Flashcards in membrane and receptors - 6 Deck (81):
What is paracrine signalling?
Signalling between cells by using a local mediator that travels in the interstitial fluid to reach its target cell.
What is endocrine signalling?
Signalling using a hormone that travels in the blood to its target cell - can travel long distances.
What is synaptic signalling?
Signalling from pre-synaptic to post-synaptic neurone by the release of neurotransmitters into the synaptic cleft.
Name three subdivision of signalling molecules. Why does this classification, alothough useful, no longer hold?
1. Local chemical mediators
Can't classify all molecules into one group - may occur in two groups.
What is the difference between cell surface and intracellular receptors?
The majority of receptors are found on the cell-surface (PM) and bind hydrophilic signalling molecules. Intracellular receptors bind to small hydrophobic molecules inside the cell. These molecules are transported extracellularly by carrier proteins and are able to diffuse through the PM.
What is a receptor?
A molecule that recognises specifically a second molecule (ligand) or family of molecules and which in response to ligand binding brings about regulation of a cellular process.
NB: In the unbound state a receptor is functionally silent.
What is a ligand?
Any molecule that specifically binds to a receptor site. It may produce activation of a receptor, in which case it is an agonist. Or it may combine with the receptor site without causing activation in which case it is an antagonist (because it opposes the action of an agonist).
What is the mechanism of action of an antagonist?
They bind to receptor sites without causing activation. They oppose the action of agonists because they cannot bind to the receptor site whilst the antagonist is bound.
List some roles of receptors in cellular physiology:
1. Signalling by hormones/ local chemical mediators
3. Cellular delivery - targeting of therapeutics
4. Control of gene expression
5. Cell adhesion
6. Modulation of immune response
7. Sorting of intracellular proteins (e.g. KDEL)
8. Release of intracellular stores (organelle receptors)
What is the function of the KDEL sequence?
Proteins with this sequence are target to the ER - this includes their retrograde transport from the golgi.
How do the binding affinities of receptors and enzymes compare?
Affinity of ligand binding at receptor sites is generally much higher than binding of substrates and allosteric regulators to enzyme sites.
How are receptors classified?
1. According to the ligand which they bind e.g. nicotinic acetylcholine receptor is activated by nicotine (agonist) as well as ACh.
2. Sub-classified by affinity of a series of antagonists e.g. M1 is blocked by the same antagonists as M2 and M3 but Pirenzipine is the most potent.
What is the difference between a receptor and an acceptor?
1. At rest - a receptor is silent but an acceptor is operating
2. Agonist binding - stimulates a biological response in a receptor but produces no response in an acceptor.
How does methotrexate work? What is it used for?
It inhibits folic acid reductase which DNA synthesis (purine and pyrimidine synthesis) and cellular replication. It is therefore used as an anti-cancer drug.
Discuss different mechanisms of signal transduction
1. Membrane-bound receptors with integral ion channels
2. Membrane-bound receptors with integral enzyme activity
3. Membrane-bound receptors which couple to effector through transducing proteins
4. Intracellular receptors
What is signal transduction?
The transmission of molecular signals from a cell's exterior to its interior.
What ions does the membrane-bound receptor with integral ion channels - nAChR - allow through?
Na+, K+ and Ca2+ (mainly Na+)
What ions does the membrane-bound receptor with integral ion channels - GABA (gamma amino butyric acid) receptor - allow through?
What ions does the membrane-bound receptor with integral ion channels - glycine receptor - allow through?
What ions does the membrane-bound receptors with integral ion channels - glutamate receptors (such as NMDA, kainate and AMPA) - allow through?
What ions does the membrane-bound receptor with integral ion channels - IP3 receptor - allow through?
Ca2+ from the ER.
What are the excitatory/ inhibitory effects of GABA and glycine receptors?
They cause an influx of Cl- and therefore hyperpolarise the cell -> inhibition of action potentials.
Describe the structure of nAChR
1. Pentameric (2x alpha, 1x beta, gamma and delta)
2. 2x alpha subunits contain a receptor for ACh each
Describe the structure of a 'classical' ligand-gated ion channel?
1. Extracellular N- and C-terminus
2. N-terminus contains the ligand binding domain
3. 2nd membrane domain lines the channel (present in each subunit)
Describe the structure of a membrane-bound receptor with integral enzyme activity
2. N-terminal domains are the binding domains (on both subunits)
3. C-terminal domains are the catalytic domains (on both subunits)
4. Binding of ligand causes conformational change which activates catalytic domains on the inside of the cell.
How does the membrane-bound receptor with integral enzyme activity - atrial natriuretic peptide (ANP) receptor - work?
It is linked directly with guanylyl cyclase which converts GTP to cGMP. cGMP then goes on the be a second messenger.
How does the membrane-bound receptor with integral enzyme activity - growth factor receptors (e.g. insulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF) - work?
Linked directly to tyrosine kinase - becuase they all want to illicit the same response = growth.
