Signalling

Steroids are very hydrophobic - what are the signalling and pharmacological consequences of this?

• Requires plasma protein binding to travel in blood

• Can exist in blood for hours - days

Simply, what are two examples of non-transduction based signalling

• Steroids

• NO

Describe the general pathway of steroids

• Plasma protein bound, travel in blood

• Diffuse across membrane

• Bind cytoplasmic nuclear receptor

• Releases inhibitory factor

• Translocates to nucleus

• Homodimerizes

• Binds hormone response element on DNA (HRE)

• Recruits transcriptional trans-factors

• Regulates transcription

Describe the pathway of oestrogen signalling

• Plasma protein bound, travel in blood

• Diffused across membrane

• Bind cytoplasmic ERalpha

• Release of inhibitory protein

• Translocates to nucleus

• Homodimerizes

• Binds hormone response element on DNA (HRE)

• Recruits activating trans-factors, including src

• Upregulates transcription of pro-proliferative and differentiation genes

Describe the action of elecestrant

• Ostreogen nuclear receptor antagonist

• At very high concentrations causes degradation of the nuclear receptor

• Used in HER2-, receptor + breast cancer treatment

Describe the structure of the cytoplasmic nuclear receptors

• 3 binding domains: inhibitor, DNA, ligand

Steroids and nitric oxide are an example of…

• Non-transduction based signaling receptors

What is the synthesis pathway of NO?

• L-arginine modified by NOS to replace -H with -OH

• Inhibited by calcium-activated calmodulin

• Cleaved into citrulline and NO

How does NO cross lipid bilayers?

• Small and very lipid soluble so passively diffuses

What are the three main pathways for NO after it has crossed the bilayer?

• Activation of guanylyl cyclases, increases cGMP, increases PKG activation, increases smooth muscle relaxation and vasodilation

• Activation of PDE, decreases cGMP, decreases PKG activation, autoinhibitory but effects are delayed

• Half life of a few seconds, rapid conversion to nitrites / nitrates or sequestering by haem

Describe the action of sildenafil

• Guanine homology

• Binds substrate cleft of PDE5

• Prevents cGMP breakdown, even with NO stimulation

• Maintains vasodilation (blood flow) and smooth muscle relaxation

Draw a diagram showing specifically how NO can activate guanylyl cyclase. What can increase activation?

https://ibb.co/9m54XTDv

Describe the full NO signalling pathway, including self-termination

• Passive diffusion across bilayer

• Displace his105 in guanylyl cyclase

• Restores haem polarity in enzyme, activates

• GTP —> cGMP

• cGMP activates PKG

• Increased smooth muscle relaxation and vasodilation

• Slowly activates PDE

• cGMP —> GMP

• Decreases smooth muscle relaxation and vasodilation

• Degrades in a few seconds to nitrites/nitrates

What is the thrombin receptor?

• PAR1

• Galphaq/i coupled

• Protease activated

• Activates platelets and their aggregation

Describe how the thrombin PAR1 receptor is activated

• Has long extracellular N terminus

• Thrombin cleaves short peptide from it, new N terminus revealed

• Binds to activation helix of GPCR

• G proteins activated

• Signalling activates platelets and their aggregation

What is the purpose of thrombin?

• Cleaves fibrinogen → fibrin to bind platelets in place and begin formation of extracellular matrix

• Cleaves PAR1 receptor to activate platelets and their aggregation for the extracellular matrix

How can the PAR1 receptor be activated experimentally?

• TRAP sequence

• Synthetic protein derived from cloning the receptor

• Binds where the ‘revealed’ N terminus would bind

Compare voltage gated vs ligand gated ion channels

• Open in response to electrical changes across plasma membrane vs ligand binding

• Single polypeptide with 4 homologous domains containing alpha helices vs different families with 3/4/5 subunits each

• Small extracellular domain as doesn’t require specificity vs large extracellular domain for more structure variation and ligand specificity

Describe the AChR family

• Pentameric, each domain has 4 TM alpha helices

• Hydrophobic residues (ala, leu) line pore to repel water

• nAChR also have -ve aa (Asp, glu) lining pore to repel anions

Draw a nAChR

https://ibb.co/tptLBbGD

Which type of ion channel is, overall, more selective for the ions that pass through?

