messengers are able to diffuse across the plasma membrane.
- Channels: Messenger binds to and acts on ion channels, causing them to close. This triggers intracellular events either by entry of ions into the cell or changes in membrane potential.
- Allosteric proteins: Messenger binds to and acts on extracellular portion of allosteric proteins, causing conformational change that triggers intracellular events.


What is intracellular division of labour?

- Cells are divided into different intracellular compartments.
- Each compartment has a specific function and they work together in order to ensure the survival of the cell and that it carries out its normal function.


What are the principles behind intracellular transport of proteins?

- Most intracellular proteins contain specific target sequences ('address labels') recognised by chaperone proteins.
- These allow the protein to be transported between different compartments of the cell.


What is co-translational targeting?

When a protein is carried to a specific organelle by chaperones while it is still being translated.


What is the sequence of events during co-translational processing?

1. As the polypeptide sequence emerges from the ribosome, it is recognised by specific Signal Recognition Proteins (SRPs), which binds to it and halts translation.
2. The SRP carries the polypeptide, with ribosome still attached, to the ER where it binds onto an SRP receptor (SR).
3. Binding of SRP to SR causes conformational change resulting in SRP unbinding from polypeptide (only occurs if SR next to translocon and involves hydrolysis of GTP to GDP.
4. Ribosome binds to translocon and opens its channel.
5. Dissociation of SRP from polypeptide causes translation to resume. As the polypeptide elongates, it is threaded through the translocon into the ER.
6. Inside the ER lumen, folding proteins bind to the polypeptide.
7. Once translation is complete and the polypeptide is fully threaded into the ER, ribosome unbinds from translocon and translocon closes.
8. Inside the ER, transmembrane proteins are embedded into the membrane and their topologies are checked. They also undergo additional post-translational processing such as glycosylation.


What are the roles of folding molecules in the ER lumen?

1. Aids in the folding of the polypeptide chain into final protein.
2. Helps pull polypeptide chain through the translocon.


What are the functions of co-translational targeting to ER?

1. ER lumen effectively continuous with extracellular environment. Provides oxidising environment to facilitate the formation of disulfide bridges.
2. Chaperones in ER lumen aid in folding of the protein.
3. Co-translational because polypeptide chain largely unfolded so can be easily thread through translocons.


What is the 'address label' to the ER?

9-12 amino acid long sequence on the N-terminus end of a polypeptide consisting of mostly hydrophobic amino acids.


What is the structure of SRPs?

- SRPs are ribonucleoproteins containing multiple RNA and protein domains.
- The address label-binding domain consists of mainly hydrophobic amino acids, with a large number of Met residues to ensure flexibility.
- The ribosome-binding domain binds to ribosome and stops translation.
- GTPase domain important in unbinding mechanism of SRPs at ER.


What is the purpose of the ERAD pathway?

Breaking down damaged ER proteins.


What is the sequence of events in the ERAD pathway?

1. Damaged ER proteins are detected by specific recognition proteins.
2. Proteins are unfolded and threaded through retrotranslocons. This causes protein to be extruded from ER.
3. Outside ER, protein is ubiquitinated by membrane-bound ubiquitin ligase.
4. Ubiquitination targets proteins to proteolysis via proteosomes.


What are some examples of diseases caused by defects in ERAD pathway?

- In cystic fibrosis, mutated CFTR detected by recognition particles in ER and deemed damaged. They are targeted to destruction via ERAD pathway.
- The human cytomegalovirus (HCV) targets MHC complexes of infected cells to destruction by ERAD, which prevents T-cells from destroying infected cells.


What is the significance of topologies in transport between membrane-bound organelles?

Topology of transmembrane proteins are preserved so that domain facing cytoplasm always remains facing cytoplasm and domain facing inside of vesicle (effectively extracellular environment) remains so.


What are Rabs?

G-proteins that are master regulators of intracellular transport. They regulate vesicle formation and fusion.


What is the sequence of events that occur during transport of proteins from ER to Golgi?

