L26,27

Types of Hormones

• Hormones are categorized according to their chemical structure.

Amino Acid-Derived Hormones

• Synthesized from individual amino acids (e.g., tyrosine).
• Thyroid hormone is an example.
• The starting component undergoes modification by enzymes to become the active hormone.

Peptide Hormones

• Produced similarly to proteins (gene transcription & translation into a polypeptide).
• Often, the initial polypeptide is a pre-hormone (inactive state).
• Further modification may occur:
  • Cleavage by enzymes into the active peptide.
  • Addition of sugars, forming a glycoprotein or glycopeptide (e.g., TSH, erythropoietin).
  • The type of sugar is critical for hormone activity.

Lipid-Derived Hormones

• Steroid hormones (testosterone, estrogens, progesterone) are derived from cholesterol.
• Eicosanoids are derived from arachidonic acid.

Hormone Solubility and Transport

• Solubility influences release, transport, and signal transmission.
• Water-soluble hormones:
  • Hydrophilic, so they cannot passively cross the plasma membrane.
  • Require active mechanisms for secretion from cells and signal transmission.
  • Soluble in blood and lymphatic system for easy diffusion.
  • Includes peptide hormones like insulin and amino acid-derived hormones (except thyroid hormone).
• Lipid-soluble hormones:
  • Can cross the plasma membrane easily.
  • Require transporter proteins for blood transport due to insolubility.
  • Includes thyroid hormones, steroid hormones, and eicosanoids.

Function of Water-Soluble Hormones

• Synthesized in the cell.
• Released from the cell.
• Transported in the blood.
• Exert function by binding to extracellular receptors on the target cell's plasma membrane.

Adrenaline and Noradrenaline

• Water-soluble hormones derived from tyrosine.
• Tyrosine is converted to L-DOPA by tyrosine hydroxylase (TH), which is the rate-limiting step.
• L-DOPA converts to dopamine, then noradrenaline, then adrenaline.

Peptide Hormone Processing

• Polypeptides are often processed into the active form.
• Insulin starts as proinsulin, cleaved into the active form when needed.

Neurohormone Cleavage

• A pre-prohormone is generated in the anterior pituitary gland.
• Activated into corticotropin and beta-lipotropin by specific enzymes.
• In the intermediate lobe, enzymes cleave further into different neurohormones.
• Regulation occurs by compartmentalizing enzymes into different brain regions, controlling neurohormone activation.

Hormone Secretion

• Once produced, water-soluble hormones are immediately packaged into vesicles. Synthesis and packaging happens constitutively.
• Vesicles await a stimulus to release the hormone.
• Exocytosis: Vesicle fuses with the plasma membrane, releasing the hormone into the extracellular fluid.
• This is regulated secretion, occurring only upon a stimulus. The mechanics of exocytosis will be explored in a later lecture.
• Released hormones travel freely in the blood to target cells.
• Target cells must have matching receptors on their surface.

Hormone Action at Target Cell

• Secretion, transport, and binding to a specific receptor activate intracellular signal transduction pathways.
• Not all ligands are hormones; cytokines and extracellular matrix proteins can also be ligands.
• Hormones are always ligands for a receptor.
• Two key changes follow signal transduction:
  • Fast-acting, rapid changes driven by existing proteins (non-genomic responses).
  • Slower, longer-term changes affecting gene transcription and protein production (genomic responses).

Review of Water-Soluble Hormones

• Synthesized before stimulus, stored in vesicles.
• Released upon a stimulus via exocytosis.
• Transported freely in the blood. They are not transported in the blood bound to carrier proteins.
• Signaling effect occurs by binding to receptors, inducing short-term (non-genomic) and long-term (genomic) effects.

Intracellular Signal Transduction

• Focus is on what happens within the cell: receptor activation and signal transmission to the nucleus or effector proteins.

Learning Targets

• Understanding the signal transduction cascade.
• Identifying different types of extracellular receptors.
• Understanding roles of protein kinases and protein phosphatases.
• Understanding the MAP kinase cascade.
• Understanding death receptor signaling pathways.
• Understanding termination of signaling pathways.
• Understanding altered signaling cascades in the context of miscommunication and disease.