• Voltage gated

How do ions exist in cells / synapses / intracellular space etc

• Generally bound to water

Describe how voltage gated ion channels can be selective for Na+ rather than K+

• Small channel, too small for K+

Describe how volage gated ion channels can be selective for K+ rather than Na+

• Wider channel lined with C=O from peptide backbones to displace bound water

• Na+ too small to interact with enough C=O at once, remains water bound, repelled by hydrophobic residues (ala, leu) further down the channel

• K+ interacts with enough C=O, displaces water, is not repelled by hydrophobic residues

What are the four G proteins that need to be known and, simply, what do they do?

• Alphas, increase cAMP

• Alphai, decrease cAMP

• Alphaq, increase Ca2+

• Alpha-transducin, prevents inhibition of bipolar cells

Simply, an example of a key GalphaS signalling pathway

• Adrenaline binding beta adrenoreceptors in skeletal muscle and liver

Describe fully a GalphaS signalling pathway

• Adrenaline binds beta-adrenoreceptor GPCR

• GDP-GTP exchange

• Dissociation

• AlphaS activates adenylyl cyclase

• Increase cAMP

• Binds PKA, dissociates regulatory subunits

• Catalytic subunits increase glycogenolysis by activating phosphorylase kinase and inactivating glycogen synthase

• Increases flux through glycolytic pathway

Simply, state an example of a Galphai signalling pathway

• Adrenaline binding CNS alpha2 adrenoreceptors to decrease vasoconstriction

Describe fully a Galphai signalling pathway

• Adrenaline binds alpha2-adrenoreceptor GPCR

• GDP-GTP exchange

• Dissociation

• Inhibits adenylyl cyclase

• Decrease in cAMP

• Less PKA activation

• Less Ca2+ influx

• Increase vasodilation, lower BP

Simply, what is a Galphaq signalling pathway

• Adrenaline binding alpha1 adrenoreceptors on vascular smooth muscle cells

Describe fully a Galphaq signalling pathway

• Adrenaline binds alpha1-adrenoreceptor GPCR

• GDP-GTP exchange

• Dissociation

• Activates PLCbeta

• Increased PIP2 hydrolysis

• IP3 opens LGIC on ER

• Increase [Ca2+]

• Increase vascular smooth muscle contraction

• Vasoconstriction, increase BP

Simply, what is the Galpha-transducin signalling pathway?

• Rhodopsin/transducin system for vision

Describe fully the Galphaq-transducin pathway

• Light absorption coverts all cis-retinal to trans in photoisomerization

• Trans can bind and activate Rhodopsin GPCR

• GDP-GTP exchange

• Dissociation

• Binds inhibitor of PDE6, PDE6 remains active

• cGMP —> 5’ GMP

• Decrease cGMP

• cGMP-gated Na+c close

• Ca2+v close

• Ions removed via cation pumps

• Hyperpolarization

• Prevents glutamate release

• Less activation of inhibitory GPCR on bipolar cells

• Action potential generated in bipolar cell

Describe how the cholera toxin effects G protein signalling

• A-B toxin

• A subunit binds Arg close to GTP-binding domain of GalphaS, prevents intrinsic GTPase activity

• Remains active

• cAMP stays high

• Disturbs Na+/Cl- membrane pumps

• Mass water and electrolyte loss into gut lumen

Describe the principle of RTK signaling

• Ligand binding brings two RTKs closer together

• Conf change and dimerization

• Trans-autophosphorylation activates kinase domains within the receptor

• Intracellular pTyr sites generated on RTK for binding of SH2 domains from signalling proteins

• Series of activated signalling proteins

What is a good example of RTK signalling

• Her family

• All receptor TKs with extracellular dimerization domains, can hetero or homodimerize

