Cell signalling and pharmacology

What is cell signalling?

* The ability of a cell to: 
* Detect or receive information 
* Process the information 
* Respond to generate events fundamental to living! 
* Also allows for:  
* Specialist functions 
* Co-ordination with other cells 

Why are signalling molecules and their receptors the main targets for therapeutic drugs?

Because abnormal cell signalling underpins most disease processes

How do pathogenic organisms use cell signalling?

Pathogenic organisms and viruses modify the host’s signalling pathways to use to their own advantage e.g. cholera, peptic ulcers, TB, dysentery  

How do prairie voles use cell signalling?

* The breeding behaviour of prairie voles is controlled by the action of related peptides oxytocin (females) and vasopressin (males) 
* Act through their specific receptors found in regions of the brain concerned with mating 

How do cells communicate with each other?

Via extracellular signalling molecules (first messengers) 

What are the features of intracellular signalling?

* Signalling cell produces a signalling molecule (ligand) which binds to a specific receptor found on the surface of the target cell. 
* Can travel short or long distances 
* Allows for control and specialised functions 

What are 2 classes of extracellular signalling molecules?

* Large/hydrophilic - bind to cell surface receptors 
* Small/hydrophobic - enter cell and bind to intracellular receptors. 
* Majority of signalling molecules are hydrophilic so most cell signalling is via surface receptors. 

What is paracrine signalling?

* Released signal affects cells in close proximity 
* Act as local mediators by staying close to point of origin 
* Limit travel availability due to biochemistry 
* E.g. some growth factors, histamine, nitric oxide 

What is autocrine signalling?

* Sender and target cell are the same. 
* E.g., molecules regulating development, some growth factors 

What is endocrine signalling?

* Usually the signal acts on distant cells (but can act on nearby cells) 
* Hormones 
* E.g., insulin, glucagon, testosterone, oestrogen, adrenaline (epinephrine) 

What is synaptic signalling?

* Axon of neurone transmits an electrical signal over long distances 
* At axon terminal, electrical signal causes the release of neurotransmitter messenger molecules into the synapse, e.g., acetylcholine, GABA 
* Neurotransmitter travels short distance only to specific target cell. 

What is juxtacrine signalling?

* The signalling cell is in direct contact with target cell 
* E.g., integrins 
* Signalling molecule on surface of target cell or via gap junction 
* Only very small molecules 

What is signal transduction?

* One type of signal is changed to a different type of signal 
* Linked with cell surface receptors – not intracellular receptors 
* Begins when receptors on the cell surface recieves the signal and convert or relay the "message" to a molecule inside the cell. 
* Signal is subsequently transduced along many intracellular molecules (second messengers) - intracellular signalling. 

What happens in a fast response cell?

* Altered protein function
* Altered cytoplasmic machinery
* Altered cell behaviour
* Secs/mins

What happens in a slow response cell?

* Altered DNA/RNA
* Altered protein synthesis
* Altered cytoplasmic machinery
* Altered cell behaviour
* Mins to hours

How can the same signal molecule induce different responses in different target cells?

* Variants or isoforms of the same receptor 
* Similar receptors use different intracellular signal transduction pathways. 

How do cell surface receptors relay extracellular signals?

* Via intracellular signalling molecules or pathways


* Acts like molecular relay as ‘message’ is transduced from molecule to molecule  
* Final molecule in sequence interacts/activates an effector protein 
* Cellular response 

What does a metabolic enzyme do?

Alter metabolism

What does a cytoskeletal protein do?

Altered cell shape or movement

What does a transcription regulator do?

Alter gene expression

How are messages relayed?

* Information is transferred in the signal transduction pathway __mainly__ by changes in the state of proteins (results in a change of function of the protein).  
* A change in protein in the pathway is subsequently detected by the next molecule in the sequence,  which itself in turn becomes altered…..and so on and so on…...  

What is a common alteration in shape induced by?

* Molecules simply binding with each other 
* Addition/removal  of a phosphate to the molecule 
* Molecule binds to a phosphate on another molecule 

What is the point of a signal transduction cascade?

* Amplify the original signal
* Integrate and distribute signals coming from other signal transduction pathways

What do scaffold proteins do?

Allow for some signalling components to be activated more efficiently

What type of molecules are signal transduction pathways made up of?

