1. Haploid a- and  -cells form shmoos in response to chemical signals Shmoos mate to form diploid a/  cell Examples of: - “differentiated” cell types (a-,  -, and a/  -cells) cell-cell adhesion -cell-cell signaling Human body consists of trillions of cells, 200+ specialized cell types that must differentiate (next time) and communicate (today) with one another Cell-cell communication required to coordinate: - physiology and metabolism - behavior -growth, proliferation, and differentiation ECB 16-1 Mating dance of a budding yeast (S.cerevisiae)… Multicellularity: From cells to tissues to organisms shmoos

2. “Neuronal” Cell body of neuron Post-synaptic target (muscle, neuron, etc) Axon Synapse Action potential Basic categories of cell-cell signaling in animals ECB 16-3 “Paracrine” (local) ex. inflammation Signaling cell Target cells “Autocrine” “Contact-mediated” (short range) ex. - nerve cell production Signaling cell Target cell (ex.-hormones) “Endocrine” (long distance) Endocrine (signaling) cell Target cells Bloodstream hormone

3. Cellular response depends on specific combination of signals ECB 16-6 No signal often results in activation of apoptosis

4. Common features of cell-cell signaling pathways Other signals ECB 16-7

5. Receptors for diffusible signals can be intracellular or surface Small non-polar molecules Large polar molecules Plasma membrane cross plasma membrane by simple diffusion And bind to intra- cellular receptors …cannot cross membrane They bind cell surface receptors Membrane receptors for hydrophilic signaling molecules activate a wide variety of intracellular “signal transduction” pathways, including gene regulation Most receptors for hydrophobic signaling molecules act in nucleus to regulate gene transcription ECB 16-9 Transcription Intracellular signals Intracellular receptors Cell surface receptors

6. A few examples of hydrophobic hormones ECB 16-11 Responses mediated by a conserved family of “steroid” receptors HO OH Estradiol OH O Testosterone HOOCOO - CH 2 C H NH 3 + II II Thyroid hormone HO Cholesterol CH 2 OH O HO C=O OH Cortisol (not hormone)

7. Responses to hydrophobic hormones are mediated by intracellular receptors ECB 16-12 Transcription Translation Cytoplasm Nucleus Nuclear envelope Plasma membrane Lipophilic hormone carried in blood Hormone binds intracellular receptor inducing receptor dimerization and activation Complex is imported into nucleus Binds to “hormone response element” to regulate gene expression Intracellular receptor PromoterTarget gene “Hormone response element” Target cell

8. ReceptorG-protein (inactive) Target (inactive) G-protein linked receptor Cell-surface receptors - three classes ECB 16-14 Receptor (active) G-protein (active) Target (inactive) Signaling ligand Catalytic domain (active) Signaling ligand Ions Catalytic domain (active) Enzyme-linked receptor Ion channel-linked receptor Receptor (active) G-protein (active) Target (active)

9. Activation of surface receptor can cause fast (cytoplasmic) or slow (transciptional) changes

10. Review: phosphorylation and GTPases as molecular switches ECB 16-15 ADP ATP Pi PhosphataseKinase Pi GAPGEF GTP On P Energy (in the form of ATP or GTP hydrolysis) used to activate (or inactivate) signaling molecules Energy use allows transient, high affinity/specificity interactions Signaling with GTPasesSignaling with phosphorylation Signal in Signal activates protein kinase Signal activates GEF Signal out GTP GDP Off Signaling GTPase Off Signaling protein

11. “Heterotrimeric G-proteins” mediate many cell signals   GDP  See ECB 16-17 G , G  subunits G  binds guanine nucleotide Receptor acts as GEF, activating G-protein Activated G  - and G  regulate targets G  inactivated by GTP hydrolysis, subunits reassociate   GTP  + GDP Pi G  (inactive GDP form) Active  G  and G  (GTP form) Heterotrimeric G-proteins Downstream targets Multiple G-proteins with distinct  -,  -, and  -subunits (>20 known) “G s ” stimulates or activates effectors “G i ” inhibits effectors “G q” mediates Ca 2+ signaling

