External signal is received and converted to another form to elicit a response

1. Cell Communication – Signal Transduction External signal is received and converted to another form to elicit a response

2. Signal Transduction & G Protein-coupled Receptors • Topics • Signal Trans.: From Extracellular Signal • to Cellular Response • Cell-Surface Receptors & Signal • Transduction Proteins • G Protein-coupled Receptors (GPCRs): • Structure and Mechanism • GPCRs That Regulate Ion Channels • GPCRs That Regulate Adenylyl Cyclase • GPCRs That Regulate Cytosolic Calcium

3. Learning Objectives • Learn the general properties of signaling molecules (ligands), cell-surface receptors, & intracellular signal transduction components. • Learn the G protein cycle of reactions involved in GPCR signaling. • Learn the epinephrine receptor signal trans pathway used for control of glycogen degradation. • Learn about the GPCR-stimulated IP3/DAG signaling pathway.

4. General Principles of Signal Transduction Signal transduction refers to the overall process of converting extracellular signals into intracellular responses . Key players in signal transduction are signaling molecules, receptors, signal transduction proteins and second messengers, and effector proteins.

5. Cells respond to signals by changing the activity of existing enzymes (fast) and/or the levels of expression of enzymes and cell components (slower) by gene regulation (Steps 7a & 7b). Receptors and signal transduction systems have evolved to detect and respond to hormones, growth factors, drugs , neurotransmitters.

6. Intercellular Communication

7. Intercellular Communication Communication between cells requires: ligand: the signaling molecule receptor protein: the molecule to which the receptor binds -may be on the plasma membrane or within the cell

9. Structure and function of receptors • Globular proteins acting as a cell’s ‘letter boxes’ • Located mostly in the cell membrane • Receive messages from chemical messengers coming from other cells • Transmit a message into the cell leading to a cellular effect • Different receptors specific for different chemical messengers • Each cell has a range of receptors in the cell membrane making it responsive to different chemical messengers

10. Mechanism • Receptors contain a binding site (hollow or cleft in the receptor surface) that is recognised by the chemical messenger • Binding of the messenger involves intermolecular bonds • Binding results in an induced fit of the receptor protein • Change in receptor shape results in a ‘domino’ effect • Domino effect is known as Signal Transduction, leading to a chemical signal being received inside the cell • Chemical messenger does not enter the cell. It departs the receptor unchanged and is not permanently bound

11. M M M R E R E R Signal transduction Overall process of receptor/messenger interaction • Binding interactions must be: • - strong enough to hold the messenger sufficiently long • for signal transduction to take place • - weak enough to allow the messenger to depart • Implies a fine balance • Drug design - designing molecules with stronger binding interactions results in drugs that block the binding site - antagonists

12. vdw interaction H-bond Binding site ionic bond H Phe O Ser CO2 Asp Receptor Messenger binding Bonding forces • Ionic • H-bonding • van der Waals • Example:

13. Phe Phe H O H O Ser Ser CO2 Induced Fit CO2 Asp Asp How does the Binding Site Change Shape? Substrate binding • Bonding forces • Induced fit - Binding site alters shape to maximise intermolecular bonding • Intermolecular bonds not optimum length for maximum binding strength • Intermolecular bond lengths optimised

14. External signals are converted to Internal Responses • Cells sense and respond to the environment Prokaryotes: chemicals Humans: light - rods & cones of the eye sound – hair cells of inner ear chemicals in food – nose & tongue • Cells communicate with each other Direct contact Chemical signals

15. General principles: 1. Signals act over different ranges. 2. Signals have different chemical natures. 3. The same signal can induce a different response in different cells. 4. Cells respond to sets of signals. 5. Receptors relay signals via intracellular signaling cascades.

16. 1º messenger Cells detect signal & respond Effector Enzymes Target Enzymes 2º messengers Signal transduction: ability of cell to translate receptor-ligand interaction into a change in behavior or gene expression

17. EXTRACELLULAR FLUID CYTOPLASM Plasma membrane 3 2 1 Reception Transduction Response Receptor Activation of cellular response Relay molecules in a signal transduction pathway Signal molecule Primary Messenger Target Enzymes Secondary Messengers Cascade Effect

18. - activates an enzyme activity, processes 100 substrates /second primary signal Each protein in a signaling pathway • Amplifies the signal by activating multiple copies of the next component in the pathway Primary enzyme activates 100 target enzymes Each of the 100 enzymes activates an additional 100 downstream target enzymes Each of the 10,000 downstream targets activates 100 control factors so rapidly have 1,000,000 active control factors

19. Reception Binding of epinephrine to G-protein-linked receptor (1 molecule) Transduction Inactive G protein Active G protein (102 molecules) Inactive adenylyl cyclase Active adenylyl cyclase (102) ATP Cyclic AMP (104) Inactive protein kinase A Active protein kinase A (104) Inactive phosphorylase kinase Active phosphorylase kinase (105) Inactive glycogen phosphorylase Active glycogen phosphorylase (106) Response Glycogen Glucose-1-phosphate(108 molecules) 1 A Signal Cascade amplification 102 104 105 106 108

20. Receptors relay signals via intracellular SIGNALING CASCADES amplification

21. Main Types of Receptors ION CHANNEL RECEPTORS G-PROTEIN-COUPLED RECEPTORS KINASE-LINKED RECEPTORS INTRACELLULAR RECEPTORS

