Cell-to-Cell Communication and Integration: Signals, Receptors, and Pathways

1. Chapter 6 Communication, Integration,and Homeostasis

2. About this Chapter • How cells communicate • Electrical and chemical signals • Receptor types and how they function • Local regulation of cells • Modification of receptors and signals • Homeostatic balance depends on communication • Feedback regulates integration of systems

3. Overview of Cell to Cell Communication: • Chemical • Autocrine & Paracrine: local signaling • Endocrine system: distant, diffuse target • Electrical • Gap junction: local • Nervous system: fast, specific, distant target

4. Gap Junctions and CAMs • Protein channels - connexin • Direct flow to neighbor • Electrical- ions (charge) • Signal chemicals • CAMs • Need direct surface contact • Signal chemical Figure 6-1a, b: Direct and local cell-to-cell communication

5. Fig 3-6

6. Mucin-like glycoproteins (Sialyl-Lewis X PSL-1 & ESL-1) Integrins CAMS Selectins f

8. Adhesion • Mediated by integrins ICAM-1 and VCAM-1

9. Paracrines and Autocrines • Local communication • Signal chemicals diffuse to target • Example: Cytokines • Autocrine–receptor on same cell • Paracrine–neighboring cells Figure 6-1c: Direct and local cell-to-cell communication

10. Long Distance Communication: Hormones • Signal Chemicals • Made in endocrine cells • Transported via blood • Receptors on target cells Figure 6-2a: Long distance cell-to-cell communication

11. Long Distance Communication: Neurons and Neurohormones • Neurons • Electrical signal down axon • Signal molecule (neurotransmitter) to target cell • Neurohormones • Chemical and electrical signals down axon • Hormone transported via blood to target Figure 6-2 b: Long distance cell-to-cell communication

12. Long Distance Communication: Neurons and Neurohormones Figure 6-2b, c: Long distance cell-to-cell communication

13. Signal Pathways • Signal molecule (ligand) • Receptor • Intracellular signal • Target protein • Response Figure 6-3: Signal pathways

14. Receptor locations • Cytosolic or Nuclear • Lipophilic ligand enters cell • Often activates gene • Slower response • Cell membrane • Lipophobic ligand can't enter cell • Outer surface receptor • Fast response Figure 6-4: Target cell receptors

15. Membrane Receptor Classes • Ligand- gated channel • Receptor enzymes • G-protein-coupled • Integrin

16. Membrane Receptor Classes Figure 6-5: Four classes of membrane receptors

17. Signal Transduction • Transforms signal energy • Protein kinase • Second messenger • Activate proteins • Phosporylation • Bind calcium • Cell response Figure 6-8: Biological signal transduction

18. Signal Amplification • Small signal produces large cell response • Amplification enzyme • Cascade Figure 6-7: Signal amplification

19. Receptor Enzymes • Transduction • Activation cytoplasmic • Side enzyme • Example: Tyrosine kinase Figure 6-10: Tyrosine kinase, an example of a receptor-enzyme

20. G-Protein-coupled Receptors • Hundreds of types • Main signal transducers • Activate enzymes • Open ion channels • Amplify: • adenyl cyclase-cAMP • Activates synthesis

21. G-Protein-coupled Receptors Figure 6-11: The G protein-coupled adenylyl cyclase-cAMP system

22. Many enzymes are regulated by covalent attachment of phosphate, in ester linkage, to the side-chain hydroxyl group of a particular amino acid residue (serine, threonine, or tyrosine).

23. A protein kinasetransfers the terminal phosphate of ATP to a hydroxyl group on a protein. • A protein phosphatase catalyzes removal of the Pi by hydrolysis.

24. Protein kinases and phosphatases are themselves regulated by complex signal cascades. For example: • Some protein kinases are activated by Ca++-calmodulin. • Protein Kinase A is activated by cyclic-AMP (cAMP).

25. Adenylate Cyclase (Adenylyl Cyclase) catalyzes:  ATPàcAMP + PPi Binding of certain hormones (e.g., epinephrine) to the outer surface of a cell activates Adenylate Cyclase to form cAMP within the cell. Cyclic AMP is thus considered to be a second messenger.

26. Phosphodiesteraseenzymes catalyze: cAMP + H2OAMP The phosphodiesterase that cleaves cAMP is activated by phosphorylation catalyzed by Protein Kinase A. Thus cAMP stimulates its own degradation, leading to rapid turnoff of a cAMP signal.

27. G Protein Signal Cascade Mostsignal molecules targeted to a cell bind at the cell surface to receptors embedded in the plasma membrane. Only signal molecules able to cross the plasma membrane (e.g., steroid hormones) interact with intracellular receptors. A large family of cell surface receptors have a common structural motif, 7 transmembrane a-helices. Rhodopsin was the first of these to have its 7-helix structure confirmed by X-ray crystallography.

28. The signal is usually passed from a 7-helix receptor to an intracellular G-protein. • Seven-helix receptors are thus called GPCR, or G-Protein-Coupled Receptors. • Approx. 700-1000 different GPCRs are encoded in the human genome.

29. G-proteins are heterotrimeric, with 3 subunits a, b, g. • A G-protein that activates cyclic-AMP formation within a cell is called a stimulatory G-protein, designated Gs with alpha subunit Gsa. • Gs is activated, e.g., by receptors for the hormones epinephrine and glucagon. The b-adrenergic receptor is the GPCR for epinephrine.

30. Stimulatory and Inhibitory G Proteins

31. The asubunit of a G-protein (Ga) binds GTP, & can hydrolyze it to GDP + Pi. a & g subunits have covalently attached lipid anchors that bind a G-protein to the plasma membrane cytosolic surface. Adenylate Cyclase (AC) is a transmembrane protein, with cytosolic domains forming the catalytic site.

