cell signalling

1. Overview of G Protein-Coupled Receptors (GPCRs)

• GPCRs are the largest family of receptors in the human genome.

• Target of nearly 25% of clinically used medicines.

• Function: Convert extracellular signals into physiological responses.

• Also known as serpentine receptors or seven transmembrane domain (TMD) receptors due to their structure.

2. Structure of GPCRs

• Composed of seven α-helical transmembrane domains.

• Structural variation allows for recognition of distinct hormones and neurotransmitters.

• Example: β₂-adrenoceptor is the target for both beta-blockers and anti-asthma medications.

3. Catecholamine Binding to GPCRs

• Catecholamines: Noradrenaline, Adrenaline, Dopamine.

• All share a catechol ring with two adjacent hydroxyl (-OH) groups.

• Binding interactions:

  • Aspartate (TMDIII): Conserved in all catecholamine-binding GPCRs.

  • Serine residues (TMDVI): Form hydrogen bonds with catechol ring.

  • Phenylalanine (TMDVI): Forms π-π interactions with benzene ring.

• Dopamine can bind both dopamine-specific GPCRs and adrenoceptors due to structural similarity.

4. Relationship Between Receptor Occupancy and Response

• Agonist-Receptor Interaction:

  • [A] = Agonist concentration.

  • [AR] = Agonist-receptor complex concentration.

  • [RT] = Total receptor concentration.

  • KD (Dissociation Constant): Determines ligand affinity.

  • Magnitude of response: [AR]=[A][RT]KD+[A][AR] = \frac{[A] [RT]}{KD + [A]}[AR]=KD+[A][A][RT]​

    • Follows a hyperbolic relationship.

5. GPCR Activation Mechanism

• Agonists promote enrichment of active receptor state (R).*

• Structural changes during activation:

  • Small distance between TMDIII and TMDVI in inactive state.

  • Activation → Large outward movement of TMDVI.

  • Creates space for G-protein α5 helix to bind deeply into receptor core.

6. Antagonists and Their Mechanisms

• Antagonists block agonist action by:

  • Competing for the same receptor site (orthosteric binding).

  • Acting at a different site in the signaling pathway.

  • Activating opposing physiological mechanisms.

  • Chemically interacting with the agonist.

Types of Antagonists

1. Competitive Reversible Antagonists:

   • Bind at the same site as the agonist (orthosteric).

   • Examples: Propranolol, Alprenolol, Atenolol.

   • Increase agonist concentration required for the same effect.

2. Non-Competitive Antagonists:

   • Bind at a different site or act through alternative mechanisms.

   • Can alter receptor conformation or downstream signaling.

   • Example: Proteinase-Activated Receptor 2 antagonist.

7. Orthosteric vs. Allosteric Binding

• Orthosteric ligands: Bind at the natural ligand site (e.g., neurotransmitter binding site).

• Allosteric ligands: Bind at other receptor sites, modifying orthosteric ligand effects.

  • Do NOT produce effects alone but alter agonist activity.

  • Advantages:

    • Greater selectivity (not evolutionarily conserved).

    • Saturable (safer in overdose).

Allosteric Ligands' Effects

• Can increase or decrease:

  1. Potency (affinity for receptor).

  2. Efficacy (maximal response).

• Offer cooperative effects with orthosteric ligands.

8. Inverse Agonism

• GPCRs exhibit some level of constitutive activity (active without ligand binding).

• Inverse agonists: Bind and suppress constitutive activity by stabilizing the inactive (R) state.

• Clinical Relevance:

  • Mutations causing high constitutive activity → Disease association.

Types of Ligands:

1. Full agonist: Fully activates receptor (R* state).

2. Partial agonist: Partially activates receptor.

3. Full inverse agonist: Completely stabilizes inactive state (R).

4. Partial inverse agonist: Reduces, but does not eliminate, constitutive activity.

9. Bitopic Ligands & GPCR Selectivity

• Bitopic ligands: Bind to both orthosteric and allosteric sites.

• Advantages:

  • Higher specificity for receptor subtypes.

  • Potential signal bias (preferentially activating specific pathways).

• Example:

  • Dopamine, Acetylcholine, and Serotonin receptors have multiple subtypes → Bitopic ligands can improve selectivity between closely related receptors.

Lecture 3: receptors 1

1. Introduction to Cell Signaling

• Cell signaling is essential for maintaining homeostasis in multicellular organisms.

• Communication occurs through extracellular signals that regulate cell function.

2. Six Steps of Cell-Cell Communication

1. Synthesis of the signaling molecule.

2. Release of the signaling molecule.

3. Transport to the target cell.

4. Detection by specific receptors.

5. Alteration in cell function.

6. Removal of the signal (termination).

3. Modes of Cell-Cell Communication

A. Autocrine Signaling

• Cell releases a signal and responds to it itself.

• Example: Epinephrine acting on presynaptic α2-adrenoceptors (inhibits neurotransmitter release).