The Signal Transduction Cascade

• Signal molecule binds to a receptor protein.
• The receptor protein undergoes a conformational change.
• Activation of intracellular signaling molecules.
• Relay of the message to target proteins, leading to genomic and non-genomic responses.
• Radio wave analogy: Signal finds a receptor (antenna), is transduced and amplified into a response (sound waves).
• Cellular responses: Changes in metabolism, cell structure, gene transcription.

Amplification and Multiple Pathways

• Amplification: A single signaling molecule activates one receptor, which then activates multiple downstream intracellular signaling proteins.
• Multiple pathways can be activated by one receptor.

Differential Cellular Response

• Different cells respond differently to the same external signal.
• Example: Acetylcholine, synthesized and released from nerve terminals, causes:
  • Heart pacemaker cells: Decreased firing rate.
  • Salivary gland cells: Secretion from preformed granules.
• Same ligand, same receptor, different effects due to cell type.
• Different Receptor: Voltage gated ion channel induces contraction of the smooth muscle cell.

Key Steps of Signal Transduction

• First messenger: Hormone or neurotransmitter.
• Specific receptor.
• Intracellular signal transduction via relay molecules or second messengers.
• Response/change in effector proteins.

Extracellular Receptors

• Three types:
  • Enzyme-linked receptors.
  • G protein-coupled receptors (GPCRs).
  • Ion channel-coupled receptors.
• All lead to activation of intracellular signal transduction pathways and cellular response.

Signal Complexity and Integration

• Extracellular ligand binding changes receptor confirmation, inducing binding of scaffold proteins that bring intracellular proteins together.
• Amplification of signal: Multiple copies of intracellular signaling proteins.
• Multiple pathways converging on one protein.
• Signal spread into other pathways.
• Complex crosstalk between pathways.

Ways Signals Can Be Integrated

• One receptor activates two pathways (e.g., MAP kinase and PI3 kinase).
• Multiple receptors activate the same pathway, amplifying the signal.
• Crosstalk: One receptor activates a protein that activates pathway #1 (e.g., MAP kinase pathway) and links into pathway #2.

Experimental Approaches

• Western blotting: Measures the activation of specific intracellular proteins like MAP kinase or ERK via phosphorylation.
• Mass spectrometry or proteomics: Measures the amount and activity (phosphorylation/post-translational modifications) of many proteins simultaneously.

Signaling Pathways in Disease

• Altered signaling cascades are associated with miscommunication and with disease.
• Signaling pathways are often hijacked in diseases, especially cancer.

Hallmarks of Cancer Cells

• Activation of cell viability/survival pathways.
• Overexpression of survival factors/receptors.
• Downregulation of death receptors.
• Rapid proliferation: Overexpression of growth factor receptors or downstream signaling proteins.
• Migration: Changes in cell adhesion proteins or integrins.
• Inhibition of differentiation signaling pathways.

Therapeutic Targeting of Signaling Pathways

• Drugs can inhibit ligands, receptors, or signal transduction cascade proteins (kinase inhibitors).
• Examples: Antibodies that bind to growth factors, antibody-based therapies that bind to receptors, small molecule inhibitors that block receptor activation or kinase activity.

Protein Kinases and Phosphatases

• Signal transduction cascades are mediated via phosphorylation.
• Phosphorylation leads to changes in protein structure, and therefore can change its function.
• Adding a phosphate group onto a protein quickly transmits the signal.

Protein Kinases

• Enzymes that catalyze the addition of a phosphate group from ATP onto a substrate protein.
• Usually activates a protein when phosphorylated.

Protein Phosphatases

• Enzymes that remove phosphate groups, switching off the signal.

Phosphorylation Sites

• Occurs on serines, threonines, and tyrosines.
• Receptor tyrosine kinases are phosphorylated on tyrosines.

Effects of Phosphorylation

• Changes protein activity (usually activation).
• Changes how a protein interacts with other proteins.
• Causes protein translocation from one part of the cell to another (e.g., cytosol to nucleus).