1. COPII binds to receptors on the surface of ER bound to cargo protein on inside, inducing vesicle formation.
2. COPII mediates transport of vesicles from the ER to the Golgi via network of microtubules.
3. At the Golgi, Rab proteins on vesicle bind to Golgi-specific RAB proteins on the Golgi (acts as label).
4. V-SNAREs on vesicle bind to t-SNAREs on Cis-Golgi membrane. These wind around each other, pulling the vesicle towards the Golgi membrane and eventually causing vesicle to fuse with membrane.


What is the sequence of events that occur during transport of proteins from the Golgi to ER?

1. Proteins with KDEL sequences are targeted from the Golgi to the ER.
2. Proteins then bind onto KDEL receptors on the cis-Golgi membrane.
3. COPI binds to KDEL receptors and induce vesicle formation.
4. COPI mediates the transport of vesicles from Golgi to ER, along network of microtubules.
5. Vesicle fuses with ER membrane via same mechanism involving SNAREs and Rab.


What is the sequence of events that occur during transport of proteins from the Golgi to lysosomes?

1. Proteins containing mannose-6-phosphate binds to mannose-6-phosphate receptors on the trans-membrane of the Golgi.
2. Clathrin binds to mannose-6-phosphate receptors vis GGA and AP1 adaptor proteins. This induces vesicle formation and the formation of the clathrin cage.
3. Vesicle transported to the lysosomes and fuses via same mechanism involving Rab and SNAREs.
4. Mannose-6-phosphate receptors are recycled and returned to the Golgi.


What is the clinical significance of mannose-6-phosphate?

- Used to treat lysosomal storage disease that are caused by deficiencies in certain lysosomal enzymes.
- Exogenous enzymes are modified with mannose-6-phosphate and introduced to the cell. These are targeted to lysosomes, where they can restore function.


What are the common themes shared by intracellular transport systems between different membrane-bound proteins?

1. Cargo segregation: Receptors for specific address labels.
2. Vesicle formation: Via binding of coat proteins (e.g. clathrin).
3. Transport: Along microtubule networks.
4. Recognition: Rabs.
5. Fusion: Via v-SNAREs and t-SNAREs.


What is the polarity of Golgi?

- Cis-membrane is membrane where vesicles fuse and proteins enter the Golgi.
- Trans-membrane is membrane where vesicles created and proteins leave the Golgi.


What is the sequence of events that occur during intake of cholesterol via LDLs?

1. LDLs bind to LDL receptors on plasma membrane via Apo-B100 proteins found within the LDL.
2. Clathrin binds to LDLRs via intracellular domain, inducing vesicle formation via clathrin cage.
3. Vesicles transported to early endosomes, where luminal pH is low, causing LDLRs to release LDLs.
4. LDLRs reform into vesicles that are transported back to the PM and is recycled.
5. LDLs transported to late endosome and then ribosome where it is broken down and the cholesterol is released.


What is the consequence of there being a lot of noise between the origin and target cells?

The signalling molecule needs to have high specificity and affinity of its receptors.


How do ion channels function?

- They have a pore in the centre through which ions are able to diffuse.
- Their signalling involves the physical transport of matter across the PM.


What are the 2 structural features of ion channels?

1. Ionic selectivity of pore
2. Gating


What are the main functions of ion channels?

- Transmission of action potentials along neurone
- Triggers direct metabolic changes within a cell
- Maintains/changes resting potential of cells


What types of signal transduction pathways are ion channels involved in?

- Electrical-Electrical: AP → Na+ channel opening → AP
- Chemical-Electrical: Neurotransmitter → Na+ channel opening → AP
- Electrical-Chemical: AP → Ca2+ channel opening → Chemical changes
- Chemical-Chemical: Neurotransmitter → Ca2+ channel opening → Chemical changes


What is the mechanism of Ca2+ selectivity filters ?

