1. 1 Signal molecule Active MAPKKK 1 Active MAPKK 2 Active MAPK 3 Inactive MAPKKK 1 Inactive MAPKK 2 Inactive MAPK 3 Inactive protein Active protein Cellular response Receptor P P P ATP ADP ATP PP Activated Ras-GTP A G-Protein i Phosphorylation cascade P P i i P Protein Phosphatases BCOR 011 Cell Communication II Lect 19 Chapt 11 10/17/05 And they tell 2 friends And they tell 2 friends…

2. 2 Lecture Outline 1.Finish Trimeric G-Protein: Phospholipase C -DAG, IP 3 second messengers Protein Kinase C, Ca ++ release 2.Nitric Oxide, cGMP activation - consequences of failing to inactivate 3.Tyrosine Kinase Receptor - RAS G-protein, MAP Kinase Cascade - Cell Cycle Control, Gene Control 4.Internal Receptors – Steroid Receptor - Gene Regulation

3. 3 TWO subclasses of trimeric G-protein-activated signal transduction pathways: A. target protein adenylate cyclase cAMP-> PKA B. target protein phospholipase C

4. 4 target effector enzyme is Phospholipase C PLC cleaves a membrane phospholipid (Phoshatidyl inositol) to two 2nd Messengers : Inositol- 1,4,5- Trisphosphate (InsP3) & Diacylglycerol (DAG)

5. 5 InsP 3 Water Soluble DAG Lipid Soluble PIP 2

6. 6 DAG Activates Protein Kinase C (Starts Cascade) InsP 3 Ligand for ER ligand- gated Ca ++ channels  Ca ++ levels

7. 7 Figure 11.12 3 21 IP 3 quickly diffuses through the cytosol and binds to an IP 3 – gated calcium channel in the ER membrane, causing it to open. 4 The calcium ions activate the next protein in one or more signaling pathways. 6 Calcium ions flow out of the ER (down their con- centration gradient), raising the Ca 2+ level in the cytosol. 5 DAG functions as a second messenger in other pathways. Phospholipase C cleaves a plasma membrane phospholipid called PIP 2 into DAG and IP 3. A signal molecule binds to a receptor, leading to activation of phospholipase C. EXTRA- CELLULAR FLUID Signal molecule (first messenger) G protein G-protein-linked receptor Various proteins activated Endoplasmic reticulum (ER) Phospholipase C PIP 2 IP 3 (second messenger) DAG Cellular response GTP Ca 2+ (second messenger) Ca 2+ IP 3 -gated calcium channel

8. 8 Protein Kinase C phosphorylates target proteins (ser & thr) cell growth regulation of ion channels cytoskeleton increases cell pH Protein secretion Ca++ Binds & activates calmodulin Calmodulin-binding proteins activated (kinases & phosphatases ) Response:

9. 9 Shut Off - remember, signal needs to be transient -Must shut off cascade: removal of ligand, (self)-hydrolysis of GTP, remove IP 3, protein phosphatases, Ca ++ ion pumps

10. 10 Direct activation - signal: nitric oxide (NO) It’s a gas! lipid soluble, binds directly to activate enzyme made by: endothelial cells (line blood vessels) NO synthase target: guanylyl cyclase GTP->cGMP argininecitrulline +NO response: relaxes smooth muscle vessels dilate, blood flow

11. 11 Shut off by cGMP phosphodiesterase target

12. 12 1977 –Ferid Murad Nitroglycerine acts to elict release of NO, relaxes cardiac muscle Nitroglycerine – taken to relieve angina 1998 Nobel Prize – Murad, Furchgott, Ignarro

13. 13 Shut off By cGMP phosphodiesterase VIAGRA Nitro glycerine

14. 14 Sildenafil citrate (VIAGRA)

15. 15 b. G-protein-linked cell-surface receptors a. ion-channel-linked c.Protein-kinase associated (enzyme-linked)

16. 16 Mitogen Activated Protein-Kinase associated receptors receptor has enzymatic activity - only when ligand binds ligand: allosteric effector -> conformational change receptor tyrosine kinases Growth & differentiation control of cell cycle control of gene expression bad news when messed up - cancer

17. 17 Reception Transduction Response mRNA NUCLEUS Gene P Active transcription factor Inactive transcription factor DNA Phosphorylation cascade CYTOPLASM Receptor Growth factor Figure 11.14 Growth Factor “Mitogen-activated” Signaling Cascade

