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Receptor-receptor interaction:

Receptor-receptor interaction: A critical mechanism for the regulation of neurotransmitter receptor function. Fang Liu MD, PhD Centre for Addiction and Mental Health University of Toronto. Neurotransmitter Receptors:. Ionotropic: ligand-gated ion channels. Open ion channels—4-5 subunits

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Receptor-receptor interaction:

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  1. Receptor-receptor interaction: A critical mechanism for the regulation of neurotransmitter receptor function Fang Liu MD, PhD Centre for Addiction and Mental Health University of Toronto

  2. Neurotransmitter Receptors: Ionotropic: ligand-gated ion channels Open ion channels—4-5 subunits Fast neuro-transmission e.g. for NMDA, AMPA and GABA-A receptors Metabotropic: G-protein coupled receptors Activate G proteins 7 transmembrane domains Couple to many effectors: e.g. adenylyl cyclase Slow neuro-transmission - e.g. for dopamine receptors

  3. Regulation of neurotransmitter receptors function PKA, PKC, PTK… Direct protein-protein interaction The classic pathway: phosphorylation—PKA, PKC…

  4. GAPDH and GluR2 interactionare essential in AMPA receptor-mediated excitotoxicity

  5. The AMPA Receptor * Fast synaptic transmission * neurotoxicity NMDAr NMDAr mGlur AMPAr Ca2+ Na2+ PLC DAG Ca2+ nNOS NO IP3 CaM Mito ONOO- O2- Calcineurin GluR1/2 in hippocampus H2O2 IκB NFκB

  6. NH2 NARP, N-cadherin I III IV II COOH NSF, PICK1, GRIP1/2… GluR2 Subunit * Impermeable to Ca2+ * Interact with intracellular/extracellular proteins

  7. Affinity purification: Use specific region of one protein to “pull down” another protein from a pool of proteins X GST GST GST GST ? ? ? ?

  8. 37.1 GluR1NT GST GluR2NT GAPDH GluR1NT GST GluR2NT 1 MVKVGVNGFG RIGRLVTRAA FSCDKVDIVA INDPFIDLNY MVYMFQYDST 51 HGKFNGTVKA ENGKLVINGK PITIFQERDP VKIKWGDAGA EYVVESTGVF 101 TTMEKAGAHL KGGAKRVIIS APSADAPMFV MGVNHEKYDN SLKIVSNASC 151 TTNCLAPLAK VIHDNFGIVE GLMTTVHAIT ATQKTVDGPS GKLWRDGRGA 201 AQNIIPASTG AAKAVGKVIP ELNGKLTGMA FRVPTPNVSV VDLTCRLEKP 251 AKYDDIKKVV KQAAEGPLKG ILGYTEDQVV SCDFNSNSHS STFDAGAGIA 301 LNDNIVKLIS WYDNEYGYSN RVVDLMAYMA SKE GAPDH GST-GluR2CT GST GST-GluR2NT GluR2-NT interacts with GAPDH GAPDH (glyceraldehyde-3-phosphate dehydrogenase)

  9. GAPDH Function: ENDOCYTOSIS NITRIC OXIDE APOPTOSIS PHOSPHOTRANFERSE NEURONAL DISORDERS MICROTUBULE BUNDLING GAPDH VIRAL PATHOGENESIS DNA REPLICATION NUCLEAR RNA EXPORT DNA REPAIR PROSTATED CANCER TRANSLATIONAL REGULATION

  10. NH2 GAPDH I III IV II COOH GluR2-NT1-3-2 interacts with GAPDH GAPDH GST GST GST GluR2NT1 GluR2NT1-4 GluR2NT1-1 GluR2NT1-3-1 GluR2NT3 GluR2NT2 GluR2NT1-5 GluR2NT1-2 GluR2NT1-3 GluR2NT1-3-2

  11. ? D2 ? ? Co-immunoprecipitation

  12. Agonist promotes GAPDH-GluR2 complex formation HEK293T Cells Hippocampal Neurons IP IP:GluR2 IP:GluR2 Extract GluR2 IgG GAPDH GAPDH GAPDH GluR2 GluR2 KA CTRL GluR2NT1-3-2 +Glutamate GluR2NT1-3-2 +KA Control Glutamate

  13. Extracellular GAPDH-GluR2 complex formation CM IP: GAPDH Fresh Media AMPAR+Glut IP: GluR2 Fresh Media AMPAR lysates Extracts NT CM NB B IgG IgG Tubulin GAPDH GAPDH GAPDH

  14. GluR2-GAPDH coupling in AMPA receptor-mediated toxicity 120 120 100 100 80 80 *** *** 60 60 Fraction Dead (Percent +KA) (Percent Glutamate) FractionDead 40 40 20 20 0 0 KA Glutamate GluR2NT1-3-2 GluR2NT1-3-2 +Glutamate GluR2NT1-3-2 +KA Cells Neurons

