Cell Signalling Notes 2

Cell Signalling

Introduction

• This lecture builds upon several key concepts:

  • Eukaryotic gene expression.

  • Phosphorylation.

  • Intracellular pathways connecting receptor activation to response.

  • Receptor Tyrosine Kinases (RTKs).

  • The Fibroblast Growth Factor (FGF) activation pathway leading to gene expression.

• Understanding these concepts is crucial for:

  • Further studies in developmental biology.

  • Exploration of neurobiology.

Learning Objectives

• LO1: Understand how Receptor Tyrosine Kinases couple ligand binding to phosphorylation.

• LO2: Describe the experimental evidence for EGFR/RTK dimer formation.

• LO3: Explain the function of small GTPases as molecular switches in signalling pathways.

• LO4: Describe RAS mutations in cancer and their implications for understanding small GTPase function.

• LO5: Discuss the FGF family of secreted signalling molecules.

• LO6: Describe the mechanisms by which extracellular FGF signalling leads to changes in gene transcription.

Receptor Tyrosine Kinases (RTKs)

• RTKs are a large family of cell surface receptors.

• They function by dimerization upon ligand binding, leading to activation.

• RTKs directly link the cell surface to intracellular enzymes that phosphorylate tyrosine residues.

• Activation occurs in two stages:

  • Dimerization.

  • Autophosphorylation.

Mechanism of Activation

1. Ligand Binding: A signal protein (ligand) binds to inactive RTKs.

2. Dimerization: Ligand binding promotes the dimerization of RTKs.

3. Trans-Autophosphorylation:

   • The tyrosine kinase domains of the RTK phosphorylate each other.

   • This activates the kinase domains, increasing their activity.

   • Autophosphorylation generates binding sites for downstream signalling proteins/adaptor proteins.

4. Signal Relay: Activated signalling proteins relay the signal downstream, initiating an intracellular response.

The Importance of Dimerization

• Experiment: A chimeric receptor using insulin receptor external domains (which dimerize constitutively) and EGF receptor intracellular domains was created.

• Observation: The hybrid receptor only transmitted signals when bound to insulin.

• Conclusion: Dimerization alone is not sufficient for signal transduction; ligand binding is also required.

Detection of EGFR Dimers using FRET

• Technique: Fluorescence Resonance Energy Transfer (FRET).

  • FRET is a short-distance process where energy is transferred from one fluorescent dye (donor) to another (acceptor) when they are in close proximity.

• Experiment:

  1. EGF-att0532 (donor): An EGFR ligand with a green dye.

  2. EGF-Cy5 (acceptor): An EGFR ligand with a red dye.

• Observation:

  • EGF-att0532 (Donor) is seen both near and on the membrane.

  • EGF-Cy5 (Acceptor) is only seen at the membrane.

  • FRET occurs (green light transfer to red light) when the red and green dyes are close together, energy is transferred from one fluorescence dye to another. This leads to loss of emission of atto532 and increase of Cy5 emission.

• Conclusion: EGFR is only a dimer at the membrane.

Adaptor Proteins

• Adaptor proteins bind to the phosphorylated tyrosines on the RTK.

• These proteins may:

  • Be enzymes that phosphorylate other proteins.

  • Initiate other forms of messaging, such as activating membrane lipids.

Small GTPases: Molecular Switches

• Small GTPases are proteins that bind GTP (guanosine triphosphate).

• They act as molecular switches, existing in an 'on' and 'off' state.

States

• 'On' State: Bound to GTP (active).

• 'Off' State: Bound to GDP (inactive).

Activation and Inactivation

• Activation:

  • GTPases are activated by the exchange of GDP for GTP.

  • This exchange alters the protein's conformation, turning it 'on'.

  • GEFs (Guanine nucleotide exchange factors) are required for GDP to GTP exchange.

• Inactivation:

  • GTPases intrinsically hydrolyze GTP to GDP, inactivating themselves.

