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Prof. Kristin Scott291 LSA
OFFICE HOURSM 11 AM-12 NOONW 11 AM-12 NOON,
F 3 PM-4 PM and by appointment
POWERPOINT SLIDES ON http://mcb.berkeley.edu/courses/mcb160/
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The language of neurons: Initiating a signal
how a chemical signal is detectedhow cellular activity changeshow electrical and chemical signals are produced
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Outline
signal transduction pathways **G protein-coupled cascades
receptor tyrosine kinase cascades
Concepts **how outside signals change cellular activity **how proteins are turned ‘on’ and ‘off’
how signaling cascades amplify signalshow cascades change neural excitability
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Cells need to recognize and respond to a large variety of signals
Signals• Steroid hormones
• Peptide hormones
• Neurotransmitters and neuropeptides
• Cytokines and Growth Factors
• Morphogens and other developmental signals
• Antigens
• Cell surface molecules
• Extracellular matrix components
• Gaseous molecules, UV radiation and other physical and chemcial stresses (e.g. hypoxia)
Biological Effects• Ion and nutrient transport
and/or secretion
• Membrane depolarization
• Morphological/cytoskeletal changes
• Metabolic changes
• Gene expression changes
• Cell migration
• Cell proliferation and/or differentiation
• Cell survival and/or apoptosis
• Development and Morphogenesis
• Learning and Memory
?
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What is Signal Transduction ?
Receptor activation
Intracellular signaling events
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1. IONOTROPIC Heteromultimer (GABAa, nAChR)
2. METABOTROPIC Monomer? Dimer? (mGluR, peptides)
3. RECEPTOR Dimer TYROSINE KINASE
(growth factors)
Families of Receptors
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Receptor G Protein Effector 2nd messenger 2nd Effector
Sensory Receptors Enzymes EnzymesPeptide Receptors phosphodiesterase cyclic nucleotides kinasesHormone Receptors adenylate cyclase lipids phosphatasesNeurotransmitter phospholipaseA calcium Receptors phospholipaseC Ion Channels
Ion Channels
GPCR SIGNALING CASCADES
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G Protein Coupled Receptors (GPCRs)
• largest family of receptors• hydrophobic/ hydrophilic
domains• seven transmembrane regions• Ligand-binding domain in plane
of membrane (TM3,5,6)• G protein binding domain in
loop 3 (btwn TM 5 and 6)
and C-terminus
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Where does the ligand bind?
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Are GPCRs dimers???
Expt: One GPCR can’t bind ligand, another can’t bind G protein,what happens when you put them together?
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G protein classes
• Gs stimulates Adenylate Cyclase (AC)
• Gi inhibits Adenylate Cyclase
• Gq stimulates Phospholipase C
• Gt transducin (activates PDE by binding inhibitor)
• Go opens/closes different ion channels
G proteins amplify the signal (1R=10G)G proteins can affect the timing of responses
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Discovery of G proteinsRodbell and Gilman 1994 Nobel prize
Background: epinephrine degrades glycogen to glucose in the liverproduces cAMP (stress provides energy!)
Old model: receptor for epinephrine causes activation of adenylate cyclase, converting ATP to cAMP
Rodbell’s discovery: GTP is needed for adenylate cyclase activity
Gilman’s discovery: purified the G protein
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Gilman’s experiment
- starting point: tissue culture cells that die when they make cAMP - screen for mutants don’t die - mutant A does not bind ligand, does not produce cAMP
- mutant B binds ligand, does not produce cAMP
Model 1: mutant A lacks receptor, mutant B lacks cyclase
- Gilman’s first expt: mix A and B, cyclase activity
noAR
cAMP!!!
A B
no cAMP
A+B
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cAMP!!!
A Bno cyclase
no cAMP
noAR
A+B
noAR
cAMP!!!
A B
no cAMP
A+B
Control Expt: Eliminate cyclase activity from A, mix A and B,still get cyclase activity!!!
Why does the mixing expt work?
This means that B has normal cyclase and normal receptor!!! something else is missing……G protein
Model: Mutant A has normal cyclaseand mutant B has normal receptor
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What G protein residues are important for receptor binding?
Adrenoreceptor (AR) binds Gq (activates PLC) and Gi (inhibits AC)
Dopamine receptor (DR) binds Gi (inhibits AC)
Is it possible to make a chimera that binds Dr but activates PLC?
Gq
Gi
Gq/Gi chimera
Gq binds AR activates PLC
Gi binds DR inhibits AC
Gq/Gi binds DR activates PLC
Expt: Replace Gq residues with Gi
Only 3 amino acids in C terminus switch receptor specificity!!!18
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Receptor G Protein Effector 2nd messenger 2nd Effector
Sensory Receptors Enzymes EnzymesPeptide Receptors phosphodiesterase cyclic nucleotides kinasesHormone Receptors adenylate cyclase lipids phosphatasesNeurotransmitter phospholipaseA calcium Receptors phospholipaseC Ion Channels
Ion Channels
PROTOTYPICAL GPCR SIGNALING CASCADE
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Receptor G Protein Effector 2nd messenger 2nd Effector
Adrenergic Gs adenylate cAMP protein kinaseA receptor(AR) cyclase (AC) (PKA)
metabotropic Gq phospho- IP3 and DAG Ca releaseGlutamate lipaseCReceptor (mGluR) (PLC)
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1 G protein activates 1 cyclase1 cyclase produces 100-1000 cAMP
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Another signaling cascade: PLC-mediated cascade
phospholipaseC
PIP2 DAG + IP3
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General features of second messengers:
• Their presence is a signal in the intracellular space• Small, chemically diverse, stable molecules• Synthesized or released from storage
(low amounts in resting state, regulated synthesis and destruction)
• Travel long distances• Modulate the activity of many other proteins!
Amplification and Diversification
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Second messengers activate many targetsActions of cAMP
1. Activates Protein Kinase A (phosphorylates ion channels, enzymes,transcription factors)
2. Activates cyclic nucleotide gated channels (change membrane potential)
3. Activates transcription factors (change gene expression)
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Actions of IP3
Release of calcium from internal storesGates ion channelsCo-activator of protein kinase C
Actions of DAG
Gates ion channels?Co-activator of protein kinase C
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If two pathways activate the same signaling molecule,how do they generate different cellular responses?
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