Signalling vocabulary

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Signalling vocabulary

• Signal/stimulus
• Effector
• Receptor
• Messenger
• Ligand
• Cascade

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Types of Receptors

• 7-TMS receptors (G protein receptors)
• extracellular site for hormone (ligand)
• intracellular site for GTP-binding protein
• Single-transmembrane segment receptors
• extracellular site for hormone (ligand)
• intracellular catalytic domain - e.g. kinase or
  guanylyl cyclase
• Oligomeric ion channels

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G-protein coupled receptors

• Receptors that interact with G proteins
• Seven putative alpha-helical transmembrane
  segments
• Extracellular domain interacts with hormone
• Intracellular domain interacts with G proteins
• Adrenergic receptors are typical
• Note desensitization by phosphorylationby protein
  kinase A

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Heterotrimeric G Proteins

• A model for their activity
• Binding of hormone, etc., to receptor protein in
  the membrane triggers dissociation of GDP and
  binding of GTP to ?-subunit of G protein
• G?-GTP complex dissociates from G?? and migrates
  to effector sites, activating or inhibiting
• But it is now clear that G?? also functions as a
  signalling device

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cAMP and Glycogen Phosphorylase

• Earl Sutherland discovers the first second
  messenger
• In the early 1960s, Earl Sutherland showed that
  the stimulation of glycogen phosphorylase by
  epinephrine involved cyclic adenosine-3',5'-monoph
  osphate
• He called cAMP a "second messenger"
• cAMP is synthesized by adenylyl cyclase and
  degraded by phosphodiesterase

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Signalling Roles for G(??)

• A partial list
• Potassium channel proteins
• Phospholipase A2
• Yeast mating protein kinase Ste20
• Adenylyl cyclase
• Phospholipase C
• Calcium channels
• Receptor kinases

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Stimulatory and Inhibitory G

• G proteins may either stimulate or inhibit an
  effector.
• In the case of adenylyl cyclase, the stimulatory
  G protein is known as Gs and the inhibitory G
  protein is known as Gi
• Gi may act either by the Gi? subunit binding to
  AC or by the Gi?? complex complexing all the Gi?
  and preventing it from binding to AC

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Figure 19-13 Activation/deactivation cycle for
hormonally stimulated AC.
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Figure 19-16 Mechanism of receptor-mediated
activation/ inhibition of AC.
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Phospholipases Release Second Messengers

• Inositol phospholipids yield IP3 and DAG
• PLC? is activated by 7-TMS receptors and G
  proteins
• PLC? is activated by receptor tyrosine kinases
  (via phosphorylation)
• Note PI metabolic pathways and the role of lithium

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Phospholipase targets
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Phosphotidyl inositols as secondary messengers
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Other Lipids as Messengers

• Recent findings - lots more to come
• More recently than for PI, other phospholipids
  have been found to produce second messengers!
• Phosphatidyl choline can produce prostaglandins,
  diacylglycerol and/or phosphatidic acid
• Sphingomyelin and glycosphingolipids also produce
  signals such as ceramide, a trigger of apoptosis
  - programmed cell death

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Many different activators of phospholipase
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Phospholipase C isozymes

• src-homology domains (SH)
• SH2 mediates interactions with phosphotyrosinated
  proteins
• SH3 interacts with cytoskeletal proteins

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Protein kinase C integration of two second
messenger signals

• PKC is activated by DAG and Ca2
• Most PKC isozymes have several domains, including
  ATP-binding domain, substrate-binding domain,
  Ca-binding domain and a phorbol ester-binding
  domain
• Phosphorylates Ser,Thr
• Phorbol esters are apparent analogues of DAG
• Signals terminated/modulated by cellular
  phosphatases dephosphorylate target proteins

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Ca2 as a Second Messenger

• Several sources of Ca2 in cells!
• Ca2 in cells is normally very low lt 1?M
• Calcium can enter cell from outside or from ER
  and calciosomes (plants store Ca2 in oxalate
  crystals)
• CICR - Calcium-Induced Calcium Release
• see animation

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Calcium Oscillations!

• M. Berridge's model of Ca2 signals
• Ca2 was once thought to merely rise in cells to
  signal and drop when the signal was over
• Berridge's work demonstrates that Ca2 levels
  oscillate in cells!
• The purpose may be to protect cell components
  that are sensitive to high calcium, or perhaps to
  create waves of Ca2 in the cell

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Patch clamp
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Ca2-Binding Proteins

• Mediators of Ca2effects in cells
• Many cellular proteins modulate Ca2 effects
• 3 main types protein kinase Cs, Ca2-modulated
  proteins and annexins
• Kretsinger characterized the structure of
  parvalbumin, prototype of Ca2-modulated proteins
  
• "EF hand" proteins bind BAA helices

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Calmodulin
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Protein Modules in Signal Transduction

• Signal transduction in cell occurs via
  protein-protein and protein-lipid interactions
  based on protein modules
• Most signaling proteins consist of two or more
  modules
• This permits assembly of functional signaling
  complexes

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MAP Kinase Cascade
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Single transmembrane receptors

• Receptor tyrosine kinases
• Dimerization and cross-phosphorylation
• Insulin receptor
• Epidermal growth factor receptor

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Insulin receptor binding of peptide causes
dimerization and cross- phosphorylation on Tyr
residues
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Insulin signaling

• Binding ? Dimerization ? Phosphorylation
• ?
• Binding and phosphorylation of IRS1,2
• ?
• Binding and activation of PI-3-K by IRS
• ?
• Formation of PIP3
• ?
• Activation of PIP3 dependent protein kinase
• and kinase cascade ? Ca2 release, activation of
  glycogen synthase kinase, etc.

