Receptorreceptor interaction A critical mechanism for the regulation

Receptor-receptor interaction: A critical mechanism for the regulation of neurotransmitter receptor function Fang Liu MD, Ph. D 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 Direct protein-protein interaction PKA, PKC, PTK… The classic pathway: phosphorylation—PKA, PKC…

GAPDH and Glu. R 2 interaction are essential in AMPA receptormediated excitotoxicity

The AMPA Receptor m. Glur PLC NMDAr AMPAr Na 2+ DAG IP 3 Ca 2+ n. NOS NO * Fast synaptic transmission Ca 2+ * neurotoxicity Ca. M Calcineurin ONOO- O 2 - Glu. R 1/2 in hippocampus H 2 O 2 NFκB Mito IκB

Glu. R 2 Subunit NH 2 NARP, N-cadherin * Impermeable to Ca 2+ I II IV COOH NSF, PICK 1, GRIP 1/2… * Interact with intracellular/extracellular proteins

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

Glu. R 2 -NT interacts with GAPDH 37. 1 1 NT R u. R Glu Gl 2 NT T GS GAPDH 1 NT R 2 NT ST G Glu R Glu GAPDH T T 2 CT GS lu. R 2 N T-G GS GS GAPDH (glyceraldehyde-3 -phosphate dehydrogenase) 1 51 101 151 201 251 301 MVKVGVNGFG RIGRLVTRAA FSCDKVDIVA INDPFIDLNY MVYMFQYDST HGKFNGTVKA ENGKLVINGK PITIFQERDP VKIKWGDAGA EYVVESTGVF TTMEKAGAHL KGGAKRVIIS APSADAPMFV MGVNHEKYDN SLKIVSNASC TTNCLAPLAK VIHDNFGIVE GLMTTVHAIT ATQKTVDGPS GKLWRDGRGA AQNIIPASTG AAKAVGKVIP ELNGKLTGMA FRVPTPNVSV VDLTCRLEKP AKYDDIKKVV KQAAEGPLKG ILGYTEDQVV SCDFNSNSHS STFDAGAGIA LNDNIVKLIS WYDNEYGYSN RVVDLMAYMA SKE

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

N T 1 N -5 ST G ST -2 -3 T 1 lu R 2 G lu NT 1 R 2 -3 -1 G III G II T 1 I N G ST lu G R 2 lu NT R G 2 N 1 -1 lu T 1 R 2 -2 G NT lu 1 R 2 3 G NT lu 1 -4 R 2 G lu R 2 G NT lu 3 R 2 G NT lu 2 R 2 G Glu. R 2 -NT 1 -3 -2 interacts with GAPDH NH 2 GAPDH IV COOH GAPDH

Co-immunoprecipitation ? D 2 ? ?

Agonist promotes GAPDH-Glu. R 2 complex formation HEK 293 T Cells -2 -3 Glu. R 2 T 1 Glu. R 2 G KA +K lu. R A 2 N GAPDH RL GAPDH Co G ntro lu l t G am +G lu. R ate lu 2 N ta T 1 m -3 at 2 e GAPDH R 2 Extr u G l Ig G IP: Glu. R 2 act CT IP Hippocampal Neurons

Extracellular GAPDH-Glu. R 2 complex formation IP: GAPDH CM dia e GAPDH h. M s e CM Fr Ig. G Tubulin GAPDH lut dia G e R AR+ h M s A P MP AM Freslysate NT A GAPDH IP: Glu. R 2 cts a r NB B Ig. G Ext

m at ta 0 Cells 0 T 1 +K 3 -2 A 20 2 N 40 R *** A 60 lu 100 K 80 Fraction Dead (Percent +KA) 120 G e R +G 2 N lu T 1 G tam -3 lu 2 R ate 2 N T 1 -3 -2 lu G Fraction Dead (Percent Glutamate) Glu. R 2 -GAPDH coupling in AMPA receptormediated toxicity 120 100 80 *** 60 40 20 Neurons

0 0 100 60 40 20 0 ro l ta m at e 100 lu 120 G 120 Cell Surface GAPDH (Percent Control) 80 Co nt e 20 at 40 Cell Surface GAPDH (Percent Control) 60 m 20 80 ta 40 * ro l 60 100 # lu * Glu. R 1/2 G 100 120 Co nt 80 Glu. R 1/2 Cell Surface GAPDH (Percent Control) 120 Co G ntro lu ta l m G at e +G lu. R 2 lu N ta T m 1 at 3 e 2 Co n G trol lu ta m at e Cell Surface Glu. R 2 (Percent Control) GAPDH-Glu. R 2 complex co-internalization Glu. R 1/3 80 60 40 20 0