• Homodimerization of Her2 for ligand-independent signalling

• Her2 heterodimerization allows EGF binding

• Transautophosphorylation

• GRB2 binds pTyr by SH2

• GRB2 SH3 binds Sos

• Sos activates Ras

• MAPK

• Cell growth, proliferation, survival

Describe c-src activation by PDGF

• PDGF binds, homodimerization and transautophosphorylation

• Src SH2 binds pTyr on receptor rather than pTyr-527 on N terminus

• PDFGR has other phosphorylated sites that are now active for competitive binding with linker segment for src SH3

• Conf change activates kinase domain

• Tyr416 of activation loop phosphorylated, catalytic site of src revealed

• Transduces pro-proliferative signals

What is a good example of an indirect TK signaling pathway?

• Ethylene receptors

Describe ethylene receptor signaling

• Indirect Ser/Thr RTK - CTR1 receptor

• Active in absence of ligand, where ligand binding domain is occupied by coordinated Cu2+

• The RKs are phosphorylated, which recruits proteins that continually ubiquitinate EIN3 for targeted degradation by proteasomes

• Prevents transcription of ethylene target genes

• Ethylene displacement of copper causes conf. change in RTKs that stops phosphorylation of signaling proteins

Describe CaM kinase II

• Crucial in memory - transgenic mice w/o CaMK autophosphorylation abilities have impaired memory and increased fear

• Self-inhibited, auto phosphorylates in the presence of activated CaM, activates kinase properties

How is calcium signaling an example of convergence

• PLCbeta activated by GPCRs

• PLCgamma activated by RTKs

• Both hydrolyse PIP2 into IP3, opens LG Ca2+

How does calmodullin respond to Ca2+

• Binds Ca2+ with EF-hand domains

• Conformational chagne

• CaM can now interact with target

What does activated Calmodulin activate?

• Adenylyl cyclases I / II

• NO synthase

• CaM kinase II (crucial in memory)

• Phosphorylates liver pyruvate kinase into inactive form, increasing flux through gluconeogenesis pathway in liver

What is the resting state of calcium signalling? How can this progress?

• Sometimes IP3R open spontaneously, and small Ca2+ leakage causes opening of adjacent IP3R so the Ca2+ influx is now transient and measurable

• If sufficient external stimulus is also added, the transient opening has decreased the stimulus required to reach threshold value, so high Ca2+ levels can be sustained

What is the general electrical potential change across membranes due to an action potential?

• -60 → + 30 mV in less than a millisecond

Draw a diagram showing how ion channel opening / closing propagates action potentials down a membrane

https://ibb.co/6Jwj9xmD

Describe how ion channel opening / closing propagates action potentials down a membrane

• Rapid sequential opening/closiggn of Na+v/K+v

• Na+v opens, makes inside of membrane positive, -60 → + 30mV

• Propagates, adjacent Na+ open

• Slight delay in outward K+v opening, K+ leave inner membrane, rectifies membrane potential +30 —> -60

How does localisation of Ca2+v enable accurate action potential transmition?

• Localized at neruone terminals

• Incoming AP can cause Ca2+ influx where the vesicles are required to be exocytosed

• Neurotransmitter release localised to neurone terminals

How can cAMP production be measured using radiolabelling

• Anti-cAMP bound to radiolabelled cAMP added to cell lysate

• Sample cAMP competes for antibody CDR

• Radialabel signal decreases inversely proportion to amount of cAMP in sample

• Only detects amount, not sample, and cannot happen in real time

How can cAMP production be measured in vitro?

