Prof. Kristin Scott291 LSA

kscott@uclink.berkeley.edu

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?

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

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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