M2L5: Cell signalling

What are possible stimuli for sensors in signalling pathways?

• Stimuli for sensors can be generic or cell type specific

  • Metabolites (amino acids, lipids, nucleotides)

  • Small molecules (NO, CO₂, xenobiotics)

  • PRRs (dsRNA, ssRNA, DNA, LPS)

  • Mechanical (ECM, cell to cell pressure)

  • Hormones

  • Light

  • Smell

What are the components of a signalling pathway?

• Sensors - receptors (plasma membrane, organelle), TFs (cytosolic, organelle, inner PM)

• Transducer (signalling cascade, subcellular relocalisation)

• Effectors (activity of a protein eg. IP3R and GLUT4, TFs)

What is the role of adaptor proteins?

Adaptor proteins act as scaffold to help bring together multiple proteins - assists in relaying the signal downstream for better efficiency and specificity

What are some examples of adaptor proteins?

• Grb2: binds to phospho-tyrosine on RTK through SH2 and the GEF Sos through SH3 domain

• Shc: binds to phospho-tyrosine on RTK through phospho-tyrosine binding (PTB) domain and to Grb2 via SH2

• Insulin receptor substrate (IRS): Binds insulin receptor through PTB and PIP2/3 though PH domain

• Integrins: structural, scaffolding and catalytic function

autocrine

signalling involving the cell self

intracrine

signalling without secretion, within the cell

paracrine

signalling to nearby cells

juxtacrine

signalling to physically connected cells

endocrine

signalling to distant cells via the circulation

What are the modes of contact-dependent signalling?

• Cell surface signalling (tight junction and immune cells activation)

• Cell adhesion molecule (ECM)

What is the role of small molecule metabolites in signalling?

• Small molecule metabolites (NO, Trp, ATP, succinate, alpha-KA, OA, PA, lactate) can act in intracrine and paracrine signalling molecule and and even endocrine (lactate)

• Evolution of small molecule metabolites as signalling molecules as opposed to proteins in some circumstances may be to save energy (they do not need to be specifically synthesised from scratch for signalling as they are already available) and to communicate about the status/needs of the cell more easily based on the pathways that trigger certain metabolites to be produced

How is signalling used for development?

• Signalling molecules secreted from a single source (ventral side) can create morphogen gradient that enables cellular patterning during development

• Opposing gradients of signalling molecules can endow crypt cells with positional identity to adopt a specific cell lineage

• Cell-cell contacts also contribute to positional identity

• Differential receptor expression further reinforce positional boundaries

What are the different types of receptors?

• Cell surface transmembrane receptors - extracellular, transmembrane, intracellular domains

• Intracellular receptors - for ligands originated extracellularly (passive diffusion or active uptake) or intracellularly

  • Nuclear receptor - steroid or thyroid hormone receptors, retinoic acid and orphan receptors

  • IP3 receptor (intracellular calcium channels located in the ER, release Ca²⁺ into the cytosol which activates various cellular processes)

first messenger

extracellular signalling molecule, eg. hormones, neurotransmitters

second messenger

small, easily diffusible intracellular molecules that amplify and distribute the signal within the cell

What are some examples of secondary messengers?

• Cyclic nucleotides (cAMP —> PKA, cGMP —> PKG)

• Ions (Ca²⁺, Na⁺)

• Phospholipid derived (IP3, DAG —> PKC)

• Gases (NO)

secondary effector

intracellular protein activated by the second messenger which performs the functional cellular response

Outline the function of GPCRs

• Largest family of transmembrane receptors which are coupled to a trimeric G protein (guanine nucleotide binding protein) consisting of α, β, and γ subunits

• Binding of extracellular ligand induces conformational changes in receptors —> Gα GDP-to-GTP exchange for activation

• Depending on which type of G protein is coupled to the receptor, different downstream pathways amay be activated

What are some different types of G proteins bound to GPCRs and how do they target different downstream signalling pathways?

• Gs, Gi/o —> activates/inhibits cAMP pathway

• Gq/11 —> IP3/DAG

• G12/13 —> Rho GEF

How is β-arrestin involved in GPCR signalling?