* Proteins
* Lipids (particularly membrane lipids)
* Small chemical mediators
* Ions 
* Gases

How do intracellular proteins act as molecular switches and what are the 2 classes?

* Toggle between inactive and active states 
* Comprised of two broad classes which are activated/deactivated by: 
* Binding to guanine nucleotides – GTP and GDP 
* Phosphorylation  

How does GTP effect cell activity?

* G proteins do this
* Regulated by binding to guanine nucleotides 
* Inactive when bound to GDP 
* Active when bound to GTP 
* Exchange GDP for GTP to activate  

What does intrinsic GTPase activity do?

* Removes phosphate from GTP, returning to GDP 
* Hydrolysis of GTP to GDP switches off protein 

What are the 2 forms of GTP?

* Within trimeric complex (used by GPCR's) 
* As a single monomeric protein 

What are the features of monomeric G-Proteins?

* Superfamily (150 members), activation/inactivation required 


* GEFs aid GDP/GTP exchange – binds to inactive G protein, changes its shape 
* GAPs aid GTP hydrolysis – loss of phosphate group 

What are key GTPase members?

Ras (cell division and growth), Rab (membrane transport and vesicular transport), Rac and Rho (cytoskeleton organisation and migration) 

How does phosphorylation alter cell activity?

* Undertaken by protein kinases 
* Add phosphate from ATP to specific amino acids on target protein 
* Tyrosine kinases (TKs) - add phosphate to tyrosine 
* Serine/threonine kinases (STKs) - add phosphate to serine or threonine 
* Covalent modification reversed by protein phosphatases 
* Protein kinases are also switch proteins themselves i.e. activated / deactivated by phosphorylation 
* Once activated, can  in turn phosphorylate and activate the next protein kinase in the sequence 

What do lipid kinases and lipid phosphatase do?

Modify lipids in the same manner as protein kinases

How is production of cAMP induced?

* cAMP is produced from ATP by the enzyme adenylyl cyclase (found in plasma membrane, 12 transmembrane regions), ATP is cyclized. 
* Adenylyl cyclase:- consists of two transmembrane domains, joined by a catalytic intracellular domain 
* cAMP is degraded from a cyclic nucleotide to a 5’ monophosphate (AMP) by a cAMP phosphodiesterase   

What are responses to cAMP regulated by?

Most responses to cAMP are mediated via cAMP-dependent protein kinase A i.e. Protein kinase A (PKA) 

What does inactive PKA consist of and what does cAMP do to this?

* Two regulatory (R) subunits and two catalytic (C)  kinase subunits  
* cAMP binds to the regulatory subunits causing the molecule to dissociate 
* Two resulting monomeric kinase units are active and can bind and phosphorylate target proteins 

What is the intracellular signalling pathway sequence of adenylyl cyclase?

Adenylyl cyclase → cAMP → PKA

What is the intracellular signalling pathway sequence of PI3-kinase?

PI3-Kinase → Pip2 → Pip3 → PDK1 → PKB (Akt)

What is the intracellular signalling pathway sequence of phospholipase c?

Phospholipase C → PIP2 → DAG → IP3 → Ca2+ → Calmodulin

What is PIP2?

* Phosphatidylinositol 4,5-bisphosphate 
* Cell membrane phospholipid 
* Found in inner leaflet of lipid bilayer 
* Phosphoinositide comprised of: 
* Hydrophobic diacylglycerol (DAG) lipid tail 
* Hydrophilic inositol sugar as head group, inositol triphosphate (IP3) 

How is PIP2 phosphorylated by PI3-Kinase?

* Ligand binds to receptor causes PIP2 to become PIP3 this is don’t by PI3 kinase which phosphorylates PIP2 into PIP3 by adding another phosphate into the inositol sugar


* PDK1 and PKB have a PH domain that bind to molecules of PK3
* This allows PDK1 to activate and phosphorylate PKB
* PKB is now active
* Pathway controlled by PTEN which dephosphorylates PIP3 back to PIP2 which shuts down the signalling pathway.

How is PIP 2 broken down in the lipid bilayer?

* Activation of phospholipase C (PLC) 
* Cleaves PIP2 into DAG and IP3 
* DAG activates Protein kinase C (PKC) (important in growth) 
* IP3 triggers release of Ca2+(also required for PKC activation) 

How does calcium act as a signalling molecule?

