cell signalling and pharmacology

Question 1

Explain cell signalling

Answer 1

ability for a cell to:
Detect or receive information
Process the information
Respond to generate events fundamental to living!

allows for:
Specialist functions
Co-ordination with other cells

Abnormal cell signalling underpins MOST disease processes, therefore signalling molecules and their receptors are the MAIN targets for therapeutic drugs

Question 2

general principle of cell signalling:
intercellular and intracellular signalling

Answer 2

Cells communicate with each other via extracellular signalling molecules (also known as ‘first messengers’)

INTERCELLULAR SIGNALLING:
*Signalling cell produces a signalling molecule (LIGAND)
➢Can travel short or long distances (or no distance at all!)
*Signalling molecule is detected by a receptor on (or in) the target cell
*Receptor is specific for that signalling molecule
➢Allows for control and specialized functions

INTRACELLULAR SIGNALLING
Two broad classes of extracellular signalling molecules exist:
*Large and/or hydrophilic (water soluble)
➢Bind to cell surface receptors
*Small and/or hydrophobic
➢Enter cell and bind to intracellular receptors

Note:
The majority of signalling molecules are hydrophilic so most cell signalling is via cell surface receptors

Question 3

Describe the methods/mechanisms of intercellular communication

Answer 3

Cells communicate through extracellular messenger molecules- can travel short or long distances

Paracrine:
* Released signal affects cells in close proximity
–‘ Local mediators’
Limited travel ability

Autocrine:
* Sender and target cell are the same
Examples: molecules regulating development; some growth factors

Endocrine:
* Usually, the signal acts on distant cells (but can act on nearby cells)
* Hormones
Examples: insulin, glucagon, testosterone, oestrogen, adrenaline (epinephrine)

Synaptic:
* 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

Juxtacrine signalling (or contact dependent): the signalling cell is in direct contact with target cell

Question 4

general principle of cell signalling:
explain signal transduction

Answer 4

*Linked with cell surface receptors (and not intracellular receptors)
➢Begins when receptors on the cell surface receive the signal and convert or relay the ‘message’ to a molecule inside the cell
Signal is subsequently transduced along many intracellular molecules (also known collectively as ‘second messengers’) i.e. INTRACELLULAR SIGNALLING

Signal transduction : process whereby one type of signal is converted into another type

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

Question 5

general principle of cell signalling:
responces can be fast or slow

Answer 5

fast - altering protein function
slow - altering the synthesis of proteins(mins-hours)

both result in altered cytoplasmic machinery, therefore altered cell behaviour

Question 6

general principle of cell signalling:
The same signal molecule can induce different responses in different target cells via:

Answer 6

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

Question 7

signal transduction: how is the message relayed

Answer 7

Information is transferred in the signal transduction pathway mainly by changes in the state of proteins:
* 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,
A common alteration is shape change, induced by:
* Molecules simply binding with each other
* Addition/removal of a phosphate to the molecule
* Molecule binds to a phosphate on another molecule

Question 8

Explain the role of signal transduction cascades

Answer 8

Amplify the original signal

Integrate and distribute signals coming from other signal transduction pathways

Note: scaffold proteins allow for some signalling components to be activated more efficiently

Question 9

Discuss the varied types of molecules that comprise intracellular signalling molecules

Answer 9

Signal transduction pathways are comprised of many different types of molecules
Proteins: includes enzymes
Lipids: e.g. phospholipids, ceramides, diacylgycerol (DAG)
Small chemical mediators: e.g cAMP, cGMP, inositol triphosphate (IP3)
Ions: e.g. Ca2+ , Zn2+
Gases: e.g. nitric oxide

Many intracellular proteins that act as signal transduction molecules act as molecular switches
Toggle between inactive and active states
Comprised of two broad classes which are activated/deactivated by:
Binding to guanine nucleotides – GTP and GDP
Phosphorylation

Question 10

binding to GTP - monomeric G proteins

Answer 10

Known as G proteins - regulated by binding to guanine nucleotides
* Inactive when bound to GDP
* Active when bound to GTP
*Intrinsic GTPase activity
Hydrolysis of GTP to GDP switches off protein
*Exist in two forms:
Within trimeric complex (used by G-protein coupled receptors - will cover in detail later)
As a single monomeric protein