B. Plasma Membrane-Attached Proteins

• Signal remains tethered to the membrane of the signaling cell.

• Example: Ephrins binding Ephrin receptors.

C. Paracrine Signaling

• Soluble signals act on nearby target cells.

• Example: Acetylcholine at the neuromuscular junction.

D. Endocrine Signaling

• Hormones travel through the bloodstream to distant target cells.

• Example: Insulin secreted by pancreatic β-cells.

4. Types of Receptor Systems

A. Intracellular Receptors

• Examples: Steroid and thyroid hormone receptors.

• Mechanism:

  • Hydrophobic molecules cross the plasma membrane.

  • Bind receptor in the cytosol.

  • Receptor-ligand complex translocates to the nucleus.

  • Acts as a transcription factor.

B. Plasma Membrane Receptors

• Examples: Epinephrine (adrenaline) and histamine receptors.

• Mechanism:

  • Hydrophilic ligands bind to surface receptors.

  • Triggers intracellular signaling via second messengers.

  • Leads to metabolic, gene expression, or structural changes.

5. How Cells Respond to Signals

• Receptors translate extracellular signals into intracellular responses.

• Three key functions of receptors:

  1. Detect and bind agonists.

  2. Couple to intracellular signaling pathways.

  3. Amplify signals for a strong intracellular response.

• Response time varies:

• Skeletal muscle (milliseconds).

• Heart muscle (seconds).

• Signal termination is crucial to prevent continuous activation.

6. Plasma Membrane Receptor Classification

1. Ligand-Gated Ion Channels

   • Example: Nicotinic acetylcholine receptor (nAChR).

2. Receptors with Intrinsic Enzymatic Activity

   • Example: Receptor Tyrosine Kinases (RTKs) – insulin, platelet-derived growth factor.

3. Receptors Non-Covalently Associated with Enzymes

   • Example: gp130 (IL-6 receptor) associates with Janus kinases (JAKs).

4. G-Protein-Coupled Receptors (GPCRs)

   • Example: Adrenoceptors.

7. Nicotinic Acetylcholine Receptor (nAChR)

A. Function & Importance

• Fast synaptic transmission (milliseconds).

• Located at neuromuscular junctions, autonomic ganglia, and the CNS.

• Rapid response ensures complex muscle movements.

B. Activation Mechanism

1. Acetylcholine (ACh) binds to receptor on the postsynaptic membrane.

2. Receptor transiently increases Na⁺ influx and K⁺ efflux.

3. Depolarization opens voltage-gated sodium channels → Action potential → Muscle contraction.

C. Ion Selectivity

• nAChR is a non-selective cation channel.

• Relative permeability:

  • Na⁺ = 100%

  • K⁺ = 111%

  • Li⁺ = 87%

8. Structure of nAChR

• Pentameric receptor (5 subunits): (α1)₂β1γδ.

• Forms a cation-conducting pore (6.5 Å wide).

• Common structure:

  • Extracellular N-terminal domain (ligand-binding site).

  • Four transmembrane (TM) α-helices (M1–M4) per subunit.

  • M2 helix lines the channel pore and controls ion passage.

A. Molecular Basis of Channel Opening

1. Resting State:

   • Leucine residues in M2 helix form a "gate" that blocks ion flow.

2. ACh Binding:

   • Two ACh molecules bind to extracellular receptor sites.

3. Channel Activation:

   • Helices tilt outward, opening the pore (~1 ms).

   • 25,000 Na⁺ ions enter per millisecond.

   • Gate rapidly closes as ACh dissociates.

9. Other Aspects of nAChR Function

• Multiple receptor subtypes exist (α1–α9, β1–β4).

• Different nAChR combinations determine receptor properties.

• Muscle vs. Brain subtypes:

  • Muscle: (α1)₂β1γδ.

  • Brain: (α4)₂(β2)₃.

A. Neurotoxins that Bind nAChR

• α-Bungarotoxin (Kd = 1 nM).

• Cobratoxin, curare – block receptor function.

• First ion channel to be purified and cloned.

B. nAChRs and Anesthetics

• Certain anesthetics (isoflurane, halothane, nitrous oxide) bind nAChRs.

• Reduce channel opening time.

C. Nicotine Sensitivity & Addiction

• α4-containing nAChRs linked to nicotine addiction.

• β2 deletion in mice reduces addiction risk.

• Ongoing genetic studies to personalize treatments.

10. Ligand-Gated Ion Channel Superfamily

• Gene duplication led to multiple ion channel families.

• Other ligand-gated ion channels:

  • Excitatory:

    • Nicotinic ACh receptor (Na⁺/K⁺ channel).

    • Glutamate receptor (Na⁺/K⁺ channel).

    • Serotonin (5HT3) receptor (Na⁺/K⁺ channel).

  • Inhibitory:

    • GABA (GABAA) receptor (Cl⁻ channel).

    • Glycine receptor (Cl⁻ channel).

