Topic 3.2. - Cell signalling

principles of cell signalling (4)

1. binding of extracellular signalling molecule to the receptor protein will activate the protein

2. activated receptor will activate one or more intracellular signalling pathways involving a series of intracellular signalling proteins

3. one or more of the intracellular signalling proteins will alter the activity of effector proteins

4. altered activity of effector proteins will alter the activity of the cell by inducing a particular metabolic pathway

types of intercellular signalling - list (4)

contact dependent

paracrine

synaptic

endocrine

intercellular signalling - contact dependent

cells in direct membrane to membrane contact

membrane bound signal molecule on a signalling cell can bind to a receptor on the target cell

intercellular signalling - paracrine

local mediators released from signalling cell can bind to a receptor on the target cell

intercellular signalling - paracrine example

autocrine signalling

signalling and target cell are the same

intercellular signalling - synaptic

neurons transmit signals electrically along their axons and then release chemical neurotransmitters at the synapses to act on the target cell

intercellular signalling - endocrine

secrete hormones into the blood stream which act on target cells throughout the body

location of intercellular signalling receptors (2)

on the cell surface or intracellularly

location of receptor effects how the signal is transmitted into the cell

intercellular signalling receptors - hydrophilic signalling molecules

majority cannot pass hydrophobic cell membrane → bind to cell surface receptors which can direct signalling pathways within the cell

intercellular signalling receptors - small signalling molecules

can diffuse across the plasma membrane and into the cell to bind to receptors in the cytosol or int he nucleus

mostly hydrophobic so need to be transported via carrier protein in the bloodstream or within the extracellular environment and then dissociate before cross in the membrane

signalling by phosphorylation - summary

protein kinase covalently adds phosphate from ATP to the signalling protein

protein phosphatase removes the phosphate

phosphorylation and dephosphoryaltion can both activate or inactive a signalling pathway

signalling by GTP binding - summary

GTP binding protein is induced to exchange its bound GDP for GTP → always activates protein

GTP binding protein can inactivate itself by hydrolysing its bound GTP to GDP

structure of GPCRs

present in all eukaryotes with the similar structure of 7 transmembrane segments

what do GPCRs respond to (6)

hormones

neurotransmitters

local mediators

light photons

amino acids

fatty acids

true or false - the same signal can only activate one GPCR depending on cell type

false

same signal can activate multiple GPCRs depending on cell type

acetylcholine activates 5 GPCRs with different effects on different cells depending on which GPCR and ion-channel receptor it binds to

G protein subunits and location within the cell (3)

trimeric binding proteins with three subunits

alpha and gamma = membrane bound

beta = cytosolic

active and inactive state of G proteins difference

active: alpha is GTP bound

inactive: alpha is GDP bound

activation of G protein steps (5)

1. Signal molecule binds to GPCR induces conformational change of receptor

2. Activated GPCR can bind and alter the conformation of the trimeric G protein

3. AH domain of G protein opens up to expose nucleotide binding site -> promotes dissociation of GDP

4. GTP binding to AH domain promotes closure of nucleotide binding side -> triggers conformational changes that dissociates alpha subunit from receptor and activates gamma subunit

5. GTP bound alpha subunit and activated beta gamma subcomplex both regulate the activity of downstream signalling molecules

role of GPCR in amplifying intracellular respone

as signal molecule stays bound to GPCR, receptor can activate many G protein molecules

what is adenylyl cyclase targeted by and what is its role

common target for G proteins

catalyses synthesis of cyclic AMP from ATP

effect of cholera on G proteins

cholera toxin lead sot overactive G protein → increased activation of adenylyl cyclase and up-regulated chlorine channel opening which leads to diarrhoea

effect of serotonin on G proteins

serotonin acts through GPCR to cause rapid rise in intracellular concentrations of cyclic AMP

effect of GPCR on gene expression via PKA - activatation of GPCR to activation of PKA (4)

