Pharmacodynamics : Signal Transduction

1. Topic 10 Pharmacodynamics: Signal Transduction 713 311 PRINCIPLES OF VETERINARY PHARMACOLOGY Dr. Korawuth Punareewattana Faculty of Veterinary Medicine, Khon Kaen University

2. Topics on Signal Transduction • Cell signaling • General principles • Signaling molecules and their receptors • Signal transduction • General concepts • Mechanism of transduction

3. Cell Signaling: General Principles Cell Signaling (Communication between cells) • receive a signal • Recognition of stimulus at outer cell surface by specific membrane-embedded receptor • Transfer of signal across membrane to cytoplasmic surface • transmit the signal within the cell (signal transduction) • Transmission of signal to specific effector molecules on inner membrane surface or in cytoplasm that trigger cell response • alter behavior to fitsignal

4. Signaling and Signal transduction Signaling is about communication between different groups of cells and tissues…how one group of cells informs another group of cells what to do. Signal transduction refers to how the presence of an extracellular signal can produce a change in the intracellular state of the cell without the initial signal crossing the membrane. Or a step of cell signaling within target cells

5. Signal Transduction Cell Signaling

6. General Principles; signaling molecules (Ligands, Drugs) and their receptors Extracellular Signals • recognized by specific receptors. • 2 types of Signals • Hydrophilic; unable to cross the plasma membrane - bind to cell-surface receptors Example: growth factors • Hydrophobic; pass across the cell membrane and directly regulate the activity of an intracellular receptor. • Example: Steroid hormones, thyroid hormones, retinoids, and vitamin D bind to intracellular receptors that are ligand-activated transcription factors. cell surface receptors cell intra- cellular receptors cell

7. Cell surface receptors: Getting the Message Across Extracellular ligand-binding domain • Many signals (e.g. peptide growth factors) cannot cross the plasma membrane. • Cells get round this problem by utilising transmembrane receptors. • These are proteins that sit in the membrane with the ligand* binding domain outside the cell and an intracellular domain that couples to the next step in the signalling pathway • *Ligand – a molecule that binds to, and is recognised by a receptor Plasma membrane outside inside Transmembrane domain Intracellular domain – couples to next step (may have enzymatic activity)

8. Intracellular receptors Steroid hormones such as cortisol • diffuses across the plasma membrane, • binds to a intracellular receptor protein, • enters the nucleus, and • activates specific transcription.

9. Signal Transduction • Signal transduction is a process that relay signal or information or instruction from cell surface receptor to effectors inside the cells. • Steps of signal transduction • Activation of intracellular domain of receptors • Signal transduction • Cascade of phosphorylation • Use 2nd messengers • Activation of effectors • Patterns of signal transduction • Depends on the types of cell-surface receptors

10. Signal molecule binds receptor activates Intracellular signaling pathway Target protein alters cell behavior A simple intracellular signaling pathway activated by an extracellular signal molecule influences cell behavior.

11. Cell-surface receptors There are three known classes of cell-surface receptor proteins: • Ion-channel-linked receptors also known as transmitter-gated ion channels. • G-protein-linked receptors also known as serpentine receptors • Enzyme-linked receptors • enzymes; tyrosine kinases • directly associate with an enzyme that they regulate

12. Types of receptor & Signal transduction Types of receptor • Cell surface receptorLigands (Drug) • Classical receptors activate signal transduction • Ion-channel receptors activate or block ion movement • Uptake carriers block transport • Intracellular receptor • Enzymes block enzyme function • Transcription factor activate or block

13. Cell-surface receptors • act as signal transducers: they bind the signaling ligand with high affinity and convert this extracellular event into one or more intracellular signals that alter the behavior of the target cell. • Activated cell surface receptors trigger a phosphorylation cascade which results in signal relay through the cell and to the nucleus. • Some pathways using 2nd messengers (eg. cAMP) to amplify the signals

14. Phosphorylation cascade Many enzymes are regulated by covalent attachment of phosphate, in ester linkage, to the side-chain hydroxyl group of a particular amino acid residue (Ser, Thr, or Tyr). • A protein kinase transfers the terminal phosphate of ATP to a hydroxyl group on a protein. • A protein phosphatase catalyzes removal of the Pi by hydrolysis.

