M&R session 6: receptors and receptor-mediated endocytosis Flashcards
What is a receptor?
Any biological molecule to which a drug binds and produces a measurable response which regulates a cellular process. Unbound, they are functionally silent.
Specialised macromolecules on a cell surface/within cell that selectively interact with a specific ligand to cause a response
How might chemical signalling take place?
Hormones: signally between cells in different tissues via the circulation
Neurotransmitters: signalling at synapses
Local chemical mediators: signalling between adjacent cells in the same tissue
What is a ligand?
A small molecule that binds specifically to a site on a receptor protein. Can activate a receptor (agonist) or combine without causing activation (antagonist)
Could be naturally-occurring or a drug
What is an acceptor?
A receptor who’s basic function can occur without the interaction of a ligand. Ligand binding alone gives no response
Specificity of a response
If signalling molecule is hydrophilic, signal recognition site of receptor must be present on the extracellular space of the cell surface, then interaction of signalling molecule with receptor will cause activation of process
If signalling molecule is hydrophobic it will gain access to cell via lipid bilayer diffusion, but intracellular receptor still needed to transduce signal into a cellular response
Similarities and differences between receptor binding sites and enzyme active sites
Similarities:
- receptor binding governed by shape of binding side
- often reversible
- induce a conformational change and change in activity of a molecule
- no chemical modification of ligand/enzyme
Differences:
- L-R affinity usually higher than E-S
- ligand-R not chemically modified; S-E is modified in a chemical reaction catalysed by the active site
List some general roles of receptors
Signalling Neurotransmission Cellular delivery (LDL, transferrin) Control of gene expression (steroids, thyroid hormones) Release of intracellular Ca2+ stores (IP3 receptors) Immune responses Cell adhesion Absorption Pressure sensing e.g. baroreceptors Hormonal actions Antigen recognition
What is signal transduction and why is it necessary?
Hydrophobic signalling molecules can freely cross the plasma membrane and interact with intracellular receptors (e.g. steroids, thyroxine). Most receptors however are hydrophilic, so must interact with specific receptor proteins at the cell surface (can’t cross the plasma membrane)
Signal transduction involves 4 common mechanisms which transduce extracellular hydrophilic signals into an intracellular event, after which amplification can occur
Signal transduction via ligand-gated ion channels
Agonist binds to receptor, change in conformation, channel opens, so flow of ions down electrochemical gradient. This transduces the signal into an electrochemical event at the plasma membrane
Response is RAPID: a few ms
Response mediates a diverse range of functions, e.g. neurotransmission, cardiac conduction and muscular contraction
Classical or structurally distinct LGICs
Describe the classical LGIC family and give examples
Pentameric (5 subunit) structure
- 4 transmembrane domains per subunit
- of which 1 TM domain (M2) forms the channel lining
Examples:
- nAChR: a gated Na+, K+ and Ca2+ channel
- GABA R: gated Cl- channel (inhibitory)
- GlyR: gated Cl- channel (inhibitory, in spinal cord
- glutamate receptor: gated Ca2+ entry
Some LGICs used in signal transduction are distinct from the classical family strucute. Give examples
P2X ATP receptor
IP3 receptor (gated Ca2+ entry from ER)
Ryanodine receptor (intracellular Ca2+ entry)
Cyclic nucleotide receptor
Describe the general actions of enzyme-linked receptors in signal transduction
Agonist binds to extracellular domain-conformational change-activates intrinsic enzyme activity
Atrial natriuretic peptide action
ANP receptor is coupled to guanyl cyclase and growth factor receptors such as receptors for insulin, EGF and PDGF
These are directly linked to tyrosine kinase
Give features of the signal transduction by insulin receptors on binding insulin
Insulin binds to receptor, activates integral tyrosine kinase activity in cytoplasmic domain
Receptor autophosphorylation
Binding of transducing proteins via specific SH2 domains which recognise phosphotyrosine residues on the receptor
Tyrosine phosphorylation of transducing protein, activation of effector enzymes
Describe the mechanism of tyrosine kinase-linked receptors
- HORMONE binds and site and activates PROTEIN KINASE activity in the CYTOPLASMIC domain of the receptor
- receptor AUTOPHOSPHORYLATES tyrosine residues (catalyses P from ATP into its own structure)
- phosphorylated receptor tyrosine kinase residues are recognised by TRANSDUCING PROTEINS (e.g. IRS-1) or DIRECTLY by enzymes with recognition domains
- on association with receptor/transducing protein, the enzyme is activated ALLOSTERICALLY or by tyrosine phosphatase
Therefore message is transduced by an intracellular chemical event
Give some examples of GPCRs
mAChR adrenoceptors dopamine receptors 5-HT receptors opioid receptors peptide receptors purine receptors light/smell/taste receptors
Mechanism of signal transduction via GPCRs
7 TM domain receptor couples to effector molecules via a transducing molecule: a GTP-binding regulatory protein (G-protein).