1. activated GPCR activates G protein

2. activated alpha subunit activates adenylyl cyclase

3. adenylyl cyclase catalyses production of cAMP

4. cAMP binds to regualtory subunits of PKA tetramer to induce conformational change that causes dissociation of regulatory subunits and activates kinase activity of catalytic subunits

effect of GPCR on gene expression via PKA - PKA in nucleus to gene expression (2)

5. active type 1 and 2 PKA can enter nucleus via nuclear pore and phosphorylate an inactive CREB protein

6. activated CREB protein can bind associated CREB binding proteins and activate target gene expression via cyclic AMP response elements on DNA

effect of GPCR on gene expression via PKA - requirement

2 or more cyclic AMP molecules need to bind to regulatory subunits

sharpens response of kinase to changes in cAMP concentrations

types of PKA - location (2)

type 1: mainly in cytosol

type 2: bound via its regulatory subunits and anchoring proteins to the plasma, nuclear and mitochondrial outer membrane and microtubules

how are GPCRs related to vision and smell

both depend on GPCR regulated ion channels

photoreceptor structure

composed of rods and cones

rods detect dark and cones detect colour and bright light

outer segment contains photoreceptor discs which express rhodopsin GPCRs

Ion flux - reaction to light (5)

1. Light enters the outer segment of photoreceptor cells -> rhodopsin GCPRs in photoreceptor discs absorb light and become activated via conformation change to linked protein retinal

2. G alpha subunit of G protein transducin activates cyclic GMP phosphodiesterase

3. Activated cGMP phosphodiesterase hydrolyses cyclic GMP hydrolysis -> decrease in concentration of cGMP

4. Closure of cGMP gated sodium channels -> hyperpolarisation of cell and decreased rate of neurotransmitter release from synaptic region

5. neurotransmitter inhibits many of the postsynaptic retinal neurons → illumination frees neurons from inhibitions and thus excites them

ion flux - reaction to darkness (2)

1. Rhodopsin kinase phosphorylates and inhibits rhodopsin

2. arrestin binds phosphorylated rhosopsins and further inhibits its activity

3. regulators of G proteins (RGS) proteins bind transducin G protein and hydrolyse GTP to GDP to inactive it

photoreceptor cells - bipolar cells

activated bipolar cells transmit signals to retinal ganglion cells → signals to brain

photoreceptor cells on bipolar cells

can signal to ON or OFF bipolar cells

photoreceptor cells on bipolar cells - dark

photoreceptors release glutamate → inhibits ON bipolar cells and excites OFF bipolar cells

photoreceptor cells on bipolar cells - light

photoreceptor hyperpolarisation prevents inhibition of ON bipolar cells and inactivates OFF bipolar cells

structure of receptor tyrosine kinases

single transmembrane domain within plasma membrane

variable extracellular and intracellular domains → tyrosine kinase domain is always in cytosol

kinase domain can be interrupted by kinase insert region → essentially two tyrosine kinase domains

summary of RTK activation (5)

1. Signal protein binds to inactive receptor tyrosine kinases

2. Dimerization of RTKs brings kinases together

3. Trans-autophosphorylation -> each receptor phosphorylates and fully activates the other

4. Further phosphorylation generates binding sites for other intracellular signalling proteins

5. Signalling proteins bind to specific docking sites and are activated -> can relay signal to other downstream effectors

binding of signalling proteins to tyrosine kinase domains

majority do so via SH2 domains and bind to specific phosphotyrosines

usually have a SH3 domain which can bind to other intracellular signalling molecules

activated RTK to activated Ras protein - steps (4)

1. activation of RTK → adaptor proteins binds to specific phosphorytosine

2. adaptor proteins bind and activates GEF via SH3 domain

3. GEF encourages Ras-GDP to hydrolyse bound GDP and thus bind GTP → active

4. activated Ras-GTP can induce downstream signalling pathways

limitation of RTKs and Ras proteins and solution

only active for a short period of time

initial signalling event is converted into longer-term signalling event via activation of MAP kinase signalling cascade

MAP kianse signalling cascade - summary (4)