*Superfamily ~150 members
*Activation / inactivation requires:
GEFs to aid in GDP/GTP exchange
GAPs to aid in GTP hydrolysis
Key members include:
*Ras – cell division & growth
*Rab – membrane transport and vesicular transport
Rac & Rho - cytoskeleton organization, migration

Question 11

Explain the role of signal molecules as molecular switches, with focus on the ‘switch’ in activity being induced by phosphorylation or binding to GTP
—-phosphorylation

Answer 11

Undertaken by protein kinases:
* Add phosphate from ATP to specific amino acids on target protein
Tyrosine kinases (TKs)
Serine/threonine kinases (STKs)
*Covalent modification reversed by protein phosphatases

Protein kinases are also switch proteins themselves i.e. activated / deactivated by phosphorylation
Often organised in sequence in a signal transduction pathway
Once activated, can in turn phosphorylate and activate the next protein kinase in the sequence

Question 12

Overview some key intracellular signalling pathway sequences

Answer 12

adenylyl cyclase -> cAMP ->PKA(protein kinase) -> multiple specific molecules required for specific response

P13-kinase -> PIP2 to PIP3 -> PDK1 -> PKB(Akt) -> multiple specific molecules required for specific response

phospholipase C -> PIP2 to DAG&IP3 ->Ca2+ -> Calmodulin -> multiple specific molecules required for specific response

Question 13

Explain the role of cAMP in signal transduction cascades

Answer 13

cAMP is produced from ATP by the enzyme adenylyl cyclase
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

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

Question 14

Explain the role of the phospholipid PIP2 in mediating different major signalling pathways

Answer 14

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
oInositol triphosphate (IP3)

Phosphorylation of PIP2 in the lipid bilayer by PI3-Kinase( lipid kinase)

PI3-K: phosphatidylinositol 3-kinase ; PDK1: phosphoinositol-dependent kinase
PTEN: phosphatase and tensin homologue on chromosome 10
PH –pleckstrin homology
Note: The key regulatory molecule in this pathway is PTEN which dephosphorylates PIP3 back to PIP2 which shuts down the signalling via PKB

Breakdown of PIP2 in the lipid bilayer
Activation of a receptor causes:
➢ 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)

Question 15

Describe how Ca2+ acts as a signalling molecule and explain the role of calmodulin in transducing Ca2+ mediated signalling.

Answer 15

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

• [Ca2+ i] levels increase by:
  ➢ 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)
• [Ca2+ i] levels controlled/reduced via ATPase pumps in:
  – the plasma membrane (pump out Ca2+)
  – ER, SR and mitochondrial membrane (sequester Ca2+ back into organelle)

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

Question 16

Summarise the general mechanisms of signal termination

Answer 16

• Eliminate extracellular signalling molecule
  ➢ Enzymatic degradation
• Deactivate signal transduction proteins
  ➢ Dephosphorylation by phosphatases

Remove activated receptor from cell membrane by endocytosis
* Receptor and signal molecule (ligand) are 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

Question 17

Overview the role of receptors in cell signalling and highlight the different classes of cell surface receptor.

Answer 17

How does a signalling molecule exert its effects?
*A signalling molecule (or ligand) binds to its specific receptor :
➢Ensures specificity of response i.e. ‘lock and key’ – see figure
*The cell can also influence response by:
➢ Regulating the number of receptors
➢ Synthesising different isoforms of the receptor

Question 18

pharmodynamics

Answer 18

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

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

Question 19

types of cell surface receptor - ion channel-linked receptors

Answer 19

• Ion channel-linked receptor (ionotropic receptors)
  G protein coupled receptor (metabotropic receptors)
• Enzyme-linked receptor
  ➢ Intrinsic enzyme activity
  ➢ Recruit enzyme from cytoplasm

Ion channel-linked 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)

Question 20

Type of cell surface receptor: G Protein Coupled Receptor(GCPR)

Answer 20

Structure of a GPCR:
*Largest family of cell surface receptors in biology
*Bind an enormous range of extracellular signalling molecules
*~ 800 GPCRs in humans
➢At least twice as many in other mammals
*Mediate a wide array of physiological processes (including odorant detection)
*All GPCRs have a similar structure:
➢Extracellular ligand binding region
➢Seven alpha helices that span the membrane
➢Intracellular portion interacts with a trimeric G protein