22. Cell-surface receptors -large &/or hydrophilic ligands ion-channel-linked Trimeric G-protein-linked enzyme-linked (tyrosine kinase)

23. Signal transduction • Control of ion channels • Receptor protein is part of an ion channel protein complex • Receptor binds a messenger leading to an induced fit • Ion channel is opened or closed • Ion channels are specific for specific ions (Na+, Ca2+, Cl-, K+) • Ions flow across cell membrane down concentration gradient • Polarises or depolarises nerve membranes • Activates or deactivates enzyme catalysed reactions within cell

24. Gate closed Signalmolecule(ligand) Ions Ligand-gated ion channel receptor Plasma Membrane Gate open Cellularresponse Gate close Examples: Muscle Contraction Nerve Cell communication Ion channel receptors

25. Review: Remember the Na+/K+ ATPase (Na+/K+ pump)? [Na+] inside ~10mM; outside ~150mM [K+] inside ~100mM; outside ~5mM cell has membrane potential ~ -60mV Na+ Cl- -60mV K+ A- - + - - - + - + - + + +

26. Intercellular Communication There are four basic mechanisms for cellular communication: 1. direct contact 2. paracrine signaling 3. endocrine signaling 4. synaptic signaling

27. Intercellular Communication Direct contact – molecules on the surface of one cell are recognized by receptors on the adjacent cell

28. Intercellular Communication Paracrine signaling – signal released from a cell has an effect on neighboring cells local ex. nitric oxide, histamines, prostaglandins

29. Intercellular Communication Endocrine signaling – hormones released from a cell affect other cells throughout the body long distance ex. Estrogen, Thyroxine, GH Epinephrine ….

30. Intercellular Communication Synaptic signaling – nerve cells release the signal (neurotransmitter) which binds to receptors on nearby cells

31. Intercellular Communication When a ligand binds to a receptor protein, the cell has a response. signal transduction: the events within the cell that occur in response to a signal Different cell types can respond differently to the same signal.

32. Receptor Types There are 3 subclasses of membrane receptors: 1. channel linked receptors – ion channel that opens in response to a ligand 2. enzymatic receptors – receptor is an enzyme that is activated by the ligand 3. G protein-coupled receptor – a G-protein (bound to GTP) assists in transmitting the signal

34. Intercellular Communication A cell’s response to a signal often involves activating or inactivating proteins. Phosphorylation is a common way to change the activity of a protein. protein kinase – an enzyme that adds a phosphate to a protein phosphatase – an enzyme that removes a phosphate from a protein

36. Signal Transduction Components: Kinases/Phosphatases Proteins that participate in intracellular signal transduction fall into two main classes--protein kinases/phosphatases and GTPase switch proteins. Kinases use ATP to phosphorylate amino acid side-chains in target proteins. Kinases typically are specific for tyrosine or serine/threonine sites. Phosphatases hydrolyze phosphates off of these residues. Kinases and phosphatases act together to switch the function of a target protein on or off .

37. Kinases - Phosphorylation Phosphatase - Dephosphorylation Tyrosine-OH Tyr-Kinases Serine-OH Ser/Thr-Kinases Threonine-OH „dual specificity“ Kinases

39. There are about 600 kinases and 100phosphatases encoded in the human genome. Activation of many cell-surface receptors leads directly or indirectly to changes in kinase or phosphatase activity. Note that some receptors are themselves kinases (e.g., the insulin receptor).

41. GH GH binding & dimerisation Binding Activation and of kinases phosphorylation GH receptors (no kinase activity) ATP ADP PO HO OH OP OP OH OP OH kinases HO OH OH OH Growth hormone binding site Kinase active site Growth hormone receptor Tetrameric complex constructed in presence of growth hormone Kinase active site opened by induced fit

42. CO2H Steroid binding region Zinc DNA binding region (‘zinc fingers’) H2N Intracellular receptors • Chemical messengers must cross cell membrane • Chemical messengers must be hydrophobic • Example-steroids and • steroid receptors Zinc fingers contain Cys residues (SH) Allow S-Zn interactions

43. Intracellular Receptors steroid hormones -have a nonpolar, lipid-soluble structure -can cross the plasma membrane to a steroid receptor -usually affect regulation of gene expression An inhibitor blocks the receptor from binding to DNA until the hormone is present.

44. Co-activator protein Receptor DNA Messenger Receptor-ligand complex Dimerisation Cell membrane 1. Messenger crosses membrane 5. Complex binds to DNA 2. Binds to receptor 6. Transcription switched on or off 3. Receptor dimerisation Intracellular receptor Mechanism 7. Protein synthesis activated or inhibited 4. Binds co-activator protein

45. Intracellular Receptors A steroid receptor has 3 functional domains: 1. hormone-binding domain 2. DNA binding domain 3. domain that interacts with coactivators to affect gene expression

47. END PART I

48. Important Announcement • Final Exam will include 4 lectures from mid-term exam material only from L5,6,7 & 8. • The First 4 lectures are NOT required. • The distribution of marks in the final exam will be as follow : The lectures after the mid exam = 40 % The mid-term lectures will have =10 % GOOD LUCK