Other Post-Translational Modifications

• Acylation, methylation, glycosylation, ubiquitination can change activity/distribution.

Abundance of Kinases and Phosphatases

• 2-4% of protein-coding genes involve kinases/phosphatases.
• ~500 protein kinases in the human genome.
• ~90 target tyrosine residues.
• Serine/threonine kinases carry out most intracellular transduction.

Regulation of Kinases and Phosphatases

• Binding of other proteins.
• Activation of receptors.
• Addition of ions (magnesium, calcium).
• Second messengers (cyclic AMP).
• Phosphorylation or other post-translational modifications.

Key Concepts

• Kinases add phosphate groups (activating).
• Phosphatases remove phosphate groups (inactivating).
• In a quiescent cell, intracellular proteins are usually inactive.
• Upon stimulation: Phosphorylation usually activates them.

The MAP Kinase Pathway

• Also called MAPK or ERK pathway.
• Receptor activation (e.g., epidermal growth factor receptor, nerve growth factor receptor).
• Activation of RAF or MAPKKK.
• RAF phosphorylates MAPKK (MEK).
• MEK phosphorylates MAP kinase (MAPK or ERK).
• Activated MAP kinase phosphorylates target proteins, leading to early (non-genomic) and late (genomic) responses.
• A marker/biomarker of activation of this whole pathway is how much phosphorylated MAP kinase there is compared to total MAP kinase.
• PC-12 cells will be treated in the lab with nerve growth factor to activate receptor TrkA.

Orchestration of Signaling

• Scaffold, anchoring, or adaptor proteins bring components of the signal transduction cascade together in the right location.
• Adaptor proteins: Bring two different proteins together.
• Scaffold/anchor proteins: Bring together multiple proteins.
• Docking proteins: Dock to the plasma membrane, bringing components close to the receptor.
• Formation of microdomains: Scaffolding proteins present in lipid rafts, concentrating the signal in one place.

Death Receptor Signaling

• Tumor necrosis factor (TNF) receptor superfamily.
• TNF ligand forms a trimer, binding to TNF receptors (also trimers) on the cell's surface.
• Intracellular domains are called death domains.
• Adaptor proteins (FADD, TRADD) bring together death domains to activate caspases (enzymes that induce apoptosis).
• Death-inducing signaling complex (DISC) activates caspases.
• Cancer cells can evade this by blocking death domain activation and reducing adaptor protein expression.
• This is an extrinsic apoptosis pathway.* Dysregulation of the signalling is the one that leads to disease such as tumours because the body has lost the ability to switch off signalling.

Termination of Signaling

• Signals must be switched off to avoid hyperactivation.
• Mechanisms include:
  • Removal of the ligand.
  • Inactivation of the receptor:
    • Dephosphorylation (if activated by phosphorylation).
    • Binding of inhibitory proteins.
    • Internalization into an endosome.
    • Desensitization (receptor is in a state that is not ready to receive another receptor).
  • Inactivation of the intracellular cascade:
    • Dephosphorylation.
    • Binding inhibitory proteins.
    • Translocation.
    • Degradation/downregulation.
    • Sequestering it away from where it can be activated.

Summary of Signal Transduction

• Activation of cell surface receptors leads to signal transduction cascades.
• The message is relayed via signaling molecules, resulting in changes to effector proteins and a cellular response.
• Signal cascades can be highly complex.
• Dysregulation leads to different diseases.
• Phosphorylation/dephosphorylation are key mechanisms.
• Termination of the signal is essential.

Enzyme-Linked Receptors

• Enzymes themselves or linked to an enzyme.
• Transmembrane domain protein with extracellular, transmembrane, and intracellular domains.
• The intracellular domain either contains an active kinase domain or links to an enzyme via a conformational change.

Receptor Tyrosine Kinases (RTKs)

• Most common class of enzyme-linked receptors (approx. 60 in the human genome).
• Phosphorylate tyrosine residues.
• Often phosphorylate tyrosine residues within their own intracellular domain.

Key RTKs

• Epidermal growth factor receptor (EGFR).
• Insulin receptor.
• Nerve growth factor (NGF) receptor (TrkA).