• GPCR signalling is terminated via receptor phosphorylation by GPCR kinases (GRKs) which recruits β-arrestin to bind, blocking interaction with downstream signalling molecules and causing receptor desensitisation/internalisation

  

  • β-arrestin also initiates secondary GPCR signalling as it acts as a scaffold for other signalling molecules, regulating different gene expression programs

  • If the first round of signalling is not terminated properly then the receptor may remain active for longer, causing prolonged or abnormal β-arrestin recruitment, reading to secondary signalling from endosomes or pathological signalling

Explain the Wnt/β-catenin pathway

• Canonical Wnt pathway regulates cell fate specification and migration in embryonic development and tissue homeostasis

• In the absence of Wnt, GSK3 phosphorylates and targets β-catenin for proteasomal degradation without Wnt signalling

• Wnt Frizzled GPCR and LRP5/6 coreceptor proteins molecules to bind

• When Wnt binds to Fzd, Dishevelled (Dsh) gets phosphorylated at the plama membrane and inhibits GSK3

• β-catenin can thus enter the nucleus and act as a coactivator of gene expression with TCF/LEF family of transciption factors

• Non-canonical Wnt pathways do not use LRP5/6 or β-catenin and involve G protein-mediated regulation of tissue polarity/planar cell polarity (PCP) signalling and Ca²⁺ signalling

What is the importance of high β-catenin turnover rather than synethesising only when needed?

Allows rapid activation

What happens in RTK signalling?

• Ligand binding induces receptor dimerisation and reciprocal phosphorylation on tyrosine residues which act as docking sites for adaptor proteins

• Dimerised receptors recruit and activate multiple intracellular signalling molecules for signal amplification

• As it is difficult to overcome ligand-receptor binding, termination usually involves receptor internalisation via the clathrin coated pathway

How is RTK signalling terminated?

• As it is difficult to overcome ligand-receptor binding, termination usually involves receptor internalisation via the clathrin coated pathway

  

  • Sorting to lysosomes for degradation or recycling to the membrane, helps limits the signal intensity and duration

  • Following internalisation the receptor is still bound to the ligand and can still signal from the endosome until it is degraded

    • This may be exploited to give rise to therapeutic resistance

Compare RTK homodimerisation vs heterodimerisation

• Homodimerisation - identical RTKs dimerise to activate standard signalling eg. canonical MAPK, PI3K pathways

• Heterodimerisation - two different RTKs (either from the same family or different families of RTKs) dimerise which leads to signalling diversity

  • eg. EGFR/HER2, HGF/C-MET and AXL/EGFR

  • This may also be a mechanism of drug resistance by causing redundancy when honing in on one receptor in a targeted therapy 

Describe the structure and function of Ras

• Small GTPase (monomeric G protein) - HRAS, KRAS, NRAS etc

• Molecular switch that cycles between active (GTP-bound) and inactive (GDP-bound) state

• Farnesylation and palmitoylation anchors Ras to PM - critical for function

• Activates various downstream pathways involved in membrane trafficking, survival, calcium signalling, and apoptosis

What recent efforts have allowed Ras to become druggable and what are the limitations of this?

• Considered undruggable until recent efforts placed in targeting MAPK and PI3K/AKT pathways

  • Sotorasib targeting G12C (covalent inhibitors), but this is found only in a small subset of cancer (especially lung)

    • Possible because cysteine substitution at G12 position produces a side chain with a reactive sulfhydryl group, enabling covalent bonding with an electrophilic group in the inhibitor sotorasib  

  • Targeting G12D, G12V, G13D, and Q61H remain a challenge

What is the function of MAPK signalling?

• Regulates growth, differentiation and stress response, including immune response

• Signalling can be acute (proliferation or transient transcriptional activation) or sustained (differentiation, senescence, apoptosis)

What is the signal flow in MAPK signalling?

Receptor (e.g. RTK or GPCR) → Ras (small GTPase) → Raf (MAPKKK) → MEK (MAPKK) → ERK (MAPK) → transcription factors / target proteins

What are the main MAPKs?

Extracellular signal regulated kinase (ERK), p38 stress-activated protein kinase (SAPK), c-Jun N-terminal kinase (JNK)

How is MAPK signalling regulated?

Negatively regulated by dual-specificity phosphatases (DUSPs) - some influence subcellular MAPK localisation by binding and retaining them in specific cellular compartments

What is meant by ‘dual specificity’ kinases/phosphatases?

can recognise Ser/Thr and also Tyr

What is the function of the PI3K/AKT pathway?

principal pathway regulating proliferation, survival, and metabolism

What are the steps in PI3K/AKT signalling?

• Activation of RTKs and GPCRs recruit PI3K which phosphorylates PIP2 into PIP3 at the plasma membrane

• PIP3 acts as a docking site for Akt using its PH domain

• Akt is activated by PDK1 via PIP3 and mTORC2 (PM localised)

• Active Akt phosphorylates cytosolic  and nuclear substrates for growth and survival

• Tumour suppressor PTEN dephosphorylates PIP3 to PIP2

What is the function of contact-dependent RTKs?

Important for cell adhesion, migration, tissue patterning/homeostasis, angiogenesis, synaptic plasticity and immunity

How is contact-dependent RTK signalling mediated by ephrins?

