Signal transmission and signal transduction

1. Signal transmission and signal transduction Huan Ma（马欢），PhD Department of Physiology Room 515, Block C, Research Building School of Medicine, Zijingang Campus Email: mah@zju.edu.cn Tel: 88208068

2. OUTLINE • Intercellular signal transmission • Chemical transmission • Electrical transmission • Signal transduction pathway • Pathways initiated by intracellular receptors • Pathways initiated by plasma membrane receptors

3. Intercellular signal transmission • Chemical transmission • Chemical signals • Neurotransmitters:

4. Intercellular signal transmission • Chemical transmission • Chemical signals • Neurotransmitters: • Humoral factors: • Hormones • Cytokines • Bioactivators

5. Intercellular signal transmission • Chemical transmission • Chemical signals • Neurotransmitters: • Humoral factors: • Gas: NO, CO, etc.

6. Intercellular signal transmission • Chemical transmission • Chemical signals • Receptors • Membrane receptors • Intracellular receptors

7. Receptors on the surface of a cell are typically proteins that span the membrane

8. Only Cell A has the matching receptors for this chemical messenger, so it is the only one that responds Cells B & C lack the matching receptors Therefore are not directly affected by the signal

11. Intercellular signal transmission • Electrical transmission Gap junction

12. Cardiac Muscle Low Magnification View The intercalated disk is made of several types of intercellular junctions. The gap junction provides a low resistance pathway for the action potential to spread from cell to cell

13. Signal transduction pathway • Pathways initiated by intracellular receptors • Pathways initiated by plasma membrane receptors

14. This hydrophobic signal requires a carrier protein while in the plasma … … but at the target cell the signal moves easily through the membrane and binds to its receptor

15. Signal transduction pathway • Pathways initiated by intracellular receptors • Pathways initiated by plasma membrane receptors (transmembrane signal transduction)

16. Transmembrane signal transduction

17. Transmembrane signal transduction • Mediated by G protein-linked receptor • Mediated by enzyme-linked receptor • Mediated by ion channel

18. 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).

19. 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.

20. Phosphorylation may directly alter activity of an enzyme, e.g., by promoting a conformational change. Alternatively, altered activity may result from binding another protein that specifically recognizes a phosphorylated domain. • E.g., 14-3-3 proteins bind to domains that include phosphorylated Ser or Thr in the sequence RXXX[pS/pT]XP, where X can be different amino acids. • Binding to 14-3-3 is a mechanism by which some proteins (e.g., transcription factors) may be retained in the cytosol, & prevented from entering the nucleus.

21. 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).

22. 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.

23. 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.

24. Protein Kinase A (cAMP-Dependent Protein Kinase) transfers Pi from ATP to OH of a Ser or Thr in a particular 5-amino acid sequence. Protein Kinase A in the resting stateis a complex of: • 2 catalytic subunits(C) • 2 regulatory subunits(R). R2C2

25. R2C2 Each regulatory subunit (R) of Protein Kinase Acontains a pseudosubstrate sequence, like the substrate domain of a target protein but with Ala substituting for the Ser/Thr. The pseudosubstrate domain of (R), which lacks a hydroxyl that can be phosphorylated, binds to the active site of (C), blocking its activity.

26. R2C2 + 4 cAMPR2cAMP4 + 2C When each (R) binds 2 cAMP, a conformational change causes (R) to release (C). The catalytic subunits can then catalyze phosphorylation of Ser or Thr on target proteins. PKIs, Protein Kinase Inhibitors, modulate activity of the catalytic subunits (C).

27. Binding of ligands to membrane-spanning receptors activates diverse response mechanisms

28. Transmembrane signal transduction • Mediated by G protein-linked receptor • Mediated by enzyme-linked receptor • Mediated by ion channel

29. The Nobel Prize in Physiology or Medicine 1994 • "G-proteins and the role of these proteins in signal transduction in cells" Alfred G. Gilman Martin Rodbell

30. The Discovery of G Proteins Normal Lymphoma Cell Mutated Lymphoma Cell

31. 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.

32. Rhodopsin is unique. It senses light, via a bound chromophore, retinal. • Most7-helix receptors have domains facing the extracellular side of the plasma membrane that recognize & bind signal molecules (ligands). E.g., the b-adrenergic receptor is activated by epinephrine & norepinephrine. Crystallization of this receptor was aided by genetically engineering insertion of the soluble enzyme lysozyme into a cytosolic loop between transmembrane a-helices.

33. 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. 800 different GPCRs are encoded in the human genome.

34. G-protein-Coupled Receptors may dimerize or form oligomeric complexes within the membrane. Ligand binding may promote oligomerization, which may in turn affect activity of the receptor. Various GPCR-interacting proteins (GIPs) modulate receptor function. Effects of GIPs may include: • altered ligand affinity • receptor dimerization or oligomerization • control of receptor localization, including transfer to or removal from the plasma membrane • promoting close association with other signal proteins

35. 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.

36. 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.

37. 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.

38. 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).

39. 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.

40. 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.

41. 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.

42. 3. Receptordesensitization varies with the hormone. • In some cases the activatedreceptor is phosphorylated via a G-protein Receptor Kinase. • The phosphorylatedreceptor then may bind to a protein b-arrestin. • b-Arrestin promotes removal of the receptor from the membrane by clathrin-mediated endocytosis. • b-Arrestin may also bind a cytosolic Phosphodiesterase, bringing this enzyme close to where cAMP is being produced, contributing to signal turnoff. 4. Protein Phosphatase catalyzes removal by hydrolysis of phosphates that were attached to proteins via ProteinKinase A.

43. Different isoforms of Ga have different signal roles. E.g.: • The stimulatoryGsa, when it binds GTP, activates Adenylate cyclase. • An inhibitoryGia, when it binds GTP, inhibits Adenylate cyclase. Different effectors & their receptorsinduce Gia to exchange GDP for GTP than those that activate Gsa. • The complex of Gb,g that is released when Ga binds GTP is itself an effector that binds to and activates or inhibits several other proteins. E.g., Gb,g inhibits one of several isoforms of Adenylate Cyclase, contributing to rapid signal turnoff in cells that express that enzyme.

44. The family of heterotrimeric G proteins includes also: • transducin, involved in sensing of light in the retina. • G-proteins involved in odorant sensing in olfactory neurons. There is a larger family of small GTP-binding switch proteins, related to Ga.

45. 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.

46. Most GTP-binding proteins depend on helper proteins: GAPs, GTPase Activating Proteins, promote GTP hydrolysis. GEFs, Guanine Nucleotide Exchange Factors, promote GDP/GTP exchange.

47. Activation and Inactivation of the G Protein

48. G-protein-coupled Receptors

49.  G protein effector   Second messenger   Protein kinase Signaling molecules involved