Explain the mechanism of signalling via tyrosine kinase-linked receptors:
1. Binding of receptor agonist causes autophosphorylation by each of the intracellular domains of the dimer
2. Phosphorylated sites become the docking sites for enzymes/ transducing proteins containing the SH2 domain
What do SH2 regions on proteins always bind to?
Phosphorylated tyrosine residues.
What type of receptor are insulin receptors?
Tyrosine kinase-linked receptors
How many transmembrane domains do membrane-bound receptors that signal through transducing proteins have?
They are seven tranmembrane domain (7TMD) receptors
How are membrane-bound receptors that signal through transducing proteins, coupled to enzymes or channels?
Through GTP-binding regulatory proteins (G-proteins).
What is the structure of G-protein coupled receptors?
1. 7 transmembrane domains
2. N-terminal (extracellular) and intramembranous cleft ligand binding domains
3. C-terminal (intracellular) G-protein coupling
4. 7 transmembrane domains form a cleft within the body of the membrane (mutagenesis in this region changes the afinity of the receptor)
How does ligand binding to GPCR result in transduction of a signal?
1. Ligand binding causes a conformation change which allows the receptor to act as a nucleotide exchange factor.
2. It helps the G-protein exchange GDP for GTP on its alpha-subunit, activating the G-protein and causing the alpha unit and beta-gamma units to dissociate from each other
3. This alpha-subunit goes off to affect intracellular proteins or target functional proteins.
What are the two main transducing pathways of GPCRs?
1. the cAMP signal pathway
2. the phosphatidylinositol signal pathway
Explain how Gi and Gs protein coupled receptors can create integrated intracellular signally (affect the same target).
They both share the same transducing pathway - the cAMP signalling pathway - but Gs has a stimulatory affect on this pathway (increases cAMP levels) whereas Gi has an inhibitory affect (decreases cAMP levels). The level of cytosolic cAMP may then determine the activity of various ion channels as well as members of the ser/thr-specific protein kinase A (PKA) family.
Describe the structure of intracellular receptors:
They have a C-terminal hormone binding domain, a DNA binding domain (zinc-fingers) and a transcription activating domain.
What are two hormones that bind to intracellular receptors?
Thyroid and steroid hormones
How is DNA transcription controlled by intracellular receptors?
At rest inhibitory protein complexes bind to the receptor and prevent it binding to DNA. When a steroid hormone binds the inhibitory protein complex is removed and the receptor can bind and transcribe DNA.
List some steroid and thyroid hormones of the intracellualr receptor family:
Cortisol receptor, oestrogen receptor, progesterone receptor, Vitamin D receptor, Thyroid hormone receptor, retinoic acid receptor.
How does amplification occur in cellular signalling? Use G-protein cAMP signalling pathway as an example (Gs).
1. GPCR may activate 4 G-proteins before ligand dissociates
2. Each G-protein may activate a different adenyl cyclase
3. Adenyl cyclase may convert thousands of substrate whilst bound
4. cAMP goes onto activate PKA. Each PKA may phosphorylate thousands of enzymes.
5. Each of those enzymes will go on to catalyse multiple substrates etc etc...
What is the name of the process that internalises particular matter?
What is the name of the process where invagination of the plasma membrane forms a vesicle that allows uptake of impermeable extracellular solutes (not particles, just generalised capture) and retrieval of plasma membrane?
What are the two forms that pinocytosis can be sub-divided into?
2. Receptor-mediated endocytosis
Describe the process of phagocytosis
1. Particles bind to receptors in the plasma membrane.
2. The cell extends pseudopods, which permit further receptor interactions and membrane evaginations = membrane-zippering mechanism.
3. Particle is internalised forming a phagosome.
4. Phagosomes fuse with lysosomes forming a phagolysosome, in which particles are degraded.
The uptake of cholesterol is an example of what type of process?
Describe the composition of LDLs:
1. A core of esterified cholesterol esters (fatty acids)
2. Covered by a phospholipid and cholesterol monolayer, containing a single protein species = apolipoprotein B
Describe the structure of the clathrin coat:
1. Coat structures are made up of haxagons and pentagons which allows the structure to bend to form vesicles
2. These hexagons and pentagons are formed from clathrin triskelions
3. Triskelions comprise of clathrin and 2 light chains.
Describe how the clathrin coat forms vesicles and is recycled back to the membrane:
1. Clathrin coated pits form spontaneously and drive invaginations of the membrane, forming vesicles.
2. Clathrin coated vesicles are uncoated by an ATP-dependent uncoating protein.
3. Clathrin triskelions are recycled back to newly forming clathrin coated pits
4. Uncoated vesicle are then free in the cytoplasm
How can mutations affect the LDL receptor in hypercholesterolaemia?
1. Non-functional receptor - non-functional receptor so doesn't bind LDL, but normal coated pits and internalisation (but no capture)
2. Receptor binding normal - but no internalisation of LDL receptors. LDL receptors found distributed over the whole surface of the cell because deletion of C-terminal cytoplasmic domain prevents interaction with the clathrin coat.