1. Active GTP bound Ras activates MAP kinase kinase kinase (Raf in mammalian cells)

2. Raf activates MAP kinase kinase (Mek)

3. Mek activates MAP kianse (Erk)

4. Active Erk phosphorylates other proteins and gene regulatory proteins

benefit of dimerisation of RTKs

allows inhibition of signalling using domain negative receptors

if dominant negative receptor is over expressed in a cell, will replace RTKs → when ligand binds to cause dimerization trans-autophosphorylation does not occur → no signal transduction

dominant negative receptor - def

functioning extracellular domain and transmembrane domain but truncated intracellular kinase domain → no tyrosine kinase domain so no catalytic activity

TGFß composition (2)

type 2 receptor homodimer = specific for each type of ligand and constitutively active

type 1 receptor homodimer = activated via phosphorylation by type 2 receptor

effect of activated TGFß receptor complex on gene transcription (2)

1. activated TGFß binds and phosphorylates SMADs

2. Trimeric SMAD complex forms and translocates to the nucleus to form transcription regulatory complex through interactions with transcriptional regulators

common target genes mediated by TGFß signalling (7)

Inhibition of proliferation

Cell specification and differentiation

Extracellular matrix production

Epithelial to mesenchymal transformations or fibrosis (can lead to cancer metastasis)

Cell death

Tissue repair

Immune cell regulation

activated TGFß receptor and clathrin mediated endocytosis - SMAD activation

most of SMAD activation occurs in early endosomes and requires SMAD anchor for receptor activation (SARA)

inactivation of activated receptor complex requires caveolae mediated endocytosis

receptor ubiquitylation and degradation in proteosomes occurs

Wnt signalling pathways (3)

PCP signalling

ß-catenin-dependent signalling

Wnt-Ca2+ signalling

what cells use Wnt signalling

most commonly active in epithelial cells

also occurs in neurons, lymphocytes, muscles and other cells

ß-catenin role - adherens junctions

component of epithelial adherens junctions

connects actin filament bundles and anchors cells to each other

adherens junction reassembly’s effect on ß-catenin

adherenes junctions = dynamic

reassembly of adherence junctions will result in ß-catenin release into the cytoplasm

ß-catenin - Wnt signal on destruction

under normal conditions, cells does not want ß-catenin in cytosol → destroy via proteolysis

if Wnt signal via ß-catenin dependent pathway → ß-catenin stabilised

ß-catenin - destruction complex

complex of cytoplasmic proteins target ß-catenin for ubiquitylation and degradation by proteosomal enzmes

phosphorylated of ß-catenin by CK1 and CSK3-beta targets E3 ubiquitin ligase complex

ß-catenin - Wnt signal on gene transcription

no Wnt signal: Wnt responsive genes kept inactive by Groucho co-receptor protein → binds to transcription regulators Lef1/TCF

Wnt signal: unphosphoryalted ß-catenin binds to LEF1/ TCF → displaces Groucho and acts as co-activator to stimulate transcription of Wnt target genes

main regulator of cell cycle progression

rise and fall of different cyclin levels

occurs due to synthesis of cyclins via transcription of cyclin genes and translation of cyclin proteins whilst cyclins are degraded by proteosomes

activation of Cdk (2)

1. binding of cyclin partially activates Cdk through conformational change that unblocks active site on Cdk

2. full activation requires phosphorylated by a Cdk-activating kinase

activation of Cdk - inhibitory kinases

can add inhibitory phosphate to Cdk to inactivate complex

phosphotase can remove inhibitory phosphate form Cdk to activate complex

activation of Cdk - Cdk inhibitor proteins

can inhibit Cdk by wrapping aound complex and distorting Cdk active site

activation of Cdk - Cdk inhibitor protein example

p21 = Cdk inhibitor protein

transcription activated when DNA damage detected and p53 is active and binds to regulatory region of p21

cyclin levels - G1/S (3)

rise in mid G1 and usually in response to G1 cyclin → fall at end of G1

leads to formation of G1/S Cdk complexes → triggers progression through start transition

regulates G1 checkpoint

cyclin levels - S (1)

rise during end of G1 and dominant through S phase → persist into G2 and early M

cyclin levels - M (3)

rise during G2 and trigger progression of cell through G2/ M transition

remain elevated in early M

regulates G2/M checkpoint

cyclin levels - destruction of S and M (summary)

regulates metaphase-anaphase checkpoint

controlled by anaphase promoting complex/ cyclosome (APC-C)

cyclin levels - destruction of S and M (steps -2)