  • This hydrolysis is enhanced by GAPs (GTPase-activating proteins).

The Role of Ras

• Ras is a small GTPase that is activated by RTKs through the adaptor protein Grb2 and Sos (a GEF).

• Ras is not a kinase itself.

• It recruits other proteins to the cell membrane, leading to their activation.

GTPase Cycle

1. Inactive State: The monomeric GTPase is bound to GDP.

2. GEF Activation: A Guanine nucleotide exchange factor (GEF) facilitates the exchange of GDP for GTP.

3. Active State: The GTPase is now bound to GTP and is in the active state, initiating downstream signalling.

4. GAP Inactivation: A GTPase-activating protein (GAP) enhances the hydrolysis of GTP to GDP.

5. Return to Inactive State: The GTPase is now bound to GDP, returning to the inactive state.

Ras and Cancer

• Mutations in Ras are found in approximately 50% of human cancers.

• These mutations typically lock Ras in the active (GTP-bound) form.

• This prevents the attenuation of the signal, leading to uncontrolled cell growth.

Key Domains in Ras

• P-loop, Switch 1, and Switch 2: These are key domains within Ras.

• Threonine 35 (Switch 1) and Glycine 60 (Switch 2): These residues coordinate the phosphate of GTP.

• By pulling these residues in, Ras is activated and is able to activate downstream signalling through effector domain.

Examples of Mutations

The FGF Family

• FGF (Fibroblast Growth Factor) family stimulates fibroblast growth but has a wide range of roles.

• Mutations in FGF genes can affect organismal development (e.g., the Dachshund's shape).

Characteristics of FGFs

• Large Family: Composed of many peptide ligands with similar sequences.

• Human Genome: 22 FGF peptides have been identified.

• Biological Activities:

  • Regulation of cell growth and survival.

  • Regulation of cell differentiation.

  • Regulation of embryonic development.

• Hydrophilic Nature: Being peptides, FGFs are hydrophilic and cannot cross the cell membrane.

FGF Receptors

• FGFs bind to Receptor Tyrosine Kinases (RTKs) to initiate intracellular signalling.

• FGF ligands form a complex with extracellular proteoglycans (proteins with attached sugars), such as heparan sulfate proteoglycans.

• FGF does not bind the receptor without these proteoglycans.

FGF Response

• The intracellular domain of the receptor has tyrosine kinase activity and can phosphorylate proteins within the cell.

• This process starts the intracellular response which can lead to activation of Ras and the MAP Kinase cascade

Ras and the MAP Kinase Cascade

• Ras activates the MAP Kinase cascade, transmitting the signal through a series of three protein kinases:

  • Raf (MAP-kinase-kinase-kinase).

  • Mek (MAP-kinase-kinase).

  • MAP-kinase (Mitogen Activated Protein Kinase), also called Erk (Extracellular signal Regulated Kinase).

Signal Amplification and Response

• The multiple levels of kinases enable signal amplification.

• Each kinase can activate multiple downstream proteins.

• MAP-kinase is phosphorylated within minutes of stimulating a cell with FGF.

Transcriptional Changes

• MAP-kinase (Erk) translocates to the nucleus, where it phosphorylates and modulates the activity of multiple transcription factors.

• These transcription factors bind to promoters and enhancers of genes, activating them.

• This results in the transcription of at least 100 early response genes within 30-60 minutes of pathway activation.

Wrap-up

• This lecture connects to other module content, including:

  • Developmental biology.

  • Neurobiology.

  • Immunology.

• The concepts discussed feed into:

  • Cell Biology.

  • Genome Expression & Maintenance (Stage 2).

  • Cancer Cell and Molecular Biology.

  • Advanced Topics in Gene Expression (Stage 3).

• Follow-up:

  • Review the learning outcomes and lecture recording.

  • Attempt the Week 5 quiz.

  • Submit questions via the VLE.