32. The sequence of events by which a hormone activates cAMP signaling: 1. Initially Gahas bound GDP, and a, b, &g subunitsare complexed together. Gb,g, the complex of b & g subunits, inhibits Ga.

33. 2.Hormone binding, usually to an extracellular domain of a 7-helix receptor (GPCR), causes a conformational change in the receptor that is transmitted to a G-protein on the cytosolic side of the membrane. The nucleotide-binding site on Ga becomes more accessible to the cytosol, where [GTP] > [GDP]. Gareleases GDP & binds GTP (GDP-GTP exchange).

34. 3.Substitution of GTP for GDP causes another conformational change in Ga. Ga-GTP dissociates from the inhibitory bg complex & can now bind to and activate Adenylate Cyclase.

35. 4. Adenylate Cyclase, activated by the stimulatoryGa-GTP, catalyzes synthesis of cAMP. 5. Protein Kinase A (cAMP Dependent Protein Kinase) catalyzes transfer of phosphate from ATP to serine or threonine residues of various cellular proteins, altering their activity.

36. Turn off of the signal: 1. Ga hydrolyzes GTP to GDP + Pi. (GTPase). The presence of GDP on Ga causes it to rebind to the inhibitory bgcomplex. Adenylate Cyclase is no longer activated. 2. Phosphodiesterasescatalyze hydrolysis of cAMPAMP.

37. Small GTP-binding proteins include (roles indicated): • initiation & elongation factors (protein synthesis). • Ras (growth factor signal cascades). • Rab (vesicle targeting and fusion). • ARF (forming vesicle coatomer coats). • Ran (transport of proteins into & out of the nucleus). • Rho (regulation of actin cytoskeleton) All GTP-binding proteins differ in conformation depending on whether GDP or GTP is present at their nucleotide binding site. Generally, GTP binding induces the active state.

38. Cholera toxin catalyzes covalent modification of Gsa. • ADP-ribose is transferred from NAD+ to an arginine residue at the GTPase active site of Gsa. • ADP-ribosylation prevents GTP hydrolysis by Gsa. • The stimulatory G-protein is permanently activated. • Pertussis toxin (whooping cough disease) catalyzes ADP-ribosylation at a cysteine residue of the inhibitory Gia, making it incapable of exchanging GDP for GTP. • The inhibitory pathwayis blocked. • ADP-ribosylation is a general mechanism by which activity of many proteins is regulated, in eukaryotes (including mammals) as well as in prokaryotes.

39. ADP Ribosylation

40. 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.

41. { adenylate cyclase guanylate cyclase phospholipase C { cAMP cGMP - NO IP3/DAG – [Ca2+]i second messenger systems ion channels G-Protein Coupled Receptors and Second Messenger Systems neurotransmitter - hormone G-protein coupled receptor – 7 TM receptors heterotrimeric G-proteins () - GTPases enzymes change in membrane potential or resistance enzymes

42. Heterotrimeric G-Proteins and Receptors Alberts et al., Molecular Biology of the Cell, 3rd Ed., Fig. 15-23.

43. Diversity in the GPCR Signaling Pathways Receptor isoforms Adrenergic (norepinephrine)  6 isoforms  3 Muscarinic acetylcholine 5 isoforms Dopamine 5 isoforms (D1, D5 vs D2, D3, D4) tissue expression pharmacology G-protein isoforms G-protein subunit isoforms (Gs, Gi, Gq) 15  6  12  effectors  vs  effectors Rockman et al. (2002) Nature 415:206

44. G-Protein Coupled Receptors (GPCR) – 7 TM Receptors Many classification schemes for GPCRs ligands small molecules peptides proteins receptor structure sequence similarity size of extracellular domain location of ligand binding site extracellular domain membrane-spanning domain heterotrimeric G-proteins Gs, Gi, Go Alberts et al., (1994) Molecular Biology of the Cell, 3rd edn, Figure 15-17

45. Very Partial List of GPCR Ligands Peptides Huge gene superfamily 700-1000 GPCR genes in human genome ~2% of human genome angiotensin II vasopressin/ADH gastrin secretin cholecystokinin prolactin oxytocin somatostatin enkephalins/opiates Small Molecules epinephrine/norepinephrine dopamine acetylcholine - muscarinic serotonin – 5-hydroxytryptamine histamine glutamate (metabotropic) GABA type B Ca2+ adenosine ATP leukotrienes cannabinoids taste receptors – sweet, bitter odorants retinal – light receptors/rhodopsin Proteins chemokines parathyroid hormone thyroid stimulating hormone thrombin endothelin

46. Hormones with Second Messanger activity

47. GPCR’s are Major Drug Targets Partial list of diseases and conditions treated in part by drugs that target GPCRs allergies anaphylactic shock asthma cardiac arrhythmias congestive heart failure coronary artery disease/angina hypertension hypotension/shock syndromes peptic ulcer disease schizophrenia

48. Phosphatidylinositol Signal Cascades Some hormones activate a signal cascade based on the membrane lipid phosphatidylinositol.

49. Phosphatidylinositol Signal Cascade: Phospholipase C, Inositol Trisphosphate (IP3), Ca2+ and Diacylglycerol, Protein Kinase C (PKC) Ca++ A R PLC Gq DAG PKC PIP2 Protein Protein-P IP3 Endoplasmic Reticulum Ca++ DAG – diacylglycerol PKC – protein kinase C PLC – phospholipase C PIP2 – phosphatidylinositol bisphosphate IP3 – inositol trisphosphate

50. Kinases sequentially catalyze transfer of Pi from ATP to OH groups at positions 5 & 4 of the inositol ring, to yield phosphatidylinositol-4,5-bisphosphate (PIP2). PIP2 is cleaved by the enzyme Phospholipase C.