G proteins exist in 2 forms:
➢Within trimeric complex
➢As a single monomeric protein

GPCR utilise trimeric G proteins to relay the signal
Trimeric G protein:
● Three subunits i.e. a, b and g
● Ga unit binds GDP/GTP and has the GTPase activity

Question 21

signal relaying via GCPR and switching off G protein

Answer 21

Signal relay via the 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

Relaying the signal
*GTP bound activated Ga unit binds to an effector molecule altering its activity

Switching off the G protein:
*Ga subunit hydrolyses the GTP to GDP – occurs in seconds – can use RGS protein to aid hydrolysis – (not shown in diagram)

*Ga dissociates from effector molecule
a subunit having returned to its original GDP inactive conformation, can reassemble with the bg complex to form inactive trimeric G protein

Question 22

examples of classes of trimeric G proteins and their effector molecule

Answer 22

Ga exists in numerous classes:
➢ Determined by which effector molecule the Ga subunit couples with and the resulting effect

Example of a GPCR signalling pathway i.e. adrenaline mediated breakdown of glycogen:
*Rapid response (seconds)

Not shown but remember:
*Signalling pathway amplifies original signal
PKA can also enter the nucleus and effect transcription factors involved in mediating the longer term coordinated events in this biological response.

Example of dysregulated G protein signalling
*Cholera toxin binds to Gas and fixes it in GTP bound conformation
*Over stimulation of adenylyl cyclase and cAMP production
*Downstream signalling effects transporters involved in ion transport leading to water loss
Figure shows neutralization of cholera toxin with nanoparticle decoys for treatment of cholera

Question 23

Enzyme linked receptors:
Intrinsic enzyme activity
Recruit enzyme from cytoplasm

Answer 23

The most well studied receptors with intrinsic enzyme activity are the receptor tyrosine kinases (RTKs)
RTKs consist of:
* 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

Activation of a RTK:
*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

Activation of a RTK:
*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

Question 24

Regulation of glucose uptake in muscle and fat cells via activation of the insulin receptor and cytokine receptors(mechanisms)

Answer 24

• 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

Note: insulin induced signalling has multiple cellular effects which also include decreasing glycogen metabolism and promoting glycolysis

Cytokine receptors recruit Janus kinase (JAK) to help initiate signal transduction

Mechanism of action:
*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
➢Recruitment of STAT transcription factor to phosphorylated tyrosine residues on the receptor

Question 25

Discuss intracellular receptors

Answer 25

Intracellular receptors – also known as 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

Nuclear receptors are ligand-activated transcription factors:
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

Example: Mediation of gene transcription 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

Question 26

cell communication in plants

Answer 26

Similar to animal cells, extracellular communication in plants is designated by the distance travelled by the signalling molecule to reach its target cell
Long distance(endocrine)-slow via vascular system ie. Xylem+phloem
Short distance(paracrine)-most common
No distance(autocrine)-same cell
Transport into vascular systems, into cells and from cell to cell:
*Via active transport via transport proteins
*Passive - freely diffusible
*Via plasmodesmata

Juxtacrine via 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

overview of signal transduction in plants:
Respond to environmental or physiological signals
*Possess similar transduction components to animals:
➢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+
*Signal transduction can illicit rapid responses but mainly relies on altering gene expression (slow response):
➢By positive gene activation
➢Mainly involves inactivation of transcriptional repressor proteins

Question 27

electrical signalling in plants

Answer 27

*Allows relatively rapid long-distance communication e.g. in response to stress/damage
*Food capture in carnivorous plants

Electrical signalling in Venus flytrap:
*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

Question 28

Describe some of the key hormones and their signalling mechanisms used by plants to affect biological functions

auxins

Answer 28

*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

Auxin utilises the mechanism of inactivating repressor proteins to effect gene transcription

*Auxin binds to its nuclear receptors which are ubiquitin ligases.
*Binding promotes ubiquitinylation and degradation of repressor protein
*Suppression of gene transcription is relieved

Question 29

Describe some of the key hormones and their signalling mechanisms used by plants to affect biological functions
ethylene

Answer 29

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

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

Question 30

Describe some of the key hormones and their signalling mechanisms used by plants to affect biological functions

plant photoreceptors and phototropism

Answer 30

*Plants can detect the direction, intensity and wavelength (colour) of light
*Light regulates key biological events in plant growth and development
*Plants contain two major classes of photoreceptors:
➢Blue-light receptors (3 types)
Contain either cryptochromes, phototropin or zeaxanthin as photopigments
Cell surface receptor
➢Phytochromes (red light)
Intracellular receptor

Phytochromes:
Exist as two subunits and each has:
*A light detecting pigment or chromophore
*A region that has kinase activity
Modulate gene transcription by:
*Translocating to the nucleus
*Either directly binding to and activating a transcription factor
*Or indirectly by phosphorylating transcription factors.