* Variation of Ca2+ concentration in cytosol i.e. \[Ca2+ i\], constitutes the signal 

How are Ca2+ levels increased?

* Influx of Ca2+ from outside cell via Ca2+ channel proteins in the plasma membrane 
* Release of Ca2+ from intracellular stores i.e. endoplasmic reticulum (ER), sarcoplasmic reticulum (SR) and mitochondria (caused mainly via IP3) 

How are Ca2+ levels reduced?

* The plasma membrane (pump out Ca2+)  
*  ER, SR and mitochondrial membrane (sequester Ca2+ back into organelle) 

What is the structure and function of calmodulin?

* Has four Ca2+ binding sites 
* Activated when \[Ca2+ i \] increases above 500nM 
* Ca2+ -bound calmodulin binds and activity of its target proteins 

How is an extracellular signalling molecule eliminated?

Enzymatic degradation

How are signal transduction pathways deactivated?

Dephosphorylation by phosphatase

How can the activated receptor removed from the cell membrane?

Endocytosis

How can receptor and signal molecules be internalised?

* Either the receptor and signalling molecule are separated and the receptor is recycled to surface and ligand destroyed. Or the receptor and ligand are both destroyed 

What is the role of receptors in cell signalling?

* A signalling molecule (or ligand) binds to its specific receptor : Ensures specificity of response i.e. ‘lock and key’


* The cell can also influence response by: 
* Regulating the number of receptors 
* Synthesising different isoforms of the receptor 

What is an agonist?

* Agonist: A molecule that binds and activates a receptor, inducing signalling and a biological response e.g. native ligands and drugs 
* Full agonist : Full activation  
* Partial agonist : Partial activation 

What is an antagonist?

A molecule that binds to a receptor, but does NOT induce signalling and a biological response e.g. native ligands and drugs 

What are the features of ion channel like receptors?

* Act as gates 
* Ligand binding causes receptor to change shape and open gate 
*  Allows ion flow passively through channel 
*  Can be pharmacologically modified by: 
* Channel blockers (physically block channel) 
* Channel modulators (bind to channel and enhance or inhibit opening 

What is the structure of a GPCR?

* Extracellular ligand binding region 
* Seven alpha helices that span the membrane 
* Intracellular portion interacts with a trimeric G protein 

How do GPCRs use G-proteins?

* GPCR utilise trimeric G proteins to relay the signal 
* Trimeric G protein: 
* Three subunits i.e. a, b and y, b and y have lipid tails bind to the plasma membrane 
* Ga unit binds GDP/GTP and has the GTPase activity 

How is a signalled relayed via a GPCR?

* Binding of ligand alters the conformation of the receptor 
* Ga unit binds to receptor 
* Binding of Ga protein allows release of GDP and its exchange for GTP 
* a subunit is active and dissociates from the b and g units 
* Both active Ga subunit and bg complex can now interact with effector molecules to relay the signal – focus on Ga subunit 

Which effector is Gas coupled with and what does it do to it?

Stimulates adenylyl cyclase, increases cAMP.

Which effector is Gai coupled with and what does it do to it?

Inhibits adenylyl cyclase, decreases cAMP

Which effector is Gaq coupled with and what does it do to it?

Stimulates phospholipase C, increases DAG and IP3

How does GPCR signalling work in the adrenaline mediated breakdown of glycogen?

* Adrenaline binds to its adrenergic receptor
* Coupled to Gas, activates adenylyl cyclase
* ATP made into cAMP
* cAMP transducer the signal to PKA
* PKA relays the signal by phosphorylating target kinases, can also enter the nucleus and effect transcription factors involved in mediating the longer term coordinated events in this biological response

How does disregulated GPCR signalling work in the cholera toxin?

* Toxin enters the cell in the intestinal lumen
* Binds to Gas class
* Fixes the gene protein as if it was bound to GDP
* Causes excessive cAMP levels
* Induces signalling which prevents transporters and receptors for sodium retention in epithelial cells.

How does oxytocin utilise the Gaq class of G protein?

Activated PLC to induce Ca2+ mediated events to influence behavioural changes in the brain?

What are the features of RTKs?

* Extracellular domain which binds the ligand (mainly growth factors) 
* Transmembrane domain 
* Intracellular or cytoplasmic domain which contains the tyrosine kinase site 
* A tyrosine kinase adds phosphate groups from ATP to only tyrosine residues on target proteins 

How are RTKs activated?