3. Receptor deficiency - mutations that affect expression of LDL receptor
In which cells does phagocytosis occur in in mammals?
Only in specialised cells like macrophages and neutrophils.
What is receptor mediated endocytosis?
When the specific binding of cell surface receptors permits the selective uptake of substances into the cell.
How do cells requiring cholesterol procure it?
They synthesise LDL receptors which recognise apoliprotein B specifically. Within 10 minutes of binding the LDL particle is internalised and delivered to lysosomes where the cholesterol is released from cholesterol esters.
Where are LDL receptors localised?
Clathrin-coated pits (2% of cell surface)
How are LDL receptors recycled?
After the vesicles have been uncoated they fuse with larger smooth vesicles called endosomes. In the endosomes the pH is maintained at ~5.5-6.0 by an ATP-dependent proton pump. At this pH the LDL-receptor has low affinity for the LDL particle and the two dissociate. The transmembranous receptors are sequestered to a domain within the endosome membrane which buds off as a vesicle and recycles the LDL-receptor to the plasma membrane.
In which structure do the LDL receptors and LDLs get sorted from each other?
Why is clathrin coated-pit formation spontaneous?
Because the association of the coat proteins is energy-independent.
How is clathrin uncoating carried out?
By an ATP-dependent uncoating protein which binds and stabilises the freed coat proteins
How is the clathrin coat associated with the membrane?
It is attached by a number of integral membrane adapter proteins which form associations both with the clathrin and receptors, localising receptors over the clathrin coated pit.
How does receptor recycling of transferrin differ from that of uptaken LDL receptors?
Transferrin at the acidic pH of the endosome does not dissociate from its receptor, unlike LDL and its receptor. Instead the two Fe2+ which it is carrying dissociate and it returns to apotransferrin. Apotransferrin and the receptor are sorted in CURL and recycled back to the plasma membrane. Whereas only LDL receptors are returned from the endosomes to the plasma membrane.
What is another name for endosomes?
CURL = the compartment for the uncoupling of receptor and ligand
How do cells uptake Fe3+?
Apotransferrin binds two Fe3+ in the circulation becoming transferrin. Transferrin binds transferrin receptors at neutral pH and are internalised by receptor mediated endocytosis. On reaching the acidic endosome Fe3+ dissociates and the apotransferrin and receptor are recycled.
Most receptors internalised by RME are located over coated pits, however insulin receptors are not. How does their location differ?
They only cluster over coated pits when the agonist is bound. Binding insulin probably changes their conformation and allows them to be recognised by the coated pit.
Describe the uptake of insulin into cells.
Insulin binds receptors which localises them over coated pits. Endocytosis occurs and in the endosome insulin remains bound to its receptor and the complex is targeted to the lysosomes for degradation.
What is the importance of the fact that insulin receptors are not recycled?
It allows for a reduction in insulin receptors (down-regulation) which desensitises the cell to continued presence of high circulating insulin.
What is transcytosis?
When ligands that have been endocytosed remain bound to their receptors and are transported across the cell.
Give some examples of transcytosis that occurs in humans?
1. Transport of maternal immunoglobulins to the foetus via the placenta
2. Transfer of IgA from the circulation to bile in the liver
What is of note about the transcytosis of IgA from the circulation to bile in the liver?
During transport the receptor is cleaved, resulting in the release of immunoglobulin with a bound 'secretory component' derived from the receptor.
In REM what are the pathways that are common for all proteins that undergo endocytosis?
Receptors for different ligands enter the cell via the same coated pits and then pass to the endosome.
How are receptors sorted in the CURL into different cellular locations?
They are target to different cellular locations by short amino acid motifs, and sorted into discrete regions of the endosome membrane which bud-off into transport vesicles.
In the REM of LDL what is the fate of the receptor and ligand?
This is mode 1 REM. The receptor is recycles and the ligand is degraded.
In the REM of transferrin what is the fate of the receptor and ligand?
This is mode 2 REM. The receptor is recycled and the ligand is recycled.
In the REM of insulin what is the fate of the receptor and ligand?
This is mode 3 REM. The receptor and ligand are degraded.
In the REM of IgA what is the fate of the receptor and ligand?
The receptor and ligand are transported.
What is the function of mode 1 REM?
What is the function of mode 2 REM?
What is the function of mode 3 REM?
1. Receptor down-regulation (e.g. insulin, EGF)
2. Removal from circulation of foreign antigens (immune complexes)
What is the function of mode 4 REM?
Transfer of large molecules across the cell
How do membrane-enveloped viruses and some toxins exploit endocytic pathways to enter cells?
They bind to receptors in the plasma membrane. Once endocytosed, in the endosome where the pH is favourable the viral membrane fuses with the endosomal membrane, releasing the viral RNA into the cell where it can be translated and replicated to form new viral particles.
Give examples of toxins that enter via REM:
1. cholera toxin
2. diptheria toxin
Both these bind to GM1 ganglioside