Heterotrimeric GProtein Signaling

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Heterotrimeric G-Protein Signaling

• Dev Bio 231B / Bio Sci D154 (Winter 2008)
• Yi Lecture 1

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Yi Lecture Outlines

• Lecture 1 Heterotrimeric G-Protein Signaling
• Lecture 2 Chemotaxis
• Lecture 3 Rho/Rac/Cdc42
• Lecture 4 Cell Polarization
• Reading
• Xu et al. paper
• Alberts text
• Optional homework problems for each lecture
• Tested by in-class Midterm (my part will be
  open-book)

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Outline (Yi Lecture 1)

• Heterotrimeric G-protein signaling
• Introduction to Xu et al. paper
• Readings
• Alberts (p. 851, 852-861, 870)
• Xu et al. paper

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Signal Transduction Pathway
Input (Signal)
Receptor
Transducers
Regulation
Effectors
Outputs
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In Cell Biology We Are Concerned With Spatial
Characteristics
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Checklist of Signal Transduction Systems
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Heterotrimeric G-Protein Systems
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Overview of G-Protein Activation
heterotrimer (3 subunits a, b, g)
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G-Protein Cycle
Active
Guanine Exchange Factor
GTPase Activating Protein
GEF (receptor)
GAP (RGS)
Regulator of G-protein Signaling
Hydrolysis of GTP to GDP
Exchange of GTP for GDP
Inactive
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G-Protein Structure

• Lipid modifications of G-protein subunits

farnesyl
geranylgeranyl
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G-Protein Classification(Mammalian Sequences)

• In mammals
• 4 classes of a-subunits
• Gs, Gi, Gq, G12
• 17 a-subunits
• 4 b-subunits
• 6 g-subunits

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Evolutionary Conservation of G-Proteins in
Eukaryotes

• From simplest (e.g. yeast) to most sophisticated
  eukaryotes (e.g. humans)
• First among eukaryotic signal transduction
  pathways
• Conservation of sequence
• Conservation of structure
• Conservation of function
• Human G-protein subunits can substitute for yeast
  proteins
• Conservation of regulators and effectors

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G-Protein Coupled Receptors (GPCRs)

• Possess canonical 7 transmembrane helix structure
• Bind variety of ligands
• Small molecules, peptides, proteins
• 5 GPCR families by sequence alignment
• A) Rhodopsin (largest class)
• B) Secretin (hormones)
• C) Glutamate
• D) Fungal pheromone
• E) cAMP (Dictyostelium)
• 1400 human GPCRs ( 5 of human genome)
• ½ are olfactory receptors
• Primary sensors for humans (as well as other
  eukaryotes)

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Pharmacology Agonists and Antagonists

• G-protein systems (e.g. GPCRs) are common targets
  of pharmaceuticals
• An agonist binds to and activates the receptor
• An antagonist binds to the receptor and prevents
  its activation
• Partial agonists partially activate the receptor
• Inverse agonists lower the basal level of
  receptor activity

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Dose-Response Curves

• Measure the response as a function of drug dose
  (or input signal)
• EC50 is the concentration of drug that produces
  50 of maximal effect
• Often the Kd (equilibrium dissociation binding
  constant) is close in value to the EC50.
• Potency concentration (EC50) of a drug required
  to produce 50 of drugs maximal effect
• Maximal Efficacy maximal response to the drug

Greater Sensitivity
Greater Response
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Some G-Protein Effectors

• Effectors are activated (or inhibited) by Ga-GTP
  or Gbg
• Adenylyl cyclase (Gs)
• cAMP
• Ion channels (Gbg)
• Kinases
• Phospholipase C (Gq and Gbg)
• IP3 (activates Ca) and diacylglycerol
  (activates protein kinase C)
• Small G-protein exchange factors (G12/G13)
• e.g. activator of Rho
• Scaffold proteins

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Cross-Talk between GPCRs and Other Receptor
Systems
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Signal Adaptation (Alberts p. 851)

• Dynamic Range cell detect changes in signal
  over a wide range of stimulus intensities
• Broad dynamic range means wide dose-response
  curve to input signal
• Requires that the target cells undergo a
  reversible adaptation or desensitization
• Prolonged exposure to stimulus decreases cells
  response to that level of stimulus

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Regulation of Signaling

• (1) Ligand
• (2) Receptor
• (3) RGS

L
k1
k2
RL
R
k3
Ga
Ga
k4
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Receptor Regulation Phosphorylation
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Receptor RegulationEndocytosis
(late endosome)
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Experimental Tools for G-Proteins

• Genetics
• Loss-of-function mutants (e.g. dominant negative)
• Gain-of-function mutants (e.g. constituitively
  active)
• Pharmacology
• Pertussis toxin (inactivates Gi subunit)
• Receptor agonists/antagonists
• Molecular biology
• Reporters of signaling
• Biochemistry
• In vitro system
• Cell Biology
• GFP-labeled proteins and microscopy
• Physiology and behavior

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Biological (G-Protein) Sensors
Olfaction
Vision
Immune
much better than technological sensors
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Complex Dynamic BehaviorsNeutrophil Chasing
Bacterium

• http//www.biochemweb.org/fenteany/research/cell_
  migration/neutrophil.html
• Sensing (Heterotrimeric G-Protein)
• Response (Small G-Protein)

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Chemotaxing Eukaryotic Cell
Leading Edge (Pseudopod)
Ligand fMLP Formyl-methiomyl-leucine-phenylalan
ine
Trailing Edge (Uropod)
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Motivating Question for Xu et al. Paper

• Leading edge (pseudopod) depends on
  heterotrimeric G-protein signaling (Gi), PI3K,
  the small G-proteins Rac and Cdc42, and actin
  polymerization
• What signaling events (if any) produce the
  trailing edge (uropod)?

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Lecture 1 Some Questions to Ponder (Optional
Homework)

• Dose-response curves
• Draw the dose-response curve for an agonist
• On the same graph, draw the dose-response curve
  for an agonist with higher affinity for the
  receptor
• Does the higher affinity agonist have lower or
  higher potency than the original agonist?
• Rhodopsin is the light receptor in the retina
• What is the expected vision phenotype of a mouse
  in which the carboxy-terminal phosphorylation
  sites of rhodopsin are mutated from Ser/Thr to
  Ala? Would the mice be able to see well in low
  light? What about bright light?

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Summary (Yi Lecture 1)

• Basics of heterotrimeric G-protein signaling
• Dose-response curve and sensitivity
• Regulation of signaling
• Experimental tools
• Introduction to chemotaxing neutrophils