12. G-protein –GDP (inactive)    GDP Plasma membrane Cytoplasm Extracellular space See ECB 16-16 “Heterotrimeric G-proteins” are activated by a family of “Seven-pass” transmembrane receptors Inactive receptor Seven transmembrane domains (  -helices) Extracellular ligand-binding domain (N-terminal) Cytoplasmic “effector” domain Activated receptor acts as GEF to activate “heterotrimeric G-protein” Ligand binding domain Effector domain 1234567

13. Seven-pass receptor “Heterotrimeric G-proteins” are activated by a family of “Seven-pass” transmembrane receptors ECB 16-18 thru 16-18 Binding of ligand activates receptor G-protein –GDP (inactive) Inactive target    GDP

14. GTP Active receptor “Heterotrimeric G-proteins” are activated by a family of “Seven-pass” transmembrane receptors Binding of ligand activates receptor Heterotrimeric G-protein binds activated receptor Activated receptor acts as GEF for heterotrimeric G-protein Activated components (  - and  /  -) regulate downstream targets GTP hydrolysis inactivates G-protein, subunits reassociate (switches off) Activated target    GTP GDP ECB 16-18 thru 16-18

15. Activated target can be enzyme that makes “intracellular messenger” ECB 16-20

16. Ephinephrine (adrenaline) acts via heterotrimeric G protein and cAMP (intracellular messenger) Activated adenylate cyclase forms cAMP cAMP activates protein kinase A (PKA) PKA enters nucleus and phosphorylates a gene regulatory protein Result: altered transcription (slow) ECB 16-24

17. Adenylate cyclase converts ATP to 5’,3’ cAMP P O CH 2 O OH 1’ 2’3’ 4’ 5’ -O-O A -O-O O P O P O P O CH 2 O OH 1’ 2’3’ 4’ 5’ -O-O A -O-O -O-O -O-O OOO P O OH 1’ 2’ 3’ 5’ O A -O-O O CH 2 O PP i 2P i ATP Adenosine 3’,5’ cyclic monophosphate (cAMP) cAMP phosphodiesterase “Adenylate cyclase” AMP Methylated xanthines (caffiene, theophylline, and theobromine ) inhibit cAMP PDE ECB 16-21

18. cAMP levels rise rapidly in response to extracellular signal ECB 16-22 Serotonin is a neurotransmitter 5 X 10 -8 M cAMP Assay fluorescence of protein that binds cAMP 10 -6 M cAMP

19. G-protein coupled receptors also activate IP 3 and Ca 2+ -mediated signaling pathways Activate receptor acts as GEF Activated G  activates phospholipase C (PLC) Active PLC cleaves PIP 2 to IP 3 and diacylglycerol (DAG) IP 3 opens Ca 2+ channels in ER releasing Ca 2+ to cytoplasm DAG and Ca 2+ activate protein kinase C (PKC) Active PKC phosphorylates target proteins…

20. Other Ca 2+ -dependent responses are regulated by “Calmodulin” (CaM) and “CaM kinases” Ca 2+ -calmodulin activates CaM kinases, which phosphorylate and regulate target proteins CaM contains 4 Ca 2+ binding domains Ca 2+ Ca 2+ -CaM binds to regulatory domains of effector proteins (e.g. CaM kinases) see ECB 16-27 Ca 2+ Phosphorylates target proteins in cytoplasm Inactive CaM kinase P Active CaM kinase ATPADP Autophosphorylation Calmodulin (CaM) Catalytic domain Inhibitory domain

21. Cells carefully regulate “free” Ca 2+ levels in their cytoplasm [Ca 2+ ] >1 mM [Ca 2+ ] free ~0.2  M In a resting cell, intracellular [Ca 2+ ] free is low relative to external Ca 2+ … Ca 2+ is pumped into the ER (plant vacuole) Ca 2+ is pumped out of the cell by a Ca 2+ ATPase and antiport with Na + (antiport with H + in plants/fungi) Intracellular [Ca 2+ ] free may increase 10-30-fold during signaling… Moves in through channels and is released from internal stores (mostly from the ER, vacuole) ATPADP + Pi Ca 2+ 2Na + ATPADP + Pi Ca 2+ ER

22. ReceptorG-protein (inactive) Target (inactive) 2. G-protein coupled receptor Last class of cell surface receptors Receptor (active) G-protein (active) Target (inactive) Signaling ligand Catalytic domain (active) Signaling ligand Ions Catalytic domain (active) 3. Enzyme-linked receptor 1. Ligand gated ion channel Receptor (active) G-protein (active) Target (active)