* Requires dimerization of two receptor monomers 
* Activates the tyrosine kinase in each receptor 
* Kinase phosphorylates tyrosines on opposite receptor tail i.e. transphosphorylation 
* Recruitment/binding of adaptor and/or effector signaling molecules directly to the phosphorylated tyrosines to initiate signalling 
* RTK’s commonly utilise the monomeric G protein Ras to relay or transduce the signal: 
* Activated receptor either directly or indirectly (via an adaptor protein) binds and activates the GEF for Ras, thereby activating this key signalling molecule 

How is uptake of glucose in muscle and fat cells regulated by activation of the insulin receptor?

* Glucose transporters i.e. GLUT-4 are stored in walls of cytoplasmic vesicles
* Insulin induced IRS-1/PI-3 kinase/PKB signalling triggers vesicle translocation to the plasma membrane
*  Vesicle fuse with membrane where they take up glucose and pass it into the cell
* Insulin induced signalling has multiple cellular effects which also include decreasing glycogen metabolism and promoting glycolysis

How do cytokine receptors recruit Janus kinase (JAK) to help initiate signal transduction?

* Cytokine receptors lack intrinsic kinase activity


* Recruit soluble tyrosine kinase i.e. JAK
* Ligand binding e.g. prolactin causes: Receptor dimerisation and JAK recruitment and activation, JAKs phosphorylate each other and the receptor and Recruitment of STAT transcription factor  to phosphorylated tyrosine residues on the receptor

What are the features of nuclear receptors?

* In the absence of ligand, they can be found in the nucleus or the cytoplasm 
* After binding ligand, cytoplasmic receptors will translocate to the nucleus
* Bind lipid soluble molecules such as steroid hormones or small molecules that can pass through the lipid bilayer
* Exert effects by affecting gene transcription. i.e. activated receptor is a ligand activated transcription factor

What is the structure of a nuclear receptor?

Receptors contain: 

* A  ligand binding domain
* A DNA binding region- Bind to ‘response elements’ in the promoter region of target genes
* N terminal variable region which can be modified by other molecules to enhance transcriptional abilities

How is gene transcription mediated by cortisol?

* Cortisol is produced in the adrenal glands in response to stress
* Passes through lipid bilayer and binds to its  cytoplasmic nuclear receptor
* Ligand-bound receptor translocates to nucleus
* Binds to regulatory response elements in target gene to drive gene transcription

Where are nuclear receptors for some other steroid hormones, thyroid hormone, fatty acids  and retinoids found?

In the nucleus bound to the response element in the gene even in the absence of ligand – bound to transcriptional repressor which is released when ligand binds

What are the features of long distance (endocrine) signals in plants?

Slow via vascular system i.e. xylem and phloem

What are the features of short distance (autocrine) signals in plants?

Most common

How does transport work in the vascular systems of plants?

* Via active transport via transport proteins 
* Passive - freely diffusible
* Via plasmodesmata

How does juxtacrine signalling work via the plasmodesmata?

* Comprised of cytoplasmic channels linking adjacent cells
* 30-60 nm in diameter (*cf*. gap junctions with 1.5nm diameter)


* Allows passage of both small molecules and macromolecules
* Aids in electrical signalling between plant cells
* Allows relatively rapid long-distance communication e.g. in response to stress/damage


* Food capture in carnivorous plants

How does electrical signalling work in Venus flytraps?

* Stimulation of sensory trigger hairs activates mechano-sensitive ion channels
* Lead to depolarization  of membrane and generation of an action potential 
* Changes turgor pressure in hinge cells, causing closure of leaf lobes

How is plant signal transduction similar to animal?

* Membrane enzyme-linked receptors and intracellular receptors, with and without kinase activity (but negligible existence of GPCRs and G proteins)
* Use mainly serine/threonine kinases \*
* Intracellular signalling molecules e.g. lipid signalling molecules, Ca2+

What are auxins?

* Natural auxin in plants is indoleacetic acid (IAA)
* Produced in the seed embryo, meristems of apical buds and young leaves
* Functions include stem elongation, root growth, branching,  fruit development and apical dominance
* Actively transported from one cell to the next –long distance and local acting

How do auxins effect gene transcription?