18. 18 Monomer receptor 1 transmembrane segment EGFR – epidermal growth factor receptor EGF EGF – growth factor mitogen or mitogen “Mitosis-generator” GDP/GTP exchange Activate Ras G-protein Scaffold to Bind/activateScaffold to Bind/activate Target proteins Activates Tyr kinase autophosphorylate Ligand binds – dimerize receptors dimerize

19. 19 Receptor tyrosine kinases Signal molecule Signal-binding site CYTOPLASM Tyrosines Signal molecule  Helix in the Membrane Tyr Dimer Receptor tyrosine kinase proteins (inactive monomers) P P P P P P Tyr P P P P P P Cellular response 1 Inactive relay proteins Activated relay proteins Cellular response 2 Activated tyrosine- kinase regions (unphosphorylated dimer) Fully activated receptor tyrosine-kinase (phosphorylated dimer) 6 ATP 6 ADP Figure 11.7

20. 20 Receptor tyrosine kinases can activate ras ras is a monomeric G-protein “molecular switch”

21. 21 cascade A phosphorylation cascade Figure 11.8 Signal molecule Active protein kinase 1 Active protein kinase 2 Active protein kinase 3 Inactive protein kinase 1 Inactive protein kinase 2 Inactive protein kinase 3 Inactive protein Active protein Cellular response Receptor P P P ATP ADP ATP PP Activated relay molecule i Phosphorylation cascade P P i i P A relay molecule activates protein kinase 1. 1 2 Active protein kinase 1 transfers a phosphate from ATP to an inactive molecule of protein kinase 2, thus activating this second kinase. Active protein kinase 2 then catalyzes the phos- phorylation (and activation) of protein kinase 3. 3 Finally, active protein kinase 3 phosphorylates a protein (pink) that brings about the cell’s response to the signal. 4 Enzymes called protein phosphatases (PP) catalyze the removal of the phosphate groups from the proteins, making them inactive and available for reuse. 5 MAPKinaseMAPKinase MAPKinaseKinaseMAPKinaseKinase MAPKinaseKinaseKinaseMAPKinaseKinaseKinase Ras-GTP

22. 22 Ras activation sets off a phosphorylation cascade Mitogen Activated Protein Kinases MAPKs Controls: -Transcription Factors -Translation Factors -Cell Division MAPK MAPKKK MAPKK 10100 1,000 100,000

23. 23 How do you turn it off? phosphatases GTPase (GTP->GDP + P) molecular switch on internal timer If timer broken – on all the time

24. 24 PROBLEMS IN CANCER: - Broken receptor – thinks ligand there even when it isn’t - broken ras – won’t shut off -broken MAPK – on all the time, even when not phosphorylated RESULT: continuous signal for cell to divide

25. 25 1.receptor-mediated cell-surface 2.receptor-mediated intracellular Specific signal transduction cascades: Lipid soluble things: steroid hormones

26. 26 Hormone (testosterone) EXTRACELLULAR FLUID Receptor protein DNA mRNA NUCLEUS CYTOPLASM Plasma membrane Hormone- receptor complex New protein Figure 11.6 The steroid hormone testosterone passes through the plasma membrane. The bound protein stimulates the transcription of the gene into mRNA. The mRNA is translated into a specific protein. Testosterone binds to a receptor protein in the cytoplasm, activating it. The hormone- receptor complex enters the nucleus and binds to specific genes. Steroid Hormone Lipid soluble Crosses membranes intracellular receptor Binds intracellular receptor In cytosol steroid receptor Complex Changes shape Releases from tether protein Travels to Nucleus steroid receptor Binds DNA Turns genes ON

27. 27 The Specificity of Cell Signaling different combinationsThe different combinations of proteins in a cell –Give the cell great specificity in both the signals it detects and the responses it carries out Same hormone can give different responses in different cells

28. 28Pathway“cross-talk” Response 1 Response 4Response 5 Response 2 Response 3 Signal molecule Cell A. Pathway leads to a single response Cell B. Pathway branches, leading to two responses Cell C. Cross-talk occurs between two pathways Cell D. Different receptor leads to a different response Activation or inhibition Receptor Relay molecules Figure 11.15

29. 29 Specific pathways 1. Cell-surface receptor mediated a. ion-channel-linked b. trimeric G-protein-linked (i) adenylyl cyclase cAMP ->protein kinase A (ii) phospholipase C InsP3, DAG, Ca ++, protein kinase C Direct activation - NO c. protein kinase-associated (enzyme-linked) Receptor tyrosine kinase monomeric G-protein (ras), MAPKs 2. Intracellular receptors – steroid hormones, dioxin