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 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

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

GAPDH and GluR2 interaction are essential in AMPA receptor-mediated excitotoxicity

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

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

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

(glyceraldehyde-3-phosphate dehydrogenase) GluR2-NT interacts with GAPDH 37.1 GluR1NT GST GluR2NT GAPDH (glyceraldehyde-3-phosphate dehydrogenase) 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 GluR1NT GST GluR2NT GAPDH GST-GluR2NT GST GST-GluR2CT GAPDH

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

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

Co-immunoprecipitation D2 ?

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

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

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

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

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

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

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

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

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

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

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

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

Disruption of GAPDH-GluR2 interaction prevents/rescues neuronal death from focal ischemia (MCAo) 600 Ischemia Scram Pep(Pre) Pep(post) 2 400 Infarct Area (mm ) 200 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 Ischemia Pep(Pre) Pep(post) Pep(Pre) Pep(post) Ischemia Scram(post)

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

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

Ultimate goal: Clinical Application PEPTIDE

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