Signal Transduction

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• Signal Transduction
• BL 4010 12.07.06

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Outline

• Extracellular signals
• Signal-Transducing Receptors
• single membrane spanning receptors
• 7 TMS receptors
• Intracellular Second Messengers
• cAMP
• Calcium
• G-proteins
• Enzyme cascades

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Additional resources

• http//www.signaling-gateway.org/
• http//web.indstate.edu/thcme/mwking/signal-transd
  uction.html
• http//stke.sciencemag.org/

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Extracellular Signals

• Light
• Small molecules
• hormones
• toxins
• metabolites
• Large molecules
• oligosaccharides
• proteins

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Transcriptional activation
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Interferon
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The phosphorelay system
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Phosphorelay system
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Figure 19-1a Classification of hormones. (a)
Endocrine signals are directed at distant cells
through the intermediacy of the bloodstream.
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Figure 19-1b Classification of hormones. (b)
Paracrine signals are directed at nearby cells.
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Figure 19-1c Classification of hormones. (c)
Autocrine signals are directed at the cell that
produced them.
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Classes of Hormones
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Figure 19-3a Binding of ligand to receptor. (a) A
hyperbolic plot.
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Figure 19-3b Binding of ligand to receptor. (b) A
Scatchard plot.
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Classes of Hormones - Steroid Hormones

• Derived from cholesterol- (e.g.
  Glucocorticoids,vitamin D, sex hormones)
• regulate metabolism, salt/water balances,
  inflammation, sexual function. May bind to PM
  receptor or enter nucleus directly
• May either act at nucleus or at plasma membrane
• Steroids are hydrophobic and cannot diffuse
  freely to nucleus
• Receptor proteins carry steroids to the nucleus
• Steroid receptor proteins are all apparently
  members of a gene superfamily and have evolved
  from a common ancestral precursor

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Classes of Hormones - Steroid Hormones
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Classes of Hormones - Nonsteroid hormones

• Amino Acid Derived Hormones (e.g. epinephrine)
  bind to PM receptors
• regulate smooth muscle , blood pressure, cardiac
  rate, lipolysis, glycogenolysis

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Effects of epinephrine

• Receptor Molecule Effect
• Alpha1 Epinephrine, Norepinphrine Increased Ca2
• Alpha2 Epinephrine, Norepinphrine Decreased cAMP
• Beta1 Epinephrine, Norepinphrine Increased cAMP
• Beta2 Epinephrine Increased cAMP
• Increased rate and force of contraction of heart
  muscle predominantly an effect of epinephrine on
  beta receptors.
• Constriction of blood vessels increased blood
  pressure.
• Dilation of bronchioles assists in pulmonary
  ventilation.
• Stimulation of lipolysis in fat cells provides
  fatty acids for energy production and conserves
  dwindling blood glucose.
• Increased metabolic rate
• Inhibition of certain "non-essential" processes
  e.g. inhibition of gastrointestinal secretion and
  motor activity.
• Dilation of the pupils particularly important in
  situations where you are surrounded by
  velociraptors under conditions of low ambient
  light

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Classes of Hormones - Nonsteroid hormones

• Peptide Hormones (e.g. insulin) - bind to PM
  receptors
• regulate many processes in all tissues -
  including release of other hormones
• All secreted polypeptide hormones are synthesized
  with a signal sequence (which directs them to
  secretory granules)
• Usually synthesized as inactive preprohormones
  ("pre-pro" implies at least two precessing
  steps)
• Proteolytic processing produces the prohormone
  and the hormone

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Insulin is a peptide hormone
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Secretion of insulin
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Single TMS Receptors

• What is a receptor?
• Three main classes
• Extracellular domain to interact with hormone
• Single transmembrane segment
• Intracellular domain with enzyme activity
• Activity is usually tyrosine kinase or guanylyl
  cyclase
• Each of these has a "nonreceptor" counterpart
• src gene kinase - pp60v-src was first known
• Two posttranslational modifications

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Receptor Tyrosine Kinases

• Membrane-associated allosteric enzymes
• How do single-TMS receptors transmit the signal
  from outside to inside??
• Oligomeric association is the key!
• Extracellular ligand binding

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Protein-Tyrosine Phosphatases

• The enzymes that dephosphorylate Tyr
• Some PTPases are integral membrane proteins
• But there are also lots of soluble PTPases
• Cytoplasmic PTPases have N-term. catalytic
  domains and C-terminal regulatory domains
• Membrane PTPases all have cytoplasmic catalytic
  domain, single transmembrane segment and an
  extracellular recognition site

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Guanylyl Cyclases

• Soluble or Membrane-Bound
• Membrane-bound GCs are the other group of
  single-transmembrane-segment receptors (besides
  RTKs)
• Peptide hormones activate the membrane-forms
• Note speract and resact, from mammalian ova
• Activation may involve oligomerization of
  receptors, as for RTKs

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Soluble Guanylyl Cyclases

• Receptors for Nitric Oxide
• NO is a reactive, free-radical that acts either
  as a neurotransmitter or as a second messenger
• NO relaxes vascular smooth muscle (and is thus
  involved in stimulation of penile erection)
• NO also stimulates macrophages to kill tumor
  cells and bacteria
• NO binds to heme of GC, stimulating GC activity
  50-fold
• Read about NO synthesis and also see box on
  Alfred Nobel

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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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Second Messengers

• Many and there may be more!
• The hormone is the "first messenger"
• The second messenger - Ca2, cAMP or other - is
  released when the hormone binds to its
  (extracellular) receptor
• The second messenger then activates (or inhibits)
  processes in the cytoplasm or nucleus
• Degradation and/or clearance of the second
  messenger is also (obviously) important

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Steroid Receptor Proteins

• Hydrophobic domain near C-terminus that interacts
  with steroid itself
• Central, hydrophilic domain that binds to DNA
• Central DNA-binding domains are homologous to one
  another, with 9 conserved Cys residues
• Three pairs of Cys residues are in Cys-X-X-Cys
  sequences - as in Zinc-finger domains
• Steroid-receptor complex may bind to DNA or to
  transcription factors
• Thyroid hormone receptor proteins are similar

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Steroid Receptor Proteins
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Steroid Receptor Proteins
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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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How are the hormone receptor and AC coupled?

• Purified AC and purified receptor, when
  recombined, are not coupled.
• Rodbell showed that GTP is required for hormonal
  activation of AC
• In 1977, Elliott Ross and Alfred Gilman at Univ.
  of Virginia discovered a GTP-binding protein
  which restored hormone stimulation to AC
• Hormone stimulates receptor, which activates
  GTP-binding protein, which activates AC

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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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Figure 19-13 Activation/deactivation cycle for
hormonally stimulated AC.
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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 19-16 Mechanism of receptor-mediated
activation/ inhibition of AC.
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The ras Gene and p21ras

• An oncogene and its product
• a gene first found in rat sarcoma virus
• Normal cellular ras protein activates cellular
  processes when GTP is bound and is inactive when
  GTP has been hydrolyzed to GDP
• Mutant (oncogenic) forms of ras have severely
  impaired GTPase activity, so remain active for
  long periods, stimulating
• excessive growth and metabolic activity - causing
  tumors to form

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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