15. Major signaling mechanisms share common features In both receptor tyrosine kinase signaling and G-protein-mediated signaling, • a signaling protein is activated by the addition of a phosphate group (phosphorylation) and inactivated by the removal of the phosphate. Signaling by phosphorylation Signaling by phosphorylation of GTP-binding protein

16. Second Messengers • Several different molecules act as second messengers including: • Cyclic AMP (cAMP) • Cyclic GMP (cGMP) • Ca2+ ions • Inositol phosphates (IP3) • Lipids; eg. Diacylglycerol (DAG) Signal amplification is an important feature of signal cascades: • One hormone molecule can lead to formation of many cAMP molecules. • Each catalytic subunit of Protein Kinase A catalyzes phosphorylation of many proteins during the life-time of the cAMP.

17. G-protein-linked receptors Receptor • The interaction between the G-protein-linked receptor and the target is mediated by a trimeric GTP-binding regulator protein(G-protein). • All of the G-protein-linked receptors belong to a large superfamily of homologous, seven-pass transmembrane proteins. Trimeric GTP-binding protein

18. Enzymes activated by G-proteins catalyze the synthesis of second messengers.

19. Receptor tyrosine kinases Receptor tyrosine kinases • single-pass transmembrane proteins • ligand-binding site outside the cell • catalytic site inside • The binding of growth factors such as insulin, epidermal growth factor, and platelet-derived growth factor to the extracellular domain of their receptors switches on the kinase activity of their catalytic domain and triggers phosphate group additions to a network of intracellular proteins to transduce the signal.

21. Examples of Signal Transduction Systems • Receptor tyrosine kinase or Enzyme-link receptor • Kinase cascade • or Phosphorylation cascade • G-protein link receptor • using 2nd messenger (cAMP) • Steroid hormone receptor • Simple signaling system

22. Signal Integration • Complex cell behaviors are generally regulated by specific combinations of signals rather than by a single signal acting alone. ways to integrate combinations of signals previous example of complex cell behavior How are multiple signals understood by the cell?

23. Fast and slow responses

24. Signal Transduction: General Principles Summary • Cell signaling requires both • extracellular signaling molecules and • a complementary set of receptor proteins in each cell. • Most signaling molecules are hydrophilic and • activate cell surface receptors. • There are three main families of cell surface receptors. • The highly regulated phosphorylation cascade stimulated by activated receptors • relays the signal through the cytoplasm and to the nucleus to alter cellular behavior

25. Signaling molecules (ligands) - hydrophobic - hydrophilic Receptors - specific - intracellular - cell surface Ion-channel-linked receptors G-protein-linked receptors enzyme-linked receptors Intracellular signaling proteins - receive and transduce the signal - short half-life - molecular switches On and Off states Phosphate group additions and loss relays signal 2 types of signal transduction 1) Phosphorelation cascade 2) Phosphorelation and 2nd messengers

26. P Nucleus A Simple Signalling System INFg receptor Interferon (IFN) g Activated IFNg receptor recruits JAK kinase Phosphorylation causes STATs to dimerise and migrate to nucleus… …where they initiate transcription ADP ATP JAK phosphorylates STAT monomer STAT transcription factor JAK tyrosine kinase

27. Cascading Kinases Binding of epidermal growth factor to its receptor activates ras Ras activates the serine/threonine kinase raf Erk-1 phosphorylates the transcription factor myc and activates transcription ras raf raf ras GTP GDP ADP ATP Raf phosphorylates and activates the dual-specificity kinase Mek-1 P Mek1 Mek1 Nucleus ATP P Erk1 ATP ADP ADP P P P Erk1 Erk1 Mek-1 phosphorylates the serine/threonine kinase Erk-1 which migrates to the nucleus

28. MLK3 raf Mekk1 ATP ATP ATP ADP ADP ADP Mek1 MKK4 MKK7 ATP ATP ATP ADP ADP ADP Erk1 Jnk2 Jnk1 Cascading Kinases The raf Mek-1 Erk-1 cascade is one example of a MAP kinase cascade. A number of these cascades have been described, and although they utilise specific kinases, the pathways are very similar… N.B. Mode of activation of the first kinase in the cascade is variable and depende on the signal Growth Factor Stress Cytokines MAP kinase kinase kinase Serine/threonine kinase MAP kinase kinase Dual-specificity kinase Serine/threonine kinase MAP kinase

29. Using Second Messengers Binding of adrenaline to its receptor activates a G protein G protein a subunit mediated activation of adenylate cyclase leads to cyclic AMP (cAMP) production PKA catalytic subunit phosphorylates CREB* and activates transcription a b g ATP GTP 2Pi Inactive protein kinase A (PKA) Free PKA catalytic subunit migrates to nucleus Nucleus ATP ADP P P Regulatory subunit of PKA binds cAMP… …which dissociates from the catalytic subunit

30. How does insulin work?