Principles of Signal Transduction: Objectives

1.  Be able to compare and contrast the overall properties of endocrine and synaptic signaling mechanisms.

  1. Endocrine Signaling Mechanism:
  2. Hormone is secreted by an endocrine gland and is carried, via the bloodstream, to a distant target cell
  3. Signaling is SLOW
  4. Relies on passive diffusion and blood flow
  5. Signals are DILUTE
  6. Receptors have high specificity and affinity for the signal
  7. Chemically based specificity and affinity
  8. There is a slow termination of the response (slow dissociation rate)
  9. Endocrine cells secrete many ligands – thus the target cells must be specific with a high affinity
  10. Example: Steroids
  11. Synaptic Signaling Mechanism:
  12. Signaling is FAST
  13. Uses electrical impulses over short distance
  14. Signals reach high local concentrations
  15. Receptors have low affinity for the signal
  16. Mechanically based (based on the placement of the synapses)
  17. There is a rapid termination of the response (quick dissociation rate)
  18. Secrete few distinct ligands – the specificity comes from the precise nerve/target cell contact point
  19. Examples: Peptides and proteins; catecholamines

2.  Be able to identify the four major types of signal transduction pathways and give examples of ligands and cellular responses for each.

  1. Receptor Tyrosine Kinase
  2. Ligands: Insulin, lots of growth factors
  3. Insulin Receptor Signaling:
  4. Receptor binds two signal compounds (in this case, insulin peptide) which activates a tyrosine autokinase. This also phosphorylates IRS-1 (“Insulin Receptor Substrate 1”).
  5. IRS-1 has three tissue specific pathways
  6. Growth: IRS1 phosphorylates Shp, activating Ras.
  7. Ras activates MAP which activates Transcription factors which promote growth
  8. Glucose Uptake
  9. IRS-1 phosphorylates P13K, initiating a phosphorylation cascade
  10. Leads to GLUT4 (transporters of glucose) onto the plasma membrane
  11. Glycogen Deposistion
  12. IRS1 phosphorylates P13K, initiating a phosporylation cascade
  13. Leads to stimulation of enzymatic steps in the conversion of glucose to glycogen
  14. G Protein-Linked Receptors
  15. Ligands: a great variety use G Protein-Linked Receptors
  16. Catecholamines, NTs, Peptide hormones…
  17. 2 possible cellular responses:
  18. cAMP Signal Pathway
  19. cAMP activates cAMP-dependent Protein Kinase (PKA) by phosphorylating PKA
  20. Kinase is a tetramer
  21. 2 cAMP-binding chains
  22. 2 catalytic chains
  23. Binding causes release of activated catalytic subunits
  24. Catalytic units phosphorylate substrates (usually enzymes)
  25. Moderation or reversal of response is achieved via:
  26. Dephosphorylation of substrates
  27. By phosphatases
  28. Degradation of cAMP
  29. By phosphodiesterases (PDE)

  1. Phosphatidylinositol Pathway
  2. Hormone signal causes increase in cytosolic Ca++ and activation of PKC, leading to 2 distinct (but interacting) chain of events:
  3. Ca++ binds to Calmodulin (CaM)
  4. Myosin Light Chain Kinase (MLCK) phosphorylates myosin
  5. Causes interaction with actin and muscle contracts
  6. Nitric Oxide Synthase (NOS)
  7. Protein Kinase C phosphorylates a variety of enzymes and proteins
  8. Ion channels (like Na+/H+ pump)
  9. Phosphorylation leads to increase of cellular pH and proliferation
  10. Activation of transcription factors controlling gene expression
  11. Intracellular Receptors (steroids)
  12. Examples of ligands: Glucocorticoid, mineralcorticoid, progesterone, estrogen, androgen, vitamin D, thyroid
  13. Three domains
  14. Transcription activation Domain
  15. Responsible for interaction promoting activity by RNA polymerase
  16. Thus, Communication with Pol
  17. DNA Binding Domain
  18. Site of direct interaction with DNA in the promoter region of genes
  19. Thus, Recognition of Hormone Response Elements
  20. Ligand Binding Domain.
  21. Site of high-selectivity binding by steroid hormones
  22. Thus, Binding Site
  23. 2 Activated Steroid Receptors recruit the Histone Acetyl Transferase (HAT) Complex
  24. Leading to acetylation of histones
  25. Causes DNA to unwind
  26. Allowing the binding of the transcription apparatus.

  1. Ligand Gated Channels
  2. Converts extracellular signals into electrical impulses
  3. Occurs between nerves and target cells (“Chemical Synapse”)
  4. Nerve terminal releases neurotransmitters (Ach, GABA, etc…) by fusion with storage vesicles with plasma membrane
  5. NT binds channels present in synapse region of target cell
  6. Gated channel opens, allowing entry of ions
  7. Example: Nicotinic Acetylchnoline Receptor:
  8. Channel opening requires 2 molecules of ligand
  9. Channel opens only briefly
  10. Closes while still ligated
  11. Ligand dissociates, returning channel to resting state
  12. Dissociated ACh is hydrolyzed by cholineresterase

3.  Be able to identify or describe the key steps and molecules involved in each of the four major pathways, including structure and location of receptors, 2nd messenger molecules (including calcium and nitric oxide), effector enzymes, and substrates.