Lecture 4: receptors 2

1. Introduction to Receptor Signaling

• Receptor signaling is essential for maintaining homeostasis in multicellular organisms.

• Cellular communication occurs via auto-, para-, and endocrine signaling.

• Receptors detect extracellular signals and translate them into intracellular responses.

• Five classes of plasma membrane receptors exist (e.g., GPCRs, ligand-gated ion channels).

2. Turning Off Receptor Signaling: Desensitization Mechanisms

A. What is Desensitization?

• Definition: A receptor-mediated response initially increases but then plateaus and diminishes despite continuous agonist exposure.

• Example: Glucagon-like peptide-1 (GLP-1) elevation of cAMP in pancreatic β-cells.

• Functional Roles of Desensitization:

  • Allows cells to rapidly adapt to changes in agonist concentration.

  • Prevents excessive activation, which could be toxic.

  • Resets the system for another round of signaling.

B. Four Major Mechanisms of Desensitization

1. Receptor Modification (Seconds to Minutes)

   • Commonly involves phosphorylation (GPCRs) or dephosphorylation (RTKs).

   • Example: Protein kinase A (PKA) phosphorylates β₂-adrenoceptor to inhibit signaling.

   • Example: Protein tyrosine phosphatases (PTPs) dephosphorylate receptor tyrosine kinases (RTKs).

2. Receptor Sequestration (Minutes to Hours)

   • Receptor internalization into endosomal compartments.

   • Example: β₂-adrenoceptor sequestration following prolonged stimulation.

3. Receptor Downregulation (Hours)

   • Internalized receptors degraded in lysosomes, reducing receptor numbers.

   • Leads to long-term receptor desensitization.

4. Inhibitory Protein Induction (Hours)

   • Signaling triggers gene expression of negative regulators.

   • Example: Suppressors of cytokine signaling (SOCS) proteins inhibit JAK-STAT signaling.

3. Types of Receptor Desensitization

• Homologous Desensitization:

  • Occurs at the same receptor activated by an agonist.

  • Example: β₂-adrenoceptor phosphorylation by GRKs and arrestin binding.

• Heterologous Desensitization:

  • Occurs when activation of one receptor inhibits another receptor.

  • Example: PKA phosphorylation of β₂-adrenoceptor after activation of other GPCRs.

4. Model System 1: β₂-Adrenoceptor Desensitization

A. Cyclic AMP-Mediated Desensitization

• PKA phosphorylates Ser and Thr residues in the β₂-adrenoceptor.

• Effects:

  • Prevents G-protein coupling (reduces cAMP production).

  • Blocks receptor interaction with Gs-proteins.

B. GPCR Kinases (GRKs) & Arrestin in Homologous Desensitization

1. GRK phosphorylates activated β₂-adrenoceptor (at Ser/Thr residues near the C-terminus).

2. Arrestin binds phosphorylated receptor.

3. Results in:

   • Inhibition of receptor-G protein interaction.

   • Receptor sequestration into endosomes via clathrin-coated pits.

C. Visualizing β₂-Adrenoceptor Sequestration

• Fluorescently labeled β₂-adrenoceptors:

  • Without agonist: Exclusively at the plasma membrane.

  • After 30 min of isoprenaline exposure: Receptors internalized into endosomes.

D. Arrestin Triggers Receptor Degradation

• Arrestins recruit NEDD4, which polyubiquitinates the receptor.

• Receptor is targeted to lysosomes for degradation.

E. Time Course of β₂-Adrenoceptor Desensitization & Recovery

• Two phases of desensitization:

  1. Rapid Phase: Due to receptor phosphorylation (minutes).

  2. Slow Phase: Due to receptor degradation (hours).

• Recovery:

• Rapid phase: Receptors are dephosphorylated and recycled (minutes).

• Slow phase: Requires new receptor synthesis (hours).

5. Model System 2: Cytokine Receptor (JAK-STAT) Desensitization

A. JAK-STAT Signaling Pathway

1. Cytokine binds receptor → induces receptor dimerization.

2. Janus kinases (JAKs) phosphorylate Tyr residues on the receptor.

3. Signal transducers and activators of transcription (STATs) bind phosphorylated receptor.

4. STATs get phosphorylated, dimerize, and move to the nucleus → gene transcription.

B. SOCS Proteins: Negative Regulators of JAK-STAT Signaling

• STAT dimers trigger SOCS gene transcription.

• SOCS proteins inhibit JAK-STAT signaling by:

  1. Binding to phosphorylated receptors (blocks JAK activity).

  2. Inducing polyubiquitination of JAKs (leads to degradation by proteasomes).

C. SOCS-Mediated Desensitization

• Homologous Desensitization:

  • Example: SOCS-3 inhibits IL-6 signaling via gp130 receptor.

• Heterologous Desensitization:

  • Example 1: G-CSF induces SOCS-3 expression, inhibiting IL-6 signaling.

