The Language of Neurons: Signal Transduction and G Protein-coupled Cascades

1. Prof. Kristin Scott 291 LSA kscott@berkeley.edu OFFICE HOURS M 11 AM-12 NOON W 11 AM-12 NOON, F 2-3pm and by appointment  POWERPOINT SLIDES ON http://mcb.berkeley.edu/courses/mcb160/ 1

2. The language of neurons: Initiating a signal how a chemical signal is detected how cellular activity changes how electrical and chemical signals are produced 2

3. 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 signals how cascades change neural excitability 3

4. What is Signal Transduction ? Receptor activation Intracellular signaling events 4

5. 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 ? 5

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7. GPCR SIGNALING CASCADES Receptor G Protein Effector 2nd messenger 2nd Effector Sensory Receptors Enzymes Enzymes Peptide Receptors phosphodiesterase cyclic nucleotides kinases Hormone Receptors adenylate cyclase lipids phosphatases Neurotransmitter phospholipaseA calcium Receptors phospholipaseC Ion Channels Ion Channels 7

8. 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 8

9. Where does the ligand bind? 9

10. Are GPCRs dimers??? Expt: One GPCR can’t bind ligand, another can’t bind G protein, what happens when you put them together? 10

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12. Discovery of G proteins Rodbell and Gilman 1994 Nobel prize Background:epinephrine degrades glycogen to glucose in the liver produces 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 13

13. 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 A B A+B no AR no cAMP cAMP!!! 14

14. Why does the mixing expt work? A B A+B Model: Mutant A has normal cyclase and mutant B has normal receptor no AR no cAMP cAMP!!! no cyclase Control Expt: Eliminate cyclase activity from A, mix A and B, still get cyclase activity!!! A B A+B no AR no cAMP cAMP!!! This means that A is not supplying cyclase!!! it’s supplying something else……G protein 15

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16. 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? Expt: Replace Gq residues with Gi Gq binds AR activates PLC Gi binds DR inhibits AC Gq/Gi binds DR activates PLC Gq Gi Gq/Gi chimera Only 3 amino acids in C terminus switch receptor specificity!!! 19

17. G protein classes activate different effectors • 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 12

18. PROTOTYPICAL GPCR SIGNALING CASCADE Receptor G Protein Effector 2nd messenger 2nd Effector Sensory Receptors Enzymes Enzymes Peptide Receptors phosphodiesterase cyclic nucleotides kinases Hormone Receptors adenylate cyclase lipids phosphatases Neurotransmitter phospholipaseA calcium Receptors phospholipaseC Ion Channels Ion Channels 17

19. Receptor G Protein Effector 2nd messenger 2nd Effector AdrenergicGs adenylate cAMP protein kinaseA receptor(AR) cyclase (AC) (PKA) metabotropic Gq phospho- IP3 and DAG Ca release Glutamate lipaseC Receptor (mGluR) (PLC) 18

20. 1 G protein activates 1 cyclase 1 cyclase produces 100-1000 cAMP 20

21. Another signaling cascade: PLC-mediated cascade phospholipaseC PIP2 DAG + IP3 21

22. 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 22

23. Second messengers activate many targets Actions 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) 23

24. Actions of IP3 Release of calcium from internal stores Gates ion channels Co-activator of protein kinase C Actions of DAG Gates ion channels? Co-activator of protein kinase C 24