Effectors: enzymes (e.g. adenylyl cyclase, PIP2) or ion channels (e.g. Ca2+ channels, K+ channels). Often >1 GPCR type for a particular agonist, each with own pharmacology
Action: receptor binds-conformational change-GDP/GTP exhchange in G protein-transduces message onto enzyme or channel
Second messengers: essential in conducting and amplifying signals from GPCRs
Describe the structure and action of intracellular receptors
Entirely intracellular: ligand must diffuse into cell to interact with receptor @ C terminal
Ligands involved are therefore hydrophobic (e.g. cortisol, thyroxine, oestrogen)
- penetrate plasma membrane
- bind to monomeric receptors in the cytoplasm/nucleus
Resting state: receptors stabilised by association with heat shock proteins or chaperone proteins
Activated receptor dissociates from the chaperone protein and moves to the nucleus:
- binds to control regions in DNA (ZINC FINGERS) defined by specific sequences, so regulate gene expression
- effects relatively SLOW compared to other receptors as the onset of transcription and translation must occur
Amplification
In many mechanisms it is possible to amplify molecules, e.g. the binding of a chemical signal to a single receptor can cause the modification of thousands of substrates. A cascade of such catalytic events can produce further amplification
Cell activation and inhibition-cardiac pacemaker cells
NA acts on beta 1 adrenoceptors to increase HR
ACh acts on M2 muscarinic receptors to decrease HR
Cell activation and inhibition-hepatocytes
Insulin stimulates synthesis of glycogen from glucose
Glucagon stimulates glycogen breakdown
How are extracellular chemical signals recognised at target tissues?
Bind to receptor and initiate a response in the cell/tissue.
E.g. may cause depolarisation by activating VG Na+ channels
Receptors can also recognise light or pressure
Describe the types of cholinergic and muscarinic receptors
CHOLINERGIC:
- Nicotinic ACh receptor. Agonist=nicotine (and ACh)
- Muscarinic ACh receptr. Agonist=muscarine (and ACh)
MUSCARINIC:
- M1. Antagonist=pirenzipine
- M2. Antagonist=gallamine
- M3. Antagonist=hexahydrosiladiphenol
What is receptor-mediated endocytosis?
How large, hydrophilic molecules can enter cells by association with a cell surface receptor
Describe cell membrane recycling
Membrane moved from ER to plasma membrane via an exocytic, secretory pathway; so there must be an opposite movement of plasma membrane back into cell by endocytosis
Membrane is trafficked by vesicles which bud from the donor organelle and are moved to their destination where they fuse with the recipient organelle
What are the mechanisms of membrane internalisation?