  • Example 2: LPS (from Gram-negative bacteria) induces SOCS-3, inhibiting gp130 via NF-κB.

6. Disease Implications of Receptor Desensitization Defects

A. Nephrogenic Diabetes Insipidus (NDI)

• Cause: Mutations in the V2 vasopressin receptor (a GPCR) → failure to concentrate urine.

• Example Mutation: Arg137His leads to constitutive receptor internalization.

B. Polycythemia Vera (PV)

• Cause: JAK2 Val617Phe mutation → constitutive JAK-STAT activation.

• Effect:

  • Hyperproliferation of hematopoietic stem cells → excess red blood cells → clots, stroke, heart attack.

  • Phosphorylates SOCS-3, preventing it from inhibiting JAK2 signaling.

C. Cholangiocarcinoma (CCA)

• Highly lethal cancer of bile duct epithelium.

• Associated with chronic inflammation and SOCS-3 promoter hypermethylation.

• Effect:

  • Epigenetic silencing of SOCS-3 → sustained IL-6 signaling → cancer progression.

Lecture 5: yeast signaling

1. Introduction to Yeast Signaling

• Saccharomyces cerevisiae (Baker’s/Brewer’s yeast)

  • Model eukaryote for studying signaling pathways.

  • Complete genome sequenced (~6000 genes).

  • Experimentally tractable with available genetic and molecular tools.

• Significance of Yeast Signaling:

  • Single cells must respond to environmental stimuli.

  • Used to study evolution of signaling in eukaryotes.

  • Cells communicate via signals like hormones, cytokines, and growth factors.

• Yeast Life Cycle & Environmental Responses:

  • Proliferates by budding (asexual reproduction).

  • Undergoes meiosis to form haploid spores (a & α mating types).

  • Mating occurs in rich medium, forming diploids.

  • Sporulation occurs under nutrient starvation.

2. Yeast Mating Signaling Pathway

• Yeast mating involves communication between haploid cells (a and α types).

• Pheromone signaling initiates mating process.

A. Pheromone Receptors

• Ste2p (a-cell receptor): Binds α-factor (pheromone).

• Ste3p (α-cell receptor): Binds a-factor (pheromone).

B. G-Protein-Coupled Receptor (GPCR) Activation

1. Ste2p (GPCR) binds α-factor → Conformational change.

2. Activates heterotrimeric G-protein complex:

   • Gpa1p (Gα).

   • Ste4p (Gβ) + Ste18p (Gγ).

3. Ste4p/Ste18p recruits Ste5p (scaffolding protein) to the plasma membrane.

   • Ste5p requires a pleckstrin homology (PH) domain for membrane binding.

C. MAPK Cascade in Yeast Mating

• Ste5p organizes kinases into a signaling cascade:

  1. Ste20p: Phosphorylates Ste11p (MAPKKK).

  2. Ste11p: Phosphorylates Ste7p (MAPKK).

  3. Ste7p: Phosphorylates Fus3p (MAPK).

  4. Fus3p: Activates transcription factors (Ste12p) for mating-specific genes.

• MAPK cascades are conserved across eukaryotes (mammals, plants).

• Allows signal amplification & ultrasensitive responses.

D. Achieving Specificity in Yeast MAPK Pathways

• Scaffolding Proteins:

  • Ste5 (mating).

  • Pbs2 (HOG pathway for osmotic stress).

• Cross-Pathway Inhibition:

  • Mating pathway inhibits filamentation (Fus3 phosphorylates Tec1, causing its degradation).

3. Nutrient Sensing & Response in Yeast

A. Glucose Repression in S. cerevisiae

• Glucose is the preferred energy source.

• Genes for metabolism of alternative carbon sources (e.g., sucrose, galactose) are repressed in the presence of glucose.

• Key regulator: SNF1 kinase complex (yeast homolog of AMPK).

B. SNF1 Pathway Activation

1. SNF1 (AMPK) is activated under low glucose conditions.

2. SNF1 phosphorylates transcriptional activator Sip4p, increasing expression of glucose-repressed genes.

3. SNF1 phosphorylates transcriptional repressor Mig1p, alleviating repression.

• SNF1 complex components:

  • Snf1p (catalytic subunit).

  • Regulatory subunits: Sip1p, Sip2p, or Gal83p.

  • Activation requires phosphorylation at Thr210.

4. Target of Rapamycin (TOR) Pathway in Yeast

• TOR pathway regulates growth & metabolism in response to nutrients.

• Rapamycin (antifungal) inhibits TOR activity.

A. TOR Complexes

• Two complexes exist in yeast:

  1. TORC1 (sensitive to rapamycin, responds to nutrients).

  2. TORC2 (distinct functions, not sensitive to rapamycin).

• TORC1 Activation:

• Promotes growth, proliferation, and protein translation.

• Inhibits autophagy.

• Stimulates ribosome biogenesis.

B. Glucose & Amino Acid Sensing by TORC1

• Glucose sensing:

  • Kog1p (TORC1 subunit) is phosphorylated by SNF1 under glucose starvation, inhibiting TORC1 activity.