• FRET

• Radiate with one wavelength, can only emit different wavelength when acceptor and donor are in close proximity

• Synthesize PKA R with an acceptor and PKA C with a donor

• cAMP binding causes dissociation, decreases FRET

Describe MAP kinase cascades as second messenger signalling

• Ras/ Rho activated by RTK

• Physical interaction with MAPKKK (Ser/Thr K) e.g. Raf, activates

• Pi MAPKK (Tyr/Thr K) e.g. Mek

• Pi MAPK e.g. Erk

• Interaction with effector proteins (TFs, GFs, cytokine production, especially the TF myc)

What are four main ways of transducing signals

• Calcium signaling and activation of calmodulin

• MAPK cascades

• Propagation of action potentials

• cAMP and activation of PKA

What are the five main signaling receptors

• Non-transduction based

• Protease activated

• Ion channels

• GPCRs

• Enzyme activated

How can downstream effects of Ca2+ signaling be terminated?

• Phosphodiesterases are regulated by Ca2+/Calmodulin, and protein phosphorylation

• PDE2, activated by high cGMP, lowers Km for cAMP and cGMP to enhance rate of both cGMP/cAMP hydrolysis

• PDE5 target of viagra

Simply, what are the two ways of GPCR desensitisation?

• Hetero/homo logous

Describe heterologous GPCR desensitisation

• GPCR 1 activates PKA by increasing cAMP

• PKA phosphorylates Ser/Thr at C terminus of GPCR 2

• Conf change

• GPCR 2 now unable to interact with Gs, can interact with Gi

• If ligand binsds, Gi inhibits adenylyl cyclase and decreases cAMP

• So stimulus at GPCR 1 that originally caused activation of AC has caused caused inactivation of AC when second ligand binds

• Heterologous as G protein change on different receptor

Summarise what heterologous GPCR inactivation is

• Ligand signaling pathways also intrinsically prevent that pathway being activated by other receptors

Describe homologous GPCR desensitisation

• Beta-adrenoreceptor kinase phosphorylates Ser/Thr residues at C-terminus of its own receptor

• Recruits bet-arrestin

• Blocks signal transmission through that receptor only

Summarise what homologous GPCR inactivation is

• Signaling pathway causes inhibition of its own, individual receptor

How is RTK signaling terminated?

• Dephosphorylated by protein tyrosine phosphatases at the RTK itself

• PTPases are not very specific and dephosphorylate several different kinases

List different types of signal termination

• RTK dephosphorylation by PTPs e.g. SHP1/2

• GPCR hetero/homologous desensitization

• Bacterial inactivation of G protein e.g. Cholera CTX

• Outward rectifying K+v in action potentials

• Ca2+ pumps

• Ras intrinsic GTPase

How is Ca2+ signalling terminated?

• Outward pumps from cytosol to extracellular space

• Inward pumps from cytosol into ER

Tamoxifen

• Binds ERalpha to prevent homodimerization and binding to HRE on DNA

• Very specific so less side effects than cytotoxic drugs, but also susceptible to resistance

• Hormone receptor positive breast cancer

Compare tamoxifen and herceptin

• Small molecule inhibitor vs monoclonal antibody

• Binds ERalpha vs binds Her2 receptor

• Hormone receptor positive vs her2 over expressing

Describe hormone receptor positive breast cancer

• Over expression of transcription factor ERalpha

• Not enough inhibitory proteins in cell, oestrogen-indepndent homodimzerization and pro-proliferative signaling

Imatinib

• Small molecule kinase inhibitor highly selective for the BCR-ABL fusion oncoprotein in chronic myeloid leukaemia

Ipilimumab

• Monoclonal antibody against CTLA-4

• CTLA-4 on T cell cannot compete for CD80/86 on APC, so CD28 can bind and activate T cell

• T cell response not diminished

• Enables T cell recognition of tumour cells and killing

Describe the action of V. Cholerae’s CTX toxin

• B binds GM1-ganglioside on host GI epithelia

• Receptor mediated endocytosis

• Endosome transports to ER

• Exact mechanism unclear but could be break of DSB and then reform within A in a way that both activates it and releases it from B

• Active A translocates to cytosol

• Binds Arg on Galphas, preventing intrinsic GTPase activity

• Remains active, maintains high cAMP

• Disturbs Na+ and Cl- membrane pumps

• Ion imbalance leads to mass water and electrolyte loss into gut lumen