- In solutions, ions are usually in their hydrated states.
- Pore through the ion channel is usually to small to accommodate hydrated ions.
- There are 2 Ca2+ binding sites in the selectivity filter in which Ca2+ ions are bound to oxygen atoms in C=O groups by coordinate bonds.
- These interactions dehydrate Ca2+ ions, allowing the much smaller dehydrated Ca2+ ion to pass through the pore.
- Smaller ions such as Na+ do not interact with oxygen atoms well, so cannot be dehydrated, and so are unable to pass through the channel.
- Amino acid residues are arranged in selectivity filter so that free energy change for dehydration of Ca2+ is low and so reaction is favourable whereas it is high for Na+ and is unfavourable.


What is the mechanism of voltage-gating in voltage-gated ion channels (VGICs)?

- Voltage sensor in VGICs are the 4th transmembrane α-helix (S4).
- During depolarisation, the ECF becomes more -ve than the ICF, causing +ve Arg residues in S4 is attracted towards ECF and repelled away from ICF.
- This puts strain on the neighbouring S5 and S6 helices, splaying the S6 helices apart and opening the channel pore.


What is the structure of VGICs?

- Homotetramers, each consisting of 4 subunits.
- Each subunit is made out of 6 transmembrane α-helices connected together by linker peptides.
- Subunits form transmembrane pore through middle of channel.
- Selectivity filter is located on the extracellular end of the pore.


How are ions transferred through pore in Ca2+ channels?

- 2 binding sites for Ca2+ in selectivity filter.
- Binding of Ca2+ to proximal binding site repels Ca2+ in distal binding site and ejects it into the pore.
- Ca2+ transmitted through pore by being passed between amino acid residues lining pores.
- Minimal ΔG occurs between transfers ensuring high rate of passage through pore.


What is the mechanism of inactivation in VGICs?

- There is a ball-like structure at the N-terminus end of each subunit called the inactivation ball.
- After period of activation, the pore is blocked by one of the 4 inactivation balls within the structure of each VGIC.


What are the similarities between the functional architecture of allosteric receptors (i.e. GPCRs and RTKs)?

1. Signal translocation: Binding of signalling molecule to extracellular domain causes conformational change in intracellular domain, triggering intracellular signalling pathway. ESM itself does not physically cross PM.
2. Spatial organisation: Many components of signalling pathway are held in fixed position by scaffolding proteins to maximise speed of intracellular signal propagation.
3. Amplification: Multi-step pathways allow for multiple stages of amplification whereby activation of 1 molecule is able to activate multiple molecules downstream.
4. Integration: One molecule is able to act on multiple different types of other molecules.
5. Activation:
- Allosteric
- Covalent modification
6. Modular recognition motifs: Molecular-binding domains conserved across different proteins. Allows for integration.
7. Termination: Parts of the signalling pathway can be terminated by either allostery (activator unbinding) or reverse reaction (e.g. dephosphorylation).
8. Specificity: Specific receptors are only capable of binding to specific ligands.


What are the different modular recognition motifs?

14-3-3: Ser-P and Thr-P
SH2: Tyr-P
PH: PI(3,4,5)P3


What are the usual functions of receptor tyrosine kinases?

They are usually the receptors of growth factors and are involved in signalling pathways responsible for cell proliferation. Mutations are usually associated with cancers.


What are the steps in general RTK activation?

1. Signalling molecule binds to extracellular domain of RTKs and induces dimerisation (either because ligand also dimer and binds 2 receptors, or it causes conformational change).
2. Dimerisation causes autophosphorylation of intracellular protein tyrosine kinase domain on Tyr residues.
3. SH2 domains on different proteins bind onto phospho-Tyr residues and stimulates further steps in the intracellular signalling cascade.


What are the steps in the insulin pathway?