• Amino acid sensing:

  • Leucine, methionine, glutamine regulate TORC1 via multiple mechanisms.

C. Downstream Effects of TORC1 Activation

• Phosphorylation of Sch9p (Ser/Thr kinase):

  • Promotes ribosomal biogenesis & translation initiation.

  • Regulates entry into G0 phase.

Lecture 6: metabolic signaling

1. Introduction to Metabolic Signaling

• Metabolism is a dynamic equilibrium:

  • Organisms maintain a constant internal composition by taking in nutrients and eliminating waste.

  • Metabolism must adapt to environmental changes (nutrient availability, O₂ levels, redox status).

  • Regulation occurs at both short and long timescales.

• Cellular Energy Charge:

  • ATP is the universal energy currency.

  • Catabolic pathways generate ATP.

  • Anabolic pathways consume ATP.

  • Adenylate Kinase Reaction: 2ADP→ATP+AMP2 ADP → ATP + AMP2ADP→ATP+AMP

  • AMP:ATP ratio serves as an energy sensor for the cell.

2. AMP-Activated Protein Kinase (AMPK)

A. Overview of AMPK

• A Ser/Thr kinase that senses AMP:ATP ratio.

• Activated when AMP levels rise (low energy state).

• Phosphorylates metabolic enzymes to:

  • Inhibit anabolic, ATP-consuming pathways.

  • Stimulate catabolic, ATP-producing pathways.

B. AMPK Structure

• Heterotrimeric complex:

  • α subunit: Catalytic kinase domain.

  • β subunit: Regulatory domain.

  • γ subunit: AMP-binding domain (CBS domains).

• Evolutionarily conserved:

  • Snf1p in yeast.

  • SnRK1 in plants.

  • aak-1 & aak-2 in C. elegans.

C. AMPK Activation

1. AMP binding promotes AMPK phosphorylation at Thr172.

2. AMP prevents dephosphorylation of Thr172, sustaining AMPK activity.

3. Key AMPK kinases (AMPKKs):

   • LKB1 (constitutively active).

   • Ca²⁺/Calmodulin-dependent kinase kinase-β (CaMKKβ).

4. Inhibition by ATP:

   • ATP competes with AMP for binding to the γ-subunit, preventing activation.

D. When Does the AMP:ATP Ratio Increase?

• Hypoglycemia (low blood glucose).

• Hypoxia (low oxygen).

• Ischemia (restricted blood flow).

• Metabolic poisons (e.g., cyanide).

• Exercise (muscle ATP rapidly depleted).

3. AMPK Targets & Regulation of Metabolism

A. Acetyl-CoA Carboxylase (ACC)

• Key enzyme in fatty acid synthesis.

• Converts Acetyl-CoA → Malonyl-CoA (precursor for FA synthesis).

• Malonyl-CoA inhibits fatty acid oxidation (via CPT1 inhibition).

• AMPK phosphorylates ACC at Ser79:

  • Inhibits FA synthesis (↓ ATP consumption).

  • Stimulates FA oxidation (↑ ATP production).

B. Hydroxymethylglutaryl-CoA Reductase (HMGR)

• Rate-limiting enzyme in cholesterol synthesis.

• Converts Acetyl-CoA → Mevalonate (cholesterol precursor).

• AMPK phosphorylates HMGR at Ser871:

  • Inhibits cholesterol synthesis (↓ ATP consumption).

  • Prevents excessive cholesterol buildup.

• Ser79 (ACC) & Ser871 (HMGR) are required for AMPK regulation.

4. AMPK & mTORC1 Regulation

A. What is mTOR?

• mTOR (mechanistic target of rapamycin): A Ser/Thr kinase that regulates cell growth and metabolism.

• Two distinct complexes:

  • mTORC1: Regulates translation & cell growth.

  • mTORC2: Involved in cytoskeletal organization.

B. mTORC1 Activation & Inhibition

• mTORC1 is activated by:

  • Growth factors (insulin, IGF-1, PDGF, EGF).

  • Amino acids (Leu, Arg, Met).

  • Energy abundance (high ATP levels).

• mTORC1 promotes:

  • Protein synthesis (via S6K and 4EBP1 phosphorylation).

  • Ribosome biogenesis and cell proliferation.

• AMPK Inhibits mTORC1 via Two Mechanisms:

• Phosphorylates Raptor, reducing mTORC1 activity.

• Phosphorylates TSC2, which inactivates Rheb, a GTPase that activates mTORC1.

• Energy Deficiency → AMPK Activation → mTORC1 Inhibition → Conserving Resources.

