1. CHAPTER 15 Cell Signaling and Signal Transduction: Communication Between Cells

2. Signal transduction Extracellular ligand: 1st messenger –Autocrine: ligand binds to producing cell –Paracrine: ligand binds to a neighbor cell –Endocrine: ligand binds to a distant cell after traveling through the circulatory system

3. A Survey of Extracellular Messengers Extracellular messengers (the signal) include: –Small molecules such as amino acids, steroids, lipids and their derivatives –Gases such as NO and CO –Various peptides and proteins Edn1 serotonin

4. Signal transduction: theme 1 (GPCR) Cells respond to a ligand only if expressing the cognate receptor 1)1st messenger 2)binds to cell surface receptor 3)triggers conformation change in the intracellular domain(s) 4)Activates intracellular ‘Effector’ enzymes 5)Effector generates 2nd messengers molecules inside the cell 6)2 nd messengers alter the function of further downstream factors

5. Signal transduction: theme 2 (RTK) Cells respond to a ligand only if expressing the cognate receptor 1)1st messenger 2)binds to cell surface receptor 3)triggers conformation change in the intracellular domain(s) 4)Intracellular enzyme domain becomes activated (kinase) 5)Triggers a cascade of kinases activating additional downstream kinases Target proteins ultimately alter cell activity –E.g. TXN, TLN, Enzymes, etc

6. G protein-coupled receptors (GPCRs) BINDING –GPCRs involved in vision, smell, emotion (1000s of genes) –Respond to a wide variety of ligands Proteins, small chemical compounds, metabolites, photons

7. G protein-coupled receptors (GPCRs) BINDING –GPCRs involved in vision, smell, emotion (1000s of genes) –Respond to a wide variety of ligands Proteins, small chemical compounds, metabolites, photons –Ligands bind to extracellular side of receptor Induces a conformational change in intracellular domains –7 transmembrane (7TM) receptors serpentine structure passed through membrane 7 times 1234567 N C

8. GPCRs ACTIVATION / SIGNAL TRANSMISSION –Receptor is ‘coupled’ to a large heterotrimeric G protein “switch” Alpha, beta and gamma subunits Alpha subunit = GTP hydrolyzing enzyme (GTPase) GTP bound form is switched ‘on’ GDP bound form is switched ‘off’ The ligand-bound receptor promotes exchange of GDP for GTP 1234567 N C    G  -GTPG  -GDP GTP GDP Pi Active receptor RGS “off ” “on”

10. GPCRs ACTIVATION / SIGNAL TRANSMISSION –Four classes of G-alpha subunit –G-alpha-s increases activity of Adenylate Cyclase (AC) An “Effector” ATP --> cAMP + PPi cAMP is a 2nd messenger generated within the cytoplasm –G-alpha-i decreases activity of AC

12. ACTIVATION / SIGNAL TRANSMISSION –G-alpha-q increases activity of the effector Phospholipase C (PLC) PI(4,5)P2 --> DAG + IP3 DAG and IP3 are both 2nd messengers –G-alpha-12/13 is not well understood, but is linked to cancer

19. GPCRs TERMINATION Regulators of G protein Signaling (RGS) –Enhance GTPase activity GPCR-kinases (GRKs) –Phosphorylate internal portions of active receptors Arrestins –Compete with G-alpha for binding to phosphorylated GPCR Desensitization: loss of response to a stimulus in spite of the continued presence of the stimulus

20. GPCR: G  s Epinephrine: glucose mobilization –G-alpha-s –Increased AC activity –Increased [cAMP] –cAMP allosterically activates Protein Kinase A (PKA) PKA inhibits Glycogen Synthase PKA activates Glycogen Phosphorylase –Leads to glycogen breakdown and release of glucose PKA stimulates TXN factor activity –Phosphorylates cAMP Response Element Binding (CREB) protein –Drives TXN of various target genes

22. GPCR: G  q Increased Phospholipase C (PLC)-beta activity –Hydrolyzes specific phospholipids –Increased [diacylglycerol (DAG)] and [inositol triphosphate (IP3)]

23. GPCR: G  q DAG allosterically activates Protein Kinase C (PKC) IP3 allosterically opens a Calcium channel on the smooth Endoplasmic Retriculum –Increased [Ca2+] in cytoplasm

24. Increased [Ca2+] in cytoplasm –Ca2+ binds many cytoplasmic proteins –Calmodulin, regulator of many proteins GPCR: G  q

25. Increased [Ca2+] in cytoplasm –Ca2+ can pass through GAP junctions into neighboring cells –Ca2+ gated Ca2+ channels in neighboring cells open further increasing [Ca2+] –Propagation of Ca2+ effects through GAP junctions integrates tissue response (Ca2+ waves) Video GPCR: G  q

26. Receptor Tyrosine Kinases (RTKs) Over 90 different genes Extracellular ligand binding triggers receptor dimerization

27. Receptor Tyrosine Kinases (RTKs) Intracellular tyrosine (Y) kinase domain Dimerization allows for trans-autophosphorylation of the receptors

28. Intracellular tyrosine (Y) kinase domain Dimerization allows for trans-autophosphorylation of the receptors Phospho-Y (PY) sequences are binding sites SH2 domains in proteins such as Src and Grb2 bind to PY-receptor Receptor Tyrosine Kinases (RTKs)

29. Intracellular tyrosine (Y) kinase domain Dimerization allows for trans-autophosphorylation of the receptors Phospho-Y (PY) sequences are binding sites SH2 domains in proteins such as Src and Grb2 bind to PY-receptor Drag partner proteins along with them from the cytoplasm to the membrane Receptor Tyrosine Kinases (RTKs)

30. The Ras-MAPK pathway Ras is a small G protein “switch” (no beta or gamma) –Over 100 Ras family genes –Lipid anchor to plasma membrane –GEF = Guanine Nucleotide Exchange Factor (e.g. Sos) –GAP = GTPase Activating Protein Mitogen Activated Protein Kinases (MAPKs) –A large family of kinases subject to regulation by phosphorylation Ras-GTPRas-GDP GTP GDP Pi GEF GAP “off ” “on”

31. Disease and the Ras-MAPK pathway Ras G12V mutations –Insensitive to GAP activity –Ras stuck in “on” state Neurofibromatosis type I –NF1 gene encodes a GAP gene for Ras –NF1 mutations leave Ras stuck in the “on” state Ras-GTP Ras-GDP GTP GDP GEF GAP “off ” “on” X

32. RTK

35. RTK: Insulin receptor

36. Convergence in signal transduction Examples: Integrins: ECM --> Integrin --> Ras RTKs: EGF --> EGFR --> Ras

37. Crosstalk in signal transduction Examples: GPCR RTK –PKA-cAMP can inhibit Raf but activate CREB

38. Apoptosis (Programmed Cell Death) ~10^10 cell deaths per day in the average human

39. Extrinsic: Receptor-mediated Apoptosis Ligand: Tumor Necrosis Factor (TNF) TNF --> TNF-Receptor --> Procaspase recruitment Procaspase proteolytically activated to form “initiator” caspase Initiator proteolytically activates “executioner” caspases Caspase Activated DNase (CAD)

40. Irreparable DNA damage, sustained high [Ca2+] Activates proapoptotic factor: BAD/Bax –Punches holes in mitochondria –Release of cytochrome c from mitochondria Activates initiator caspase complex Activates executioner caspases Activates CAD Intrinsic pathway