1. Insulin binding to insulin receptor causes autophosphorylation of PTK domains and IRS proteins.
2. IRS proteins binds to SH2 domain on PI3K, which phosphorylated PIP2 to PIP3.
3. Binding of Akt2 to PIP3 via PH domain, with the simultaneous binding of mTORC2 and PDK1 (activated by PIP3 binding), activates Akt2.
4. Akt2 activates Rab-GTP which promotes translocation of GLUT4 to PM.
5. Akt2 inhibits glycogen synthase kinase 3 (GSK3), which inhibits glycogen synthase, promoting glycogen synthesis.
6. Akt2 activates FOXO, which inhibits gene expression of gluconeogenic enzymes and thus inhibits gluconeogenesis.


What allows G-protein activity to be controlled?

- Binding of GTP to G-proteins activates the G-protein.
- Binding of GDP deactivates the G-protein.
- The process of GDP dissociation and subsequent association of GTP is slow.
- Enzymes that speed up this rate of reaction significantly increases the activity of G-proteins.
- These enzymes are usually associated with GPCRs and is activated on binding of the ligand.


What are the steps in the G-protein cycle?

1. Binding of ESM on extracellular domain causes conformational change activating intracellular enzyme domain.
2. GPCR catalyses the dissociation of GDP from the G-protein and the subsequent association of GTP.
3. G-protein is activated and can subsequently activate components in the next step of the signalling pathway/
4. GTP-binding subunit on G-protein also has intrinsic GTPase activity and slowly hydrolyses GTP to GDP + Pi, deactivating the G-protein. This acts as a molecular clock determining how long a G-protein is active for.
5. The bound GDP then dissociates under mediation by GPCR and the cycle repeats.


What is the structure of G-proteins?

- G-proteins are trimers consisting of α, β and γ subunits.
- The β and γ subunits are always bound to each other so is collectively known as the βγ subunit.
- Both the α and βγ subunits are anchored to the PM.
- The α-subunit contains the GDP/GTP binding site and intrinsic GTPase activity.


What are the different types of α subunits?

- α_s: Stimulates adenylate cyclase
- α_i: Inhibits adenylate cyclase, stimulates PI 3-kinase
- α_q: Stimulates PLC
- α_12: Involved in regulation of cytoskeleton


What is the mechanism behind G-protein activation?

1. GPCR catalyses exchange of bound GDP on α-subunit for GTP.
2. Once bound, γ-subunit on GTP forms H-bond with amino acid residues in switch regions I and II. on α-subunit.
3. Switch I and II are pulled towards the γ-phosphate and are prevented from interacting with βγ-subunit.
4. This causes α-subunit to dissociate from βγ subunit.
5. Exposed surfaces of both α and βγ subunits are then able to interact with other proteins.


What properties of the binding site allows the function of G-proteins?

- The GDP/GTP binding site is very deep.
- This has 2 implications:
1. Allows GDP to bind tightly into the binding site, and making its dissociation slow.
2. GDP is unable to interact with switch regions because it does not have phosphate group in correct position. GTP on the other hand does so is able to interact with switch regions and activate G-protein.


What is the turnover rate of intrinsic G-protein GTPase activity?

2-3 min^-1


What are the functions of the βγ subunit?

- Stimulates PLC
- Inhibits Ca2+ channels
- Stimulates K+ channels


What is the structure of GPCRs?

- Consists of 7 α-helical transmembrane domains arranged in a ring-like formation similar to that of an ion channel.
- Loop regions between the α-helices make up the intracellular and extracellular binding domains.


What is the mechanism of GPCR activation?

Ligand binding induces conformation changes that cause intracellular hydrophobic G-protein binding sites to be revealed.


What are the clinical significances of GPCRs?

- Cholera toxin blocks GTPase activity of α_s subunits. This causes constant stimulation of cAMP production in small intestine epithelial cells, causing continued secretion of Na+, K+, HCO3-, H2O etc. into small intestinal lumen, causing diarrhoea.
- Pertussus toxin blocks receptors coupling to α_i.


What are intracellular messengers?

- Small diffusible molecules beyond the secondary messenger.
- Links receptor to intracellular responses.


What is the principle behind conserving intracellular messengers?