Activation of RTKs

• Formation of dimers upon ligand binding.
• Transphosphorylation: Internal kinase domain phosphorylates the closely located dimer, activating it.
• Phosphorylation of other sites that enable binding of adaptor proteins.

Variety of Signals Generated by RTKs

• Transient assemblies lead to broadcasting the signal to different pathways.

Insulin Signaling Example

• Insulin receptor is a receptor tyrosine kinase.
• Insulin activates target cells (fat, liver) to stimulate the uptake of glucose, amino acids, and fatty acids.
• Binding of insulin causes phosphorylation, change in confirmation, and transphosphorylation of the receptor.
• Phosphorylation enables docking of a protein called insulin receptor substrate (IRS).
• IRS is phosphorylated by the activated receptor tyrosine kinase, creating docking sites for more adaptor proteins.
• Result: Synthesis and storage of carbohydrates, proteins, and lipids.

RTK Signaling and Conformational Changes

• Ligand binding to a receptor tyrosine kinase induces conformational changes in the structure of the receptor that can lead to a variety of outcomes.

Growth Factor Signaling and Cancer

• Cancers often show hyperactivation of pro-proliferative growth factor receptor pathways.
• Excess growth factor, too many receptors, or too much activation of intracellular pathways.
• Epidermal growth factor receptor (EGFR) family: Four isoforms (EGFR1, EGFR2, EGFR3, EGFR4; also called HER1, HER2, HER3, HER4).

EGFR Receptor Isoforms

• EGFR1: Dimer activated by direct ligand binding.
• HER2/EGFR2: Forms heterodimers with other EGFR receptors; doesn't directly bind ligand.

Major Pathways Activated

• MAP kinase/ERK cascade.
• PI3 kinase pathway.
• JAK STAT pathway.
• Inhibiting these pathways can block cancer cell survival and proliferation.

GTP-Binding Proteins (GTPases)

• Another mechanism for activating signaling proteins.
• GTP-bound = active; GDP-bound = inactive.
• Exchange of GDP for GTP leads to protein activation.
• Hydrolysis of GTP back to GDP inactivates.

Guanine Exchange Factors (GEFs)

• Catalyze exchange of GDP for GTP.

GTPase Accelerating Proteins (GAPs)

• Catalyze GTP hydrolysis terminate signal quickly.

MAP Kinase Pathway and Ras

• Receptor tyrosine kinase activation.
• Phosphorylation of the receptor's internal side phosphoryl sites form a binding site for adaptor proteins such as group 2 protein.
• Adaptor protein brings in and recruits a protein called SOS a guanine exchanged factor.
• Recruitment of SOS protein activating guanine exchanged factor enables exchange of GDP for GTP on RAS an intracellular protein.
• Which in turn then activates RAF a kinase signaling protein and activates it by bringing it the plasma membrane.
• The RAF then phosphorylates MEC or MAP kinase, that now becomes activated and phosphorylates MAP kinase.
• Activated MAP kinase then activates and phosphorylates downstream effector proteins.

Persistent Activation of EGFR and Tumor Formation

• Cancer is often caused mutations within the above to become hyper activated and therefore always dividing.

EGFR Signaling as a Therapeutic Target

• Inhibiting EGFR signaling (receptor, ligand, or downstream) can block tumor growth.
• Key kinase inhibitors used clinically:
  • Herceptin/trastuzumab: Inhibits HER2/EGFR2 (used in breast cancer).
  • Gefitinib and erlotinib: Small molecule inhibitors of EGFR (used in lung cancer).
  • Gleevec/imatinib: Tyrosine kinase inhibitor (used in chronic myeloid leukemia).

Herceptin/Trastuzumab Mechanism

• Effective against breast cancers that have amplification of the HER2 gene, and bind HER2 blocking activation of EGF receptors.

Case Study Summary

• Anti-cancer drug Herceptin is effective against breast cancers that highly express HER2 or EGFR2.

Key Points

• RTK signaling is a key driver of cell signaling, often hijacked in cancer and other diseases.
• Key Regulatory Mechanisms: Phosphorylation and GTP binding.
• The MAP kinase pathway is key in cell division.