Question 31

 Demonstrate the similarities and differences between plant and animal signal transduction
similar: e.g. similar signalling principles using receptors and intracellular signal transduction molecules
differences: e.g. use of light as a signalling molecule by plants and reliance on alleviating transcriptional repression

Question 32

Describe the role of apoptosis or controlled cell suicide in biological processes

Answer 32

Apoptosis - (Gr. “falling” as in leaves) a process seen in multicellular organisms, by which specific cells are killed and removed for the benefit of the organism

Apoptosis is essential for animal development
Eg. Removal of redundant structures
Embryogenesis eg. Sculpting of limbs

Apoptosis maintains homeostasis in organisms via regulation of cell numbers-
Too much- degenerative diseases eg. Alzheimer’s
Too little- diseases of over-proliferation eg. Solid tumours, leukaemia

Apoptosis eliminates specific cells that are damaged beyond repair due to:
* DNA damage
➢ When repair mechanisms cannot cope with damage
* Accumulation of misfolded proteins
➢ Causes endoplasmic reticulum stress and cell death
➢ Linked with neurodegenerative disorders
* Cells infected by certain viral agents
➢ Limits spread of infection

Question 33

Describe the morphological features of apoptotic cells

Answer 33

• Cell shrinkage
• Chromatin condensation
• Fragmentation of intracellular contents and membrane blebbing
• Formation of apoptotic bodies (ABs)
  ➢ Membrane -bound - portions of cytoplasm and organelles
• Phagocytic ingestion of Abs and degradation

Question 34

Detail the role of caspases in apoptosis and explain how their action executes the demolition of the cellular contents

Answer 34

In animal cells, apoptosis is mediated by a family of suicide proteases called caspases
(Cysteinyl-aspartate-specific proteases)
Caspase :
* Cysteine at active site
* Cleaves target proteins at specific aspartic acids
* Synthesised as inactive procaspase
* Activated by proteolytic cleavage at own aspartic residues.

nuclear effects seen 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 (Fig. right : agarose gel electrophoresis of (A) viable cells and (B) apoptotic cells.

Key ‘engulf me’ signal is phosphotidylserine(PS):
* 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

Question 35

Describe the molecular basis of apoptosis
intrinsic pathway

Answer 35

Intrinsic pathway(mitochondrial):
*Lack of trophic factor-induced signalling i.e. growth factor / survival factor withdrawal
➢N.B. Cells are prevented from dying by ‘trophic’ factors’
*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.

For example:
*BCL-2 inhibits apoptosis by preventing release of cytochrome c from the mitochondria by blocking action of BAX and BAK
*BAX / BAK promote apoptosis by forming channels in the outer mitochondrial membrane to allow cytochrome c release
*BCL-2 > BAX : apoptosis prevented
*BAX > BCL-2 : apoptosis occurs

Survival factor or growth factor signalling suppresses apoptosis by:
*Increasing the transcription and translation of anti-apoptotic molecules
*Signal transduction kinases (e.g. Protein Kinase B) which are activated by stimulation of trophic receptors, phosphorylate and inactivate pro-apoptotic molecules

Question 36

Describe the molecular basis of apoptosis
extrinsic pathway

Answer 36

*Used by cells of the immune system to kill their targets
➢ Eg. Cancer cells, pathogen-infected cells
*Initiated by death ligands on/or secreted by the immune cells, binding to their receptors on the target cell
*Activated death receptors result in caspase cascade
➢Different initiator caspases used compared with intrinsic route i.e. procaspase 8 in extrinsic route

T-lymphocyte has death ligand FasL on surface which interacts with Fas (death receptor) – trimerized receptors use the adaptor FAAD to mediate autoactivation of initiator procaspase 8