5. Growth Factor-Mediated Activation of mTORC1

A. PI3K-Akt Pathway & mTORC1 Activation

1. Growth factors (e.g., insulin) bind to receptor tyrosine kinases (RTKs).

2. PI3K (phosphoinositide 3-kinase) phosphorylates PIP₂ → PIP₃.

3. PIP₃ recruits PDK1 (phosphoinositide-dependent kinase 1).

4. PDK1 phosphorylates Akt at Thr308.

5. Akt phosphorylates TSC2 at inhibitory sites, preventing TSC1-TSC2 inhibition of mTORC1.

6. mTORC1 is activated → Protein synthesis & cell growth.

B. PTEN: Negative Regulator of mTORC1

• PTEN (phosphatase & tensin homolog) dephosphorylates PIP₃ → PIP₂.

• Loss of PTEN (common in cancers) leads to hyperactive mTORC1 signaling.

6. Summary of Key Regulatory Pathways

Lecture 7: nuclear signaling

1. Introduction to Nuclear Receptors

• Nuclear receptors (NRs) are ligand-activated transcription factors.

• Bind lipophilic molecules (hormones, vitamins, lipid metabolites).

• Control gene expression by interacting with DNA.

Key Differences from Other Receptor Types:

2. Nuclear Receptor Ligands

A. Steroid Hormones (Derived from cholesterol)

• Glucocorticoids: Cortisol (stress response).

• Mineralocorticoids: Aldosterone (salt & water balance).

• Sex Hormones: Testosterone, Estrogen, Progesterone (sexual function).

B. Vitamins

• Vitamin D: Derived from 7-dehydrocholesterol (Ca²⁺ & PO₄³⁻ balance).

• Vitamin A (Retinoic Acid): From retinoids & carotenoids (skin, embryonic development).

• Thyroid Hormone (T₃): Synthesized from tyrosine residues in thyroglobulin (metabolic regulation).

C. Lipid Metabolites

• Fatty Acids & Prostaglandins

• Haem, Bile Acids, Cholesterol, Oxysterols

3. Nuclear Receptor Signaling Mechanism

• Ligand binding induces receptor conformational change.

• Triggers recruitment of co-regulatory proteins (co-activators or co-repressors).

• Modulates gene expression (upregulation or downregulation).

4. Structural Organization of Nuclear Receptors

• DNA-binding domain (DBD): Contains two zinc-finger motifs.

• Ligand-binding domain (LBD): Binds lipophilic hormones and vitamins.

• Dimerization domain: Allows receptor pairing (homo- or heterodimerization).

• Transactivation domain: Interacts with co-regulators (co-activators/co-repressors).

Hormone Response Elements (HREs)

• Located in the promoter region of target genes.

• Response elements are palindromic or direct repeats.

• Examples:

  • Oestrogen response element (ERE).

  • Glucocorticoid response element (GRE).

  • Thyroid hormone response element (TRE).

  • Vitamin D response element (VDRE).

  • Retinoic acid response element (RARE).

5. Classification of Nuclear Receptors

6. Type I Nuclear Receptors (Steroid Hormone Receptors)

• Unbound receptor forms a complex with heat shock proteins (Hsp90, Hsp70).

• Ligand binding → Dissociation of Hsp complex.

• Receptor homodimerizes → Translocates to nucleus.

• Binds hormone response elements (HREs) on DNA.

• Recruits co-activators → Transcriptional activation.

Example: Oestrogen Receptor (ER)

• Binds to oestrogen response element (ERE).

• Selective oestrogen receptor modulators (SERMs):

  • Tamoxifen: ER antagonist in breast cancer therapy.

  • Clomifene: Used in infertility treatment (PCOS).

  • Conjugated equine oestrogens: Hormone replacement therapy.

7. Type II Nuclear Receptors (Heterodimeric with RXR)

• Always in the nucleus, bound to DNA.

• In the absence of ligand → Bound to co-repressors (inhibitory complex).

• Ligand binding → Co-repressor dissociation → Co-activator recruitment.

• Activates transcription.

Examples:

• Vitamin D Receptor (VDR).

• Retinoic Acid Receptor (RAR).

• Thyroid Hormone Receptor (THR).

• Peroxisome Proliferator-Activated Receptors (PPARs).

8. Co-Regulators of Type II Nuclear Receptors

• Co-repressors:

  • Recruit histone deacetylases (HDACs) → Gene repression.

• Co-activators:

  • Contain histone acetyltransferase (HAT) activity → Gene activation.

Regulation by Chromatin Remodeling

• Co-activators open chromatin (euchromatin) → Increased transcription.

• Co-repressors tighten chromatin (heterochromatin) → Decreased transcription.

9. Orphan Nuclear Receptors

• Have no well-defined endogenous ligands.

• May act as metabolic sensors.

• Mostly function as heterodimers with RXR.

Examples:

• Bile Acid Receptor (FXR).

• Liver X Receptor (LXR).

• Peroxisome Proliferator-Activated Receptors (PPARs).

PPARs: Lipid Sensors

10. Non-Genomic Actions of Nuclear Receptors

• Some nuclear receptors act outside the nucleus via rapid, non-genomic mechanisms.