  1. Ligand-Gated Channels
  2. Key Steps:
  3. Nerve terminal releases neurotransmitters
  4. Via fusion of storage vesicles with plasma membrane
  5. NT binds channels present in synapse region
  6. Gated channel opens, allowing entry of ions
  7. Molecules involved:
  8. Neurotransmitters
  9. ACh, GABA, Serotonin, Glutamate, Glycine…
  10. Structure of receptors:
  11. Composed of 5 transmembrane polypeptides
  12. Each chain ~500 AAs and traverses membrane four (4) times
  13. 1 helix of each chain contains polar AAs: “Aqueous Pore”
  14. Negatively-charged AAs are at the mouth of the channel
  15. Prevents entry of anions (ligand specificity)
  16. Location of receptors:
  17. Plasma membrane of Target Cells (transmembrane)
  18. 2nd messenger molecules:
  19. Effector Enzymes:
  20. Cholineresterase (hydrolyzes dissociated ACh)
  21. Substrates:

  1. Receptor Tyrosine Kinases:
  2. Key Steps:
  3. Ligand binding to the insulin receptor (IR)
  4. Induces phosphorylation on tyrosine residues on the intracellular domain
  5. 2 ligands of insulin peptide are required for full activation
  6. Molecules involved:
  7. Insulin Peptide
  8. IRS1
  9. Ras (for MAP kinase cascade to promote growth factors)
  10. P13K (is phosphorylated by IRS1, initiates phosphorylation cascade)
  11. Structure of receptors:
  12. Single transmembrane alpha helix
  13. Example: Insulin
  14. 2 beta and 2 alpha chains (sulfide bond connects them)
  15. Alpha chains: contain hormone-binding site
  16. Beta chains: traverse the membrane and contain tyrosine kinase domain
  17. Location of receptors:
  18. On the plasma membrane (transmembrane)
  19. Extracellular domain
  20. Ligand-binding site
  21. Intracellular domain
  22. Tyrosine kinase activity
  23. 2nd messenger molecules:
  24. IRS1
  25. Ras(for MAP kinase cascade to promote growth factors)
  26. P13K (is phosphorylated by IRS1, initiates phosphorylation cascade)
  27. Effector Enzymes:
  28. MAP Kinase (activates transcription factors to promote growth)
  29. Substrates:

  1. Steroid (Intercellular) Receptors:
  2. Key Steps:
  3. 2 Ligand-ed Steroid Receptors recruits a co-activator that contains the enzyme, “Histone Acetyl Transferase” (HAT)
  4. That enzyme leads to acetylation of histones
  5. The acetylated histones allow the DNA to unwind
  6. The unwound DNA allows for the binding of general transcription factors (such as Pol II)
  7. Molecules involved:
  8. SR (Steroid Receptor)
  9. HSP
  10. IP
  11. CoA Complex
  12. TATA (gene expression begins here via RNA transcription)
  13. Structure of receptors:
  14. NH2 – Transcription activation domain – DNA binding domain – Ligand binding domain – COOH
  15. Receptors are always “ON”
  16. Location of receptors:
  17. Plasma membrane
  18. Cytosol
  19. Nucleus
  20. 2nd messenger molecules:
  21. Effector Enzymes:
  22. Histone Acetyl Transferase (HAT)
  23. Acetylates histones
  24. Leads to the unwinding of DNA
  25. Allows for binding of general transcription factors
  26. Substrates:

  1. G-Protein Linked Receptors
  2. Structure of receptors:
  3. “7 Pass” transmembrane receptor
  4. Generates 4 intracellular and extracellular loops
  5. Extracellular amino terminus:
  6. Ligand binding site
  7. Intracellular loops:
  8. G Protein Interaction
  9. Phosphorylation-mediated inactivation
  10. When 3rd loop is phosphorylated, the receptor is inactivated
  11. cAMP Signaling:
  12. Key Steps:
  13. Hormone stimulateion causes rapid increase in cAMP
  14. Adenylate cyclase cleaves ATP to generate cAMP and pyrophosphate
  15. IRREVERSIBLE step
  16. Degradation of cAMP by phosphodiesterase
  17. Yields 5’-AMP

  1. Molecules involved:
  2. cAMP, PKA (cAMP-dependent protein kinase)
  3. Location of receptors:
  4. On Plasma membrane
  5. Alpha chain:
  6. Binds and hydrolyzes GTP
  7. Activates adenylate cyclase
  8. Beta and Gamma chains:
  9. Anchors alpha chain to the cytoplasmic face
  10. 2nd messenger molecules:
  11. Cyclic AMP (cAMP)
  12. Effector Enzymes:
  13. Adenylate cyclase cleaves ATP to generate cAMP and pyrophosphate
  14. IRREVERSIBLE step
  15. Degradation of cAMP by phosphodiesterase
  16. Yields 5’-AMP
  17. Substrates:
  18. Ca++/Phosphoinositide Signal Pathway:
  19. Key Steps:

  1. Molecules involved:
  2. Cam (Calmodulin)
  3. Location of receptors:
  4. Plasma membrane
  5. On they cytosolic face
  6. 2nd messenger molecules:
  7. Diacylglycerol (DAG)
  8. Inositol Trisphosphate (IP3)
  9. Ca++ Ion
  10. Effector Enzymes:
  11. MLCK (phosphorylates myosin, causing muscle contraction)
  12. Nitric Oxide Synthase (NOS)
  13. Protein Kinase C (phosphorylates lots of enzymes and proteins)
  14. Substrates:

4.  Be able to describe the major mechanisms by which each signal pathways can be inhibited.

  1. Tyrosine Kinase, Steroid, and Ligand-Gated Channels:
  2. Down-regulation of Cell Surface Receptors
  3. Prolonged hormone exposure leads to receptor degradation
  5. G-Linked
  6. Inactivation of receptors via phosphorylation:

1.  Excess hormone

  1. Results in receptor that can bind hormone but not activate G protein
  2. Phosphorylation of serines and threonines on intracellular loops
  3. A-kinase first to phosphorylate when cAMP levels get too high, followed by BARK
  4. Fully phosphorylated receptor is further blocked from G protein by beta arrestin