1. APC/C activated by binding of an activating subunit

2. ubiquitylation enzymes E1 and E2 polyubiquitylate active APC/C → targets protein for degradation in proteosome

inhibitors of G1/S-Cdk (1)

DNA damage

inhibitors of S-Cdk (1)

DNA damage

inhibitors of M-Cdk (2)

unreplicated DNA

DNA damage

inhibitors of APC/C-Cdc20

chromosome unattached to spindle

DNA polymerase catalysis - summary

stepwise addition of deoxyribonucleotide to 3’ end of polynucleotide chain → gorwing strand is paired to an existing template strand

5’ to 3’ direction of growth

DNA polymerase catalysis - what drives reaction

large, favourable free energy change caused by release of pyrophosphate from nucleotide and subsequent hydrolysis into two molecules of inorganic phosphate

DNA polymerase catalysis - mechanism (4)

1. Binding of the correct nucleoside triphosphate induces a conformational change in DNA polymerase (closing around DNA), positioning substrates for catalysis

2. Covalent nucleotide addition occurs, releasing pyrophosphate

3. pyrophosphate is hydrolysed to 2 Pi, making the reaction energetically favourable and irreversible

4. DNA polymerase reopens and translocates by one nucleotide, allowing the next nucleotide to bind

DNA conservation - def

every time DNA is replicated, one strand will be form the parent DNA molecule

okizaki fragments

added in 5’ to 3’ on lagging strand

cell cycle pause before proceeding to M phase

pauses after G2 to check for DNA damage

metaphase - summary (3)

1. duplicated chromosomes join at the centromere → centromere surrounded by kinetochore protein complex

2. kinetochore microtubules attach to kinetochore from spindle poles

3. metaphase - anaphase checkpoint checks for correct attachment of spindle fibres so each daughter cell has correct number of chromosomes

metaphase-anaphase checkpoint - APC/C activation

1. activation of APC/C by Cdc20 leads to ubiquitylation and degradation of securin → allows separase to become active

2. active separate cleaves part of cohesion complex → allows sister chromatids to separate in anaphase

cause of cells entering G0 (2)

cell no longer needs to divide and proliferate

lack of mitogens or growth factors that stimilate growth etc

genes transcribed due to exposure to mitogens

transcription of immediate early genes → leads to transcription of delated response genes

immediate early response genes

tend to be transcription factors

eg. Myc

delayed response genes - examples (3)

cyclins

Cdks

E2F transcription factor

mitogen binding on cyclin transcription - steps (6)

1. extracellular mitogen or growth factor binds to transmembrane receptor (typically RTK) → activates RAS → MAP kinase cascade

2. ERK activates transcription of immedaite early genes including Myc

3. Myc promotes transcription of delayed response genes including D-cyclins

4. D-cyclins bind and activate Cdk → formation of active G1/Cdk complex → phosphorylates and inactivates Rb protein → active E2F

5. E2F activates transcription of cell-cycle genes including S-phase cyclins → S-Cdk

6. progression of the cell into S phase and DNA synthesis + active S-Cdk in positive feedback loop that keeps cell in S phase

effect of overactivation of cell cycle by excessive stimulation of mitogenic pathways (2)

cell cycle arrest or induced apoptosis

effect of overactivation of cell cycle by excessive stimulation of mitogenic pathways - Myc over-expression (3)

1. activation of Arf

2. Arf binds and inhibits Mdm2 → increased levels of p53

3. p53 causes cell cycle arrest or apoptosis depending on cell type and extracellular conditions via activation of p21 transcripton