  15. GAPDH-GluR2 complex co-internalization GluR1/2 GluR1/2 120 120 # GluR1/3 120 120 100 100 * 100 100 * 80 80 80 80 Cell Surface GAPDH (Percent Control) Cell Surface GluR2 60 60 60 (Percent Control) 60 Cell Surface GAPDH (Percent Control) Cell Surface GAPDH (Percent Control) 40 40 40 40 20 20 20 20 0 0 0 0 Control Glutamate Control Control Control Glutamate Glutamate GluR2NT1-3-2 +Glutamate Glutamate

  16. GluR2 and GAPDH Nuclear Translocation GluR2 Biotinlylated GluR2 GluR2 Biotinlylated GAPDH GAPDH GAPDH KA LaminB1 Control LaminB1 GluR2NT1-3-2 +KA Control Glutamate KA Control Siah1 siRNA +Glutamate GluR2NT1-3-2 +KA

  17. GluR2/GAPDH Nuclear Translocation depends on GluR2-Siah1 interaction Anti-Siah1 Anti-Siah1 GluR2NT GluR2CT-1 GST Extract GluR2CT-2 GST Extract GluR2CT GluR2CT-4 GAPDH GluR2CT-3 GluR2 GAPDH LaminB1 Control GluR2CT-4 +Glutamate Glutamate

  18. GST-GluR2NT P53 and GAPDH form complex in nucleus IP: p53 WB: anti-p53 IgG GAPDH Glutamate: GST - - + + GST-GAPDH GST-GluR2NT - - + - GluR2NT1-3-2: GST GAPDH 0 0.05 0.5 5 - p53-GST (μg): - - - - GST (ug): 0.5

  19. p53-GAPDH coupling in AMPA receptor-mediated toxicity GluR1/2 GluR1/3 125 120 120 100 100 100 80 80 75 ** * 60 60 Fraction Dead 50 (Percent Control) Fraction Dead Fraction Dead 40 (Percent Control) (Percent Control) 40 25 20 20 0 0 0 PFT-a: - - + + Glutamate: 0 PFT-a: 10µM 0 10µM - - + + GAPDH2-2-1:

  20. P53 and GAPDH coupling enhances p53 expression and phosphorylation anti-p53 GST GST GST GST-GAPDH1 GST-GAPDH2 GST-GAPDH2-1 GST-GAPDH2-2 GST-GAPDH2-2-1 GST-GAPDH2-2-2 GluR1/2 +GAPDH2-2-1 IP:GAPDH GluR1/2 IgG p53 p53 GAPDH pSer46 CTRL Control Control GAPDH2-2-1 Glutamate Glutamate

  21. Disruption of GAPDH-GluR2 interaction prevents/rescues neuronal death from ischemia in rat hippocampalCA1 region after transient cerebral ischemia 120 F B C SO 100 SP SR 80 ### A Area of Interest HPC G D ## Neuronal Survival 60 (Percentage of Sham) CA1 40 CA3 DG *** H E 20 0 Sham Ischemia Post-Scramble Post-Peptide Pre-Scramble Pre-peptide

  22. IP:p53 IgG GAPDH GAPDH p53 GluR2 Sham Pep (post) Ischemia Sham Pep (post) Ischemia IP:GluR2 IgG Enhanced GAPDH-GluR2/GAPDH-p53 interaction in ischemia

  23. Protective effect of GluR2NT1-3-2 in ischemia 120 120 Ischemia Ischemia 100 100 Peptide (Post) Peptide (Post) 80 80 Neuronal Survival (Percent Sham) Neuronal Survival (Percent Sham) ## # # 60 60 # 40 40 *** *** *** 20 20 0 0 10 days 5 days Sham Sham 2 hours 6 hours Ischemia

  24. Disruption of GAPDH-GluR2 interaction prevents/rescues neuronal death from focal ischemia (MCAo) Ischemia Scram Pep(Pre) Pep(post) 600 2 400 Infarct Area (mm ) 200 0 1 2 3 4 5 6 7 8 Stereotactic Coordinates(mm) 2hrs after MCAO 24hrs after MCAO 12 10 8 * Neurological Scores 6 * 4 2 0 Pep(Pre) Ischemia Pep(Pre) Pep(post) Pep(post) Ischemia Scram(post)

  25. Protective effect of GluR2NT1-3-3 in focal ischemia (MCAo) Ischemia Ischemia 250 Peptide (post) Peptide (Post) 300 200 250 * * 150 200 infarct volume (mm3) 150 Infarct volume (mm3) 100 ** 100 *** *** 50 50 0 0 3 days 5 days 3 0.3 0.03 0.001 Peptide dose (nmol/g)

  26. NH2 NH2 GAPDH GAPDH Extracellular Extracellular I III IV I III IV II II Intracellular Intracellular COOH COOH Nucleus p P53 GAPDH Summary AMPA activation Siah1 GluR2

  27. Ultimate goal: Clinical Application PEPTIDE

  28. Acknowledgement Dr. Shupeng Li Ms Ming Wang Dr. Yu-tian Wang Dr. Lin Pei Dr. Stephen Ferguson Dr. DongXu Zhai Dr. Sheng Chen CAMH CIHR pop grant BioDiscovery MaRS

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