- Many different ESMs bind to different receptors that activate the same intracellular messengers in different cell types and cause different effects.
- This is only possible as the intracellular messengers are restricted to inside the cell, where they can be 'programmed' to initiate different signalling pathways without causing any adverse side effects on other cells.


What are the principles behind intracellular messengers?

Activation → Effect → Deactivation


What are the control points for the activity of cAMP?

- Activation: Adenylate cyclase (ATP → cAMP)
- Effect: Activation of PKA
- Deactivation: cAMP phosphodiesterase


What are the control points for the activity of Ca2+?

- Activation: Opening of Ca2+ channels in PM/ER membrane
- Effect: Binds to calmodulin
- Deactivation: Extrusion of Ca2+ from the cytosol by NCX/PCMA/SERCA


What is the functional significance of cAMP activation in the cAMP signalling pathway?

- Integration: Different isoforms of adenylate cyclase are present and are activate by different stimuli. This allows information from a number of different stimuli to be 'computed' and the appropriate responses mediated.
- Amplification: Activation of one adenylate cyclase is capable of catalysing formation reaction for multiple cAMP molecules.


What are the different isoforms of adenylate cyclase?

- AC1: + Gα_s, - ↑Ca2+
- AC2: + Gα_s
- AC3: + Gα_s, + ↑Ca2+


How is the deactivation step of intracellular messengers controlled?

- Usually, there is little control over the deactivating step of intracellular messengers.
- There is usually a constant activity in the deactivating mechanism. This ensures that intracellular messenger activity is transient.
- This activity can be augmented by input from other ex


How is the activity of cAMP phosphodiesterase (PDE) regulated?

PDE1: - Caffeine, - theophylline
PDE3: - Cilostazol, + PKA (-ve feedback)


What is the structure of PKA?

- PKA is a heterotetramer.
- It consists of 2 regulatory (R) subunits bound to 2 catalytic (C) subunits.
- Each R subunit has 2 cAMP binding sites and a pesodosubstrate domain.
- Each pseudosubstrate domain binds to the active site on the C subunit and blocks binding of substrate.


What is the mechanism of cAMP activation?

1. When all 4 cAMP active sites on the R subunits are occupied, conformational change occurs whereby the C subunits dissociate from R subunits and the pseudosubstrate domains unbind from the active sites.
2. PKA is free to catalyse reactions?


What is the mechanism of PKA action?

- PKA recognises phosphorylation site via sequence Arg-Arg-Arg-X-Ser/Thr-X.
- Phosphorylation occurs on the Ser/Thr residue.


What are the functions of scaffolding proteins?

1. Keeps components of a signalling pathway together and minimises distance between each component, allowing for maximum rate of signal transduction.
2. Allows the signalling pathway to be isolated to a specific sub-compartment of a cell.


What is the sequence of events that occur in a PLC signalling pathway?

1. Binding of ligand to receptor induces confomrational change that activates intracellular catalytic domain.
2. Receptor catalyses exchange of GDP for GTP, activating G-protein and causing it to dissociate into Gα_q and Gβγ.
3. Gα_q activates PLC, which converts membrane-bound PIP2 to IP3 and DAG.
4. IP3 is a free-diffusing molecule which binds to IP3-gated Ca2+-release channels on ER/SR membrane to stimulate influx of Ca2+ into cytosol.
5. DAG is membrane bound and activates PKC.


How is the PLC pathway terminated?

- IP3 is broken down by inositol polyphosphate 5-phosphotase to inositol and phosphatidate.
- These are recombined with DAG to form PIP2.


What are the principles of intracellular Ca2+ signalling?

- Ca2+ signalling relies on transient waves of ↑[Ca2+]i induced by CICR.
- These waves propagate along the length of the cell.


What are the benefits of wave-baed Ca2+ signalling?

1. Reduces risk of cytotoxicity due to ↑[Ca2+]
2. Digital signals are less prone to interference
3. Frequency-variations can be used to induce different responses (e.g. pulsatile hormone release, transduction of different transcriptional events)
4. Can be used to direct signals towards specific intracellular targets