• Examples:

  • Steroid receptors (oestrogen, androgen, progesterone) activate kinases at the plasma membrane.

  • VDR & THR activate MAPKs and PI3K pathways.

11. Summary of Nuclear Receptor Functions

Lecture 8&9: immune signalling 1&2

1. Overview of Immune Signaling

• Immune system detects & responds to infection via signaling pathways.

• Key components:

  • Pathogen-associated molecular patterns (PAMPs): Conserved microbial structures.

  • Pattern recognition receptors (PRRs): Detect PAMPs and trigger an immune response.

  • Cytokines: Signaling molecules that regulate immune activity.

• Types of Immunity:

  • Innate Immunity: Fast, nonspecific, does not improve with exposure.

  • Adaptive Immunity: Slow, specific, improves upon repeated exposure.

2. Pathogen Recognition by Pattern Recognition Receptors (PRRs)

• PRRs recognize PAMPs to initiate immune responses.

• Four main PRR families:

• Toll-like receptors (TLRs)

  • Named after the Drosophila Toll gene.

  • Found in plasma membrane & endosomes.

  • Activate NF-κB and MAPK pathways.

A. TLR4 and Lipopolysaccharide (LPS) Signaling

• LPS (from Gram-negative bacteria) activates TLR4.

• Signaling process:

  1. LPS binds MD-2 → TLR4 dimerization.

  2. Adaptor proteins (MyD88, TRIF) recruit kinases.

  3. Activation of MAPKs & NF-κB → Cytokine production.

3. NF-κB & MAPK Activation via TLR4 Signaling

A. MyD88-Dependent Pathway (Early Response)

1. TLR4 interacts with MyD88 via TIR domains.

2. MyD88 recruits IRAK1 & IRAK4 (IL-1 receptor-associated kinases).

3. IRAK1 activates TRAF6 (E3 ubiquitin ligase).

4. TRAF6 promotes TAK1 activation → MAPK & NF-κB signaling.

5. NF-κB translocates to nucleus → Cytokine gene expression.

B. Ubiquitination in TLR4 Signaling

• K63-linked polyubiquitination (TRAF6) → Scaffold for kinases.

• K48-linked polyubiquitination (IKK activation) → Proteasomal degradation of IκB (NF-κB inhibitor).

C. Activation of MAPK & NF-κB

• MAPK Pathway:

  • TAK1 phosphorylates JNK.

  • JNK translocates to nucleus → AP-1 activation.

• NF-κB Pathway:

  • IκB phosphorylated & degraded → Releases NF-κB dimers.

  • NF-κB enters nucleus → Induces pro-inflammatory genes.

4. Cytokine Signaling & Immune Regulation

• Cytokines = Secreted proteins that regulate immune function.

• Three Major Functions:

A. Pro-Inflammatory Cytokines

1. IL-1, IL-6 & TNF-α are key inflammatory mediators.

2. Function:

   • Increase vascular permeability.

   • Stimulate adhesion molecules & neutrophil recruitment.

   • Enhance cytokine production (amplify response).

   • Induce fever & acute phase protein synthesis.

5. IL-1 Signaling via IL-1 Receptors

• IL-1 family cytokines (IL-1β, IL-18, IL-33) share common pathways.

• IL-1R signaling is highly similar to TLR4.

1. IL-1 binds IL-1R → Forms heterodimeric complex with IL-1RAcP.

2. TIR domain recruits MyD88.

3. Activation of IRAK, TRAF6, TAK1 → NF-κB & MAPKs.

4. Inflammatory gene transcription.

6. TNF-α Signaling via TNF Receptors (TNFRs)

• TNF-α: A master regulator of inflammation.

• TNFR Types:

  • TNFR1 (Ubiquitous expression).

  • TNFR2 (Restricted to immune cells).

TNFR1 Signaling Pathway

1. TNF-α binds trimeric TNFR1 → Induces receptor trimerization.

2. TRADD (TNFR-associated death domain) recruited.

3. TRADD recruits TRAF2, cIAPs, RIP1 (E3 ubiquitin ligases).

4. TAK1 activation → NF-κB & MAPK signaling.

5. Induction of inflammatory gene expression.

• TNFR1 can also trigger apoptosis.

  • TRADD recruits FADD → Caspase activation → Cell death.

7. IL-6 Signaling via JAK-STAT Pathway

• IL-6 regulates inflammation & immune cell activation.

• Receptor complex:

  • IL-6Rα (ligand binding subunit).

  • gp130 (signal transduction subunit).

IL-6R Signaling Pathway

1. IL-6 binding induces gp130 dimerization.

2. JAKs (Janus kinases) phosphorylate gp130.

3. STAT3 binds phospho-Tyr residues on gp130.

4. JAK phosphorylates STAT3 → STAT3 dimerizes & translocates to nucleus.

5. STAT3 induces expression of inflammatory genes.

• Negative Regulation:

  • SOCS (Suppressor of cytokine signaling) inhibits JAK.