- PINOCYTOSIS
- fluid-phase and receptor mediated endocytosis
- invagination of PM to form a lipid vesicle
- permits uptake of impermeable extracellular solutes, and retrieval of PM - PHAGOCYTOSIS
- internalisation of particulate matter by neutrophils and macrophages
- particle binds to receptors in PM, cell extends pseudopods to permit further receptor interactions
- particle internalisation via a “membrane-zipping” mechanism
- promotes clearance of damage cell materials and invading organisms for destruction - ENDOCYTOSIS
- the selective internalisation of molecules into the cell by binding to specific cell surface receptors
- therefore is receptor-mediated
Structure of LDLs
Core of esterified cholesterol esters covered by a phospholipid and cholesterol monolayer
Apoprotein B: LDL receptors recognise this. Receptors located in clusters of clathrin-coated pits
Structure of the coated pits
Formed spontaneously (energy-independent). Uncoating is driven (ATP-dependent uncoating protein)
Clathrin coat attached to PM by integral membrane adaptor proteins which form associates with clathrin and receptors
Minimum structure: TRISKELION
- hexagonal and pentagonal structures formed from clathrin skeletons, arranged in a 3-legged shape basket structure
- clathrin heavy chains and 3 clathrin light chains
Describe uptake of cholesterol as an example of receptor-mediated endocytosis
- LDL binds to receptor, clathrin pits invaginate to form COATED VESICLES
- Vesicles are quickly UNCOATED (requires ATP) and then fuse with larger, smooth vesicles-ENDOSOMES
- pH of endosome kept at 5.5-6.0 by H+ pump (lower than cytoplasmic pH): so LDL receptor has low affinity to LDL, so dissociate, therefore endosome known as CURL (Compartment of Uncoupling of Receptor and Ligand)
- Vesicle buds off and RECYCLES the LDL receptor to the plasma membrane
- Endosomes containign LDL FUSE with lysosomes, so cholesterol can be hydrolysed from esters and released into the cell
What mutations in the LDL receptor lead to hypercholesterolaemia?
- RECEPTOR DEFICIENCY: mutation that prevents expression of LDL receptor
- NON-FUNCTIONAL RECEPTOR: mutation of the binding site so that it can’t take up LDL (normal coated pits and internalisation)
- RECEPTOR BINDING NORMAL: no internalisation due to deletion in the C-terminal of the receptor which makes the interaction with the coated pits. In these patients there are LDL receptors over the whole cell surface instead of concentrated over 2%
Describe the uptake of ferric (Fe3+) ions as an example of receptor-mediated endocytosis
- 2 molecules of ferric bind to APOTRANSFERRIN, forming TRANSFERRIN in the circulation
- Transferrin binds to the TRANSFERRIN RECEPTOR at neutral pH and is internalised (similar mechanism to LDL)
- Reach acidic endosome, Fe3+ ions released from transferrin, but apoptransferrin remains associated with the transferrin receptor
- Complex sorted in the CURL for recycling back to the PM, where @ pH 7.4 apotransferring dissociates from the transferrin receptor
Endocytosis and insulin
- Insulin receptor only congregates over coated pits when an agonist is bound
- Insulin binds-conformational change in receptor-insulin receptor recognised by coated pit
- In endosome, insulin stays bound to receptor and complex is targeted to lysosomes for degradation
- Allows for decreased number of insulin receptors on cell surface so desensitises cells to a continued presence of high circulating insulin levels
Describe the cellular process by which cells targeted by insulin become desensitised to a prolonged insulin challenge
- Insulin receptors recognised and sequestered by adaptor proteins in coated pits
- Spontaneous invagination of coated pits to form coated vesicles
- ATP-dependent uncoating of vesicles
- Fusion of uncoated vesicles with endosomes
- Endosomal vesicles fuse with lysosome, delivering insulin-receptor complex for degradation
- Decreased no. insulin receptors on cell surface so continued high [insulin] desensitises cells to insulin
What is transcytosis? Give an example
Some ligands that remain bound to their receptor may be transported across the cell
E.g. transfer of immunoglobulin A (IgA) from the circulation to the bile in the liver. Receptor is cleaved, release of Ig with a bound “secretory component” derived from the receptor
How can membrane-enveloped viruses and toxins use RME to gain entry to a cell?
Enter cells via clathrin coated pits (RME), unfold hydrophobic domains in membrane fusion proteins in response to acidic pH of endosome
Insert membrane fusion proteins into endosome membrane and release viral RNA into cytoplasm
Uses host machinery to replicate RNA and capsid proteins to bud new viruses at the cell membrane
Toxins such as cholera and diptheria bind to GM1 ganglioside
What are the different outcomes for the ligand and receptor in RME?
Receptor recycled, ligand degraded: e.g. LDL. For metabolite uptake
Receptor and ligand recycled: e.g. transferrin. For metabolite uptake
Receptor and ligand degraded: e.g. insulin, EDF, immune complexes. For receptor downregulation/removing foreign antigens from circulation
Receptor and ligand transported: e.g. maternal IgA, secretory IgA. Transfer large molecules across cell such as maternal immunity to foetus via placenta