* Use mechanism of inactivating repressor proteins
* Auxin binds to its nuclear receptors which are ubiquitin ligases.


* Binding promotes ubiquitinylation and degradation of repressor protein
* Suppression of gene transcription is relieved

What is ethylene?

* Functions include fruit ripening and  leaf abscission
* Can pass through cell walls or diffuse through air 

What is the ethylene response?

* Ethylene receptors are found in the membrane of the endoplasmic reticulum and Golgi
* In the absence of ethylene, the ethylene receptor is activating a kinase –promoting the destruction of the transcription regulator
* Deactivation of the ethylene receptor allows the  transcription of ethylene sensitive genes

How does light link to plants?

* Plants can detect the direction, intensity and wavelength (colour) of light
* Light regulates key biological events in plant growth and development

What are blue light photoreceptors?

* (3 types)
* Contain either cryptochromes, phototropin or zeaxanthin as photopigments
* Cell surface receptor

What are red light phytochromes (Red light)?

Intracellular receptor

What is the structure of a phytochrome?

* A light detecting pigment or chromophore
* A region that has kinase activity

How do phytochromes modulate gene transcription?

* Translocating to the nucleus
* Either directly binding to and activating a transcription factor
* Or indirectly by phosphorylating transcription factors 

What is apoptosis?

A process seen in multicellular organisms, by which specific cells are killed and removed for the benefit of the organism 

Why is apoptosis essential for animal development?

* Removes redundant structures 
* Embryogenesis – e.g., sculpting of limbs 
* Maintains homeostasis 
* Regulation of cell numbers 

What does too much cell death lead to?

Degenerative diseases

What does too little cell death lead to?

Diseases of over-proliferation

What type of cells does apoptosis eliminate?

* DNA damage 
* When repair mechanisms cannot cope with damage 
* Accumulation of misfolded proteins 
* Causes ER stress and cell death 
* Linked with neurodegenerative disorders when there is a massive number of misfolded proteins 
* Cells infected by certain viral agents 
* Limits spread of infection 

What are the morphological features of apoptosis?

* Ultrastructural changes
* Cell shrinkage 
* Chromatin condensation 
* Fragmentation of intracellular contents (organelles broken down proteolytically) and membrane blebbing (perfusions) 
* Formation of apoptotic bodies (ABs) (Membrane -bound \* portions of cytoplasm and organelles)
* Phagocytic ingestion of Abs and degradation 

What is accidental cell death?

Necrosis

What are cascades?

* Apoptosis is mediated by a family of suicide proteases called caspases  
* (**C**ysteinyl-**asp**artate-specific prote**ases**) 

What are the features of caspases?

* Cysteine at active site 
* Cleaves target proteins at specific aspartic acids 
* Synthesised as inactive procaspase 
* Activated by proteolytic cleavage at own aspartic residues 
* A proteolytic cascade of caspase activity mediates apoptosis 

What do initiator caspases do?

* Undergo autoclevage
* Activates other caspases

What do effector caspases do?

* Activate other effector cascades after cleavage by initiator caspase
* Cleave cellular proteins

What are the nuclear effects seems during apoptosis?

* Hallmark cleavage of chromosomal DNA 
* Caspase cleaves a protein that normally blocks endonuclease action: 
* DNA cleavage by endonucleases cuts DNA into internucleosomal units of 180-200 base pairs 
* Apoptotic cells show DNA ‘laddering’  on electrophoresis  

What are phosphotidylserine (engulf me signals)?

* Phagocytes i.e. macrophages and neutrophils, recognize “engulf-me” or ‘cell corpse’ signals on cell surface of apoptotic bodies.  
* PS normally found in inner leaflet of plasma membrane 
* In apoptosis, some PS molecules move to outer leaflet  
* Action of caspases activate scramblase (Xkr8) which mediates PS flipping so PS head stick out of the membrane and can be recognised by phagocytes 

What are the features of the intrinsic pathway of apoptosis?

* Main way apoptosis occurs in cells  
* Lack of trophic factor-induced signalling i.e. growth factor / survival factor withdrawal 
* DNA damage (by radiation or toxins)  
* Protein misfolding (ER stress) 
* Whether the intrinsic pathway is activated depends upon the release of cytochrome c from the mitochondria 
* Regulated by a balance between molecules that promote apoptosis and those which inhibit apoptosis.