23. Many growth factors bind to receptor tyrosine kinases (enzyme-linked receptor) Receptor binds growth factor and dimerizes Kinase activity activated and receptor autophosphorylates Signaling proteins bind phosphotyrosine, activating signaling cascades

24. EGF and other growth factors activate Ras signaling “GTPase Activating Protein” (Ras-GAPs) promote GTP hydrolysis by intrinsic GTPase Pi GAP GTP Exchange Factors (GEFs) promote GDP/GTP exchange Downstream effectors Ras found to be mutated in ~30% of human tumors! GEF GDP GTP “On” RAS GTP “Off” RAS (inactive) GDP Active Ras activates downstream signaling proteins…

25. MAPKKK inactive Receptor tyrosine kinases activate intracellular Ras signaling cascades ECB 16-31, 16-32 P P P P P P DRK MAPKK inactive MAP kinase inactive Ras GEF ADP ATP ADP ATP ADP ATP Transcription factors P Other proteins P Receptor kinase (active) Growth Factors Downstream of Receptor Kinase activates Ras GEF “Mitogen-activ. protein kinase” MAP kinase MAP kinase kinase (MAPKK) MAPKKK active MAPKK active MAP kinase active P PP Regulate gene expression and protein activity RAS (inactive) GDP RAS GTP MAP kinase kinase kinase (MAPKKK)

26. Mutations in Ras signaling pathway cause uncontrolled cell proliferation: cancer MAPKKK active MAPKK active MAP kinase active P P P P P P DRK ADP ATP ADP ATP ADP ATP Transcription factors P Other proteins P Receptor kinase (active) Downstream of Receptor Kinase P PP Regulate gene expression and protein activity… RAS GTP RAS (inactive) GDP Ras GEF GTP The Ras pathway activates expression of G1 cyclins that stimulate cell proliferation Constituitive activation of pathway components results in uncontrolled cell proliferation = “cancer” Cancer causing genes = “Oncogenes” Predict effects of Ras mutations?

27. Signal transduction cascades are complex and interconnected ECB 16-38 Why? Integration Multiple inputs to a single response… Divergence Single input to multiple responses Amplification Regulation P P P P P P G-protein coupled receptors G-protein Adenylate cyclase cAMP Protein kinase A Phospholipase C IP 3 Ca 2+ Calmodulin CaM kinase Diacylglycerol Protein kinase C G-proteinAdapter Ras activator Ras Kinase I Kinase II Kinase III Receptor tyrosine kinases Gene regulatory proteinsCytoplasmic target proteins

28. Communication by direct cytoplasmic continuity between cells Cytoplasmic bridges and cell junctions

29. Communication via cell junctions: some embryonic cells and/or tissues are “dye-coupled” Membrane-impermeant dye injected into on cell passes into neighbors Cytoplasmic coupling is limited to small molecules (<1000 Da) 100 Da 1,000 Da 10,000 Da

30. “Gap junctions” are responsible for cytoplasmic coupling of animal cells Membranes of coupled cells closely apposed, separated by 2-4 nm “gap” ECB figure 19-28 MBoC figure 19-16 Large “gap jnctn” Common in developing embryo, cardiac muscle, liver, and lens TEM/Freeze fracture of gap junctions reveals “plaques” of intra-membrane particles

31. Gap junctions are composed of “connexons” made of “connexin” hexamers Cytoplasm of cell #2 Cytoplasm of cell #1 “Connexon” (2 per channel) = “connexin” x 6 ECB 21-28 Plasma membrane of cell #2 Plasma membrane of cell #1 Extracellular “gap” (2-4 nm) Channel is ~ 1.5 nm (~1000 Da cutoff) Two connexons in register form channel coupling cytoplasm of adjacent cells

32. The cytoplasm of plant cells is coupled by “plasmadesmata” Membranes continuous from cell to cell ER continuous from cell to cell thru “desmotubule” Limited to small molecules (<800 Da), but can open to let through 20,000 Da Primarily (but not exclusively) formed during cell division ECB 21-30 Nucleus Vacuole Plasmadesmata Cell wall Cytoplasm 100 nm Cell wall Plasma membrane of adjacent cells Cytoplasm Desmotubule Endoplasmic reticulum