• Activated by ligands on adjacent cells or ECM

• Eprin-A receptor - anchored to the cell membrane by glycosylphosphatidylinositol (GPI) linkage and lacks cytoplasmic domain

  • Binds EphA

• Ephrin-B receptor - attached to cell membrane by single TM domain containing a short PDZ-binding motif, Dig1 and zo-1

  • PDZ anchors receptor to cytoskeleton

  • Binds EphB

• Ephrin-Eph oligomerisation and signalling is bidirectional

integrins

heterodimeric TM proteins named according to their α/β subunit compositions, involved in mechanotransduction from ECM adhesion to cytoskeleton to nuclear envelope, all to regulate survival

Compare outside-in and inside-out integrin signalling

• Outside-in: interaction with ECM and cell surface adhesion proteins, eg collagen (outside-in), produces an intracellular signal - may involve Rho, Rac, and cytoskeletal rearrangement

• Inside out: cell-matrix adhesion is switched on by a signal generated inside the cell (eg. due to hormone binding) which acts on integrin complexes, altering the affinity of an integrin by changing the shape of the integrin complex on the outer cell surface, thereby switching to the high-affinity conformation

anoikis

cell death program induced by loss of ECM contact/integrin signalling

Explain the JAK/STAT pathway

• Cytokines (eg. TPO, IL, IFN, EPI, prolactin, growth hormones) bind to the Janus kinase (JAK) receptors, cauing receptor dimerisation and cross-phosphorylation

• Phosphorylated sites allow signal transducers and activators of transcription (STATs) to dock and get phosphorylated

• Phosphorylated STATs dimerise and translocate into the nucleus to activate gene expression

• Non-receptor protein tyrosine phosphatases (PTPNs) attenuate the signal

• 7 STATs can be recruited to different combinations of Type I/II cytokine receptors to induce a ligand specific response

Explain the TGF-β/BMP/SMAD pathway

• Dual specificity Ser/Thr and Tyr kinase receptor - tetramer of two type I and type II

• Ligands include three TGF-β, BMPs and two activins

• Ligand binding to type II receptors enable them to phosphorylate and activate type I

• Activated type I phosphorylates C terminal of receptor-activated SMAD2/3 (R-SMADs) which complex with co-SMAD (SMAD4) to enter the nucleus and activate gene expression

• Crucial role in development in development and tissue homeostasis and can promote fibrosis and metastasis in disease

• Inhibitory SMADs (I-SMADs) eg SMAD6/7 compete with R-SMADs for binding and activation by the receptor

What are some examples of proteins that can act as receptor, transducer of signal and effector

Notch receptor and ATF6

What happens in Notch signalling?

• Notch receptor interacts delta (ligand on other cell), causing proteolytic cleavage of Notch receptor and release of its intracellular domain

• Intracellular domain has co-factors of transcription factors

• Nuclear relocalisation of intracellular domain drives gene expression

What happens in the ATF6-mediated ER unfolded protein response (UPR)?

• Dissociation of chaperone protein BiP from ATF6

• ATF6 transported to Golgi

• Proteolytic cleavage by Site-1 and Site-2 proteases (S1P and S2P)

• Upon cleavage it is released as an active transcription factor p50ATF6 in the cytoplasm

• Translocated to nucleus and activates ER-associated degradation (ERAD)/BiP

How is mTORC signalling used in starvation?

• mTORC is a nexus downstream glucose and amino acid sensing

• mTORC1 promotes growth 

  

  • Promotes protein synthesis through S6K1 activation and 4E-BP1 inhibition, enabling proliferation

    • This regulation happens normally at lysosomes due to role of lysosomes in degrading/recycling proteins, allowing amino acid sensing by mTORC1

    • Glucose is sensed by ATP:ADP/AMP ratio which indicates the metabolic status

  • Inhibits autophagy through ULK1, ATG13 and TFE3/B

  • Pro proliferative signalling activates TSC2, a GTPase activating protein (GAP) which inhibits mTORC1

  • GATOR2 complex is a positive regulator of mTORC1

• mTORC2 promotes survival and regulates movement by regulating AKT

What happens in the integrated stress pathway?

• Multitude of stresses integrate at the ISR (integrated stress pathway)

  • Stresses - ER stress, infection, lack of nutrients, Cyt c release…

  • ISR downregulates global translation due to phosphorylation of eIF2α (essential for translation initiation) by stress-activated kinases (PERK, GCN2, PKR, HRI)

    

  • Phosphorylated eIF2α reduces global translation to conserve energy and activates ATF4 transcription factor which mitigates stress by activating CARE genes which promote amino acid synthesis, antioxidant response, chaperone expression, autophagy, cell survival, or autophagy for extensive damage