  • SHP phosphatases dephosphorylate receptors.

8. Clinical Applications: Targeting Immune Signaling in Disease

A. Anti-TNF Therapy (Rheumatoid Arthritis, Psoriasis, IBD)

• TNF-α antagonists reduce excessive inflammation.

• Examples:

  • Etanercept (TNFR decoy receptor).

  • Adalimumab (TNF-α monoclonal antibody).

B. Anti-IL-1 Therapy

• Anakinra (IL-1 receptor antagonist) blocks IL-1 signaling.

• Canakinumab (anti-IL-1β antibody) used in inflammatory diseases.

C. Anti-IL-6 Therapy

• Tocilizumab (anti-IL-6R monoclonal antibody).

• JAK Inhibitors (e.g., Tofacitinib) block IL-6 JAK-STAT signaling.

Lecture 10&11

1. Overview of Druggable Targets

• The "druggable genome" refers to the full set of genes encoding proteins that can be targeted by drugs.

• Early estimates (~2002): ~3,000 druggable genes.

• Recent estimates (~2017): ~4,500 druggable genes (~22% of the genome).

• Only ~700 human proteins are targeted by approved drugs.

2. Key Druggable Gene Families

A. G Protein-Coupled Receptors (GPCRs)

• Largest family of drug targets (~900 GPCR genes).

• Highly conserved across eukaryotes.

• Functions: Neurotransmission, cell growth, vision, olfaction.

• >30% of prescribed drugs act at GPCRs.

• Examples:

  • Salbutamol: β₂-adrenoceptor agonist (asthma).

  • Morphine: μ-opioid receptor agonist (analgesic).

  • Losartan: Angiotensin II receptor antagonist (hypertension).

Why are GPCRs good drug targets? ✅ Diverse biological functions.
✅ Multiple receptor subtypes → Selectivity possible.
✅ Accessible at the cell surface (no need to cross membrane).
✅ Amenable to high-throughput screening (HTS).

B. Ion Channels

• >400 ion channel types, classified as:

  • Ligand-gated channels (e.g., GABAₐ, nicotinic ACh).

  • Voltage-gated channels (e.g., NaV, CaV).

• Examples of Ion Channel Drugs:

  • Lidocaine: NaV channel blocker (local anesthetic).

  • Calcium channel blockers: Lower blood pressure.

  • Sulfonylureas: Block KATP channels → Insulin release.

Why are Ion Channels good drug targets? ✅ Crucial in neuronal signaling, muscle contraction, hormone release.
✅ No need for drugs to enter cells (extracellular binding sites).
✅ Tissue-specific expression allows for selectivity.

C. Nuclear Receptors

• 48 nuclear receptors in humans.

• Regulate gene expression in response to lipophilic molecules.

• Ligands: Steroid hormones, thyroid hormones, vitamins, fatty acids.

• Examples:

  • Tamoxifen: Estrogen receptor modulator (breast cancer).

  • Troglitazone: PPARγ activator (diabetes).

  • Cortisol: Glucocorticoid receptor activator (anti-inflammatory).

Why are Nuclear Receptors good drug targets? ✅ Central role in metabolism, homeostasis, development.
✅ Good understanding of receptor structure & function.
❌ Challenges:

• Drugs must enter cells to reach targets.

• Off-target effects due to widespread gene regulation.

D. Protein Kinases

• ~500 kinases in the human genome.

• Key role in intracellular signal transduction.

• Phosphorylate proteins on serine, threonine, or tyrosine residues.

• Examples of Kinase Drugs:

  • Imatinib (Gleevec): BCR-ABL inhibitor (chronic myeloid leukemia).

  • Trastuzumab (Herceptin): HER2 inhibitor (breast cancer).

  • Vemurafenib: B-RafV600E inhibitor (melanoma).

Why are Kinases good drug targets? ✅ Essential regulators of cell growth & survival.
✅ Dysregulation linked to many diseases (cancer, inflammation).
❌ Challenges:

• Highly conserved catalytic site → Selectivity issues.

• Off-target effects common.

3. Lead Discovery & Optimization

• After identifying a target, drugs must be optimized for:

  • Potency (low dose, strong effect).

  • Selectivity (avoiding off-target interactions).

  • Pharmacokinetics (absorption, metabolism, elimination).

  • Safety (low toxicity).

Process:

1. High-throughput screening (HTS) → Identify initial "hit" compounds.

2. Structure-Activity Relationship (SAR) analysis → Improve potency/selectivity.

3. Lead optimization → Create the most effective compound.

4. Case Study: Free Fatty Acid Receptor Agonists for Type 2 Diabetes

• Target: FFA4 receptor.

• Rationale: FFA4 knockout mice had increased obesity, inflammation, & insulin resistance.

• Approach:

  • HTS identified a "hit" compound (TUG-670).

  • SAR studies improved potency (TUG-891).

  • TUG-891 issues: Low specificity, rapid metabolism.

  • Further optimization needed for clinical use.