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Localization of Signaling Proteins

• Adaptor proteins provide docking sites for
  signaling modules at the membrane
• Typical case IRS-1 (Insulin Receptor
  Substrate-1)
• 18 potential tyrosine phosphorylation sites
• PH and PTB direct IRS-1 to receptor tyrosine
  kinase - signaling events follow!

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EGF signaling

• Binding of 2 growth factor peptides
• Dimerization
• Phosphorylation of Tyr residues at C-terminus
• Binding of adaptor protein Grb2 (SH2 binds
  phosphotyrosine)
• Recruits Sos (SH3 binds proline rich region)
• Ras G-protein binding and nucleotide exchange
• Activation of MEK ? Activation of ERK ?
  transcription, etc.
• Termination by phosphatases and ras GTPase
  activity

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When signal molecule misbehave
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Lipids Rafts

• first hypothesized in 1988
• nice review Cary, L. A. Cooper, J. A. (2000)
  Molecular switches in lipid rafts.  Nature. 404,
  945-947
• Moffett, S., Brown, D. A. Linder, M. E. (2000)
  Lipid-dependent targeting of G proteins into
  rafts. J. Biol.Chem. 275, 2191-2198.

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• Many actin binding proteins are known to bind to
  polyphosphoinositides and to be regulated by them
  
• Activation of receptor causes reorganization of
  the rafts

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• Simons, K. et al. J. Clin. Invest.
  2002110597-603

J. Fantini, N. Garmy, R. Mahfoud and N. Yahi
Lipid rafts structure, function and role in HIV,
Alzheimers and prion diseases Expert Reviews in
Molecular Medicine 20 December 2002
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Communication at the Synapse

• A crucial feature of neurotransmission
• Ratio of synapses to neurons in human forebrain
  is 40,000 to 1!
• Chemical synapses are different from electrical
• Neurotransmitters facilitate cell-cell
  communication at the synapse
• Note families of neurotransmitters in Table 34.6

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The Cholinergic Synapse

• A model for many others
• Synaptic vesicles in synaptic knobs contain
  acetylcholine (10,000 molecules per vesicle)
• Arriving action potential depolarizes membrane,
  opening Ca channels and causing vesicles to fuse
  with plasma membrane
• Acetylcholine spills into cleft, migrates to
  adjacent cells and binds to receptors
• Toxin effects botulism toxin inhibits Ac-choline
  release, black widow's latrotoxin protein
  overstimulates

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Two Classes of Ac-Ch Receptor

• Nicotinic and muscarinic
• As always, toxic agents have helped to identify
  and purify hard-to-find biomolecules
• Nicotinic Ac-Ch receptors are voltage-gated ion
  channels
• Muscarinic Ac-Ch receptors are transmembrane
  proteins that interact with G proteins
• Acetylcholinesterase degrades Ac-Ch in cleft
• Transport proteins and V-type H-ATPases return
  Ac-Ch to vesicles - called reuptake

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Other Neurotransmitters

• Excitatory and inhibitory
• Glutamate is good example nerve impulse triggers
  Ca-dependent exocytosis of glutamate
• Glutamate is either returned to neuron, or
  carried into glial cells, converted to Gln and
  taken back to the neuron from which it was
  released
• See 4 types of glutamate receptors in Fig. 34.68
• NMDA receptor is best understood for now
• Note phencyclidine (angel dust) story

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GABA and Glycine

• Inhibitory Neurotransmitters
• Inhibitory neurotransmitters diminish the actions
  of activating neurotransmitters
• See Figure 34.70 for glutamate degradation
• Excitatory glutamate is broken down to inhibitory
  GABA, which is broken down to non-signals
• GABA glycine receptors are chloride channels
• Glycine receptor is site of action of strychnine

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Catecholamine Neurotransmitters

• Epinephrine, norepinephrine, dopamine and L-dopa
  are all neurotransmitters
• Synthesized from tyrosine - see Fig. 34.72
• Excessive production of dopamine (DA) or
  hypersensitivity of DA receptors produces
  psychotic symptoms and schizophrenia
• Lowered production and/or loss of DA neurons are
  factors in Parkinsonism

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Neurological Disorders

• Depression, Parkinsonism, etc.
• Defects in catecholamine processing are
  responsible for many neurological disorders
• Norepinephrine and dopamine systems are keys
• Breakdown of NE and DA by catechol-O-methyl
  transferase and monoamine oxidase
• Reuptake by specific transport proteins
• MAO inhibitors are antidepressants
• Tricyclics - antidepressants that block reuptake
• Cocaine blocks reuptake, prolongs effects of DA

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Peptide Neurotransmitters

• Lots more to learn here!
• Likely to be many peptide NTs
• Concentrations are low purification is hard
• Roles are complex
• Endorphins and enkephalins are natural opioids
• Endothelins affect smooth muscle contraction,
  vasoconstriction, mitogenesis, tissue changes
• Vasoactive intestinal peptide stimulates AC (to
  make cAMP) via G proteins, and its effects are
  synergistic with those of other neurotransmitters

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Recap Signaling Pathways from Membrane to the
Nucleus

• The complete path from membrane to nucleus is
  understood for a few cases
• Signaling pathways are redundant
• Signaling pathways converge and diverge
• This is possible with several signaling modules
  on a signaling protein

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Module Interactions Rule!

• The interplay of multiple modules on many
  signaling proteins permits a dazzling array of
  signaling interactions
• We can barely conceive of the probable extent of
  this complexity
• For example, it is estimated that there are more
  than 1000 protein kinases in the typical animal
  cell - all signals!