Glu. R 2 and GAPDH Nuclear Translocation Glu. R 2 Biotinlylated GAPDH ol tr on Lamin. B 1 R 2 N T +K 1 -3 A 2 A K lu G Co nt ro l C KA T 1 -3 - A 2 N K u. R + l G 2 Lamin. B 1 Co nt ro l G lu ta m at Si e +G ah 1 lu si ta RN m at A e Glu. R 2

Glu. R 2/GAPDH Nuclear Translocation depends on Glu. R 2 -Siah 1 interaction Anti-Siah 1 T CT R 2 N 2 DH R u l P u l G G GA T tract S G Ex Anti-Siah 1 ct CT 2 CT 3 T-4 ST 2 a C r T 2 R R 2 C G Ext Glu lu. R G -1 Glu. R 2 GAPDH at e G l u +G R lu 2 C ta Tm 4 at e ta m lu G C on t ro l Lamin. B 1 2

P 53 and GAPDH form complex in nucleus IP: p 53 WB: anti-p 53 Ig. G GAPDH H T T GS u. R 2 N APD l T-G -G S T G GS Glutamate: - + - Glu. R 2 NT 1 -3 -2: - GST-Glu. R 2 NT + + T GS GAPDH p 53 -GST (μg): GST (ug): 0 0. 5 0. 05 - 0. 5 - - -

p 53 -GAPDH coupling in AMPA receptor-mediated toxicity Glu. R 1/2 Glu. R 1/3 120 100 * 60 40 20 0 Fraction Dead (Percent Control) 100 80 PFT-a: 125 80 60 40 20 PFT-a: 0 0 10µM Fraction Dead (Percent Control) 120 0 10µM 75 ** 50 25 0 Glutamate: - - + + GAPDH 2 -2 -1: - +

P 53 and GAPDH coupling enhances p 53 expression and phosphorylation anti-p 53 ST 2 -2 2 -1 T DH 1 DH 2 2 -12 -2 -2 T G S H 2 H G AP AP GS DH PD APD G G P A G A A T T -G T-GGSTGS GS T S G GS GS IP: GAPDH Glu. R 1/2 +GAPDH 2 -2 -1 Glu. R 1/2 Ig. G p 53 GAPDH RL CT DH 2 P GA p. Ser 46 2 -1 ol C tr on Glu te a tam C ol ate m ta Glu tr on

Disruption of GAPDH-Glu. R 2 interaction prevents/rescues neuronal death from ischemia in rat hippocampal. CA 1 region after transient cerebral ischemia B 120 C SO 100 SP 80 SR ### ## D G A Area of Interest HPC 60 CA 1 40 DG *** 20 0 S Is ham Po che st- mi Pr Pep a e t Po -pep ide st- tid S e Pr cram e. Sc ble ra m bl e Neuronal Survival (Percentage of Sham) F E H CA 3

Enhanced GAPDH-Glu. R 2/GAPDH-p 53 interaction in ischemia IP: Glu. R 2 IP: p 53 Ig. G GAPDH p 53 Glu. R 2 Sham Pep (post) Ischemia

Protective effect of Glu. R 2 NT 1 -3 -2 in ischemia *** 20 ia 0 m he Is c 10 days am 5 days Sh Sham s *** ur 20 40 ho *** # s # 40 # 60 6 ## 60 80 ur 80 Peptide (Post) ho Peptide (Post) Ischemia 100 2 100 0 120 Ischemia Neuronal Survival (Percent Sham) 120

Infarct Area (mm 2 ) Disruption of GAPDH-Glu. R 2 interaction prevents/rescues neuronal death from focal ischemia (MCAo) Ischemia Pep(Pre) 600 Scram Pep(post) 400 200 0 1 2 3 4 5 6 7 8 8 * 6 4 * 2 st po Pe p( Pr p( ) e) t) ia Pe c Is m he os Pep(post) (p 0 m Pep(Pre) 24 hrs after MCAO 10 ra Ischemia 2 hrs after MCAO 12 Sc Neurological Scores Stereotactic Coordinates(mm)

Protective effect of Glu. R 2 NT 1 -3 -3 in focal ischemia (MCAo) Ischemia 250 Ischemia Peptide (post) Peptide (Post) Infarct volume (mm 3) 200 150 * 100 ** 50 0 3 days 5 days infarct volume (mm 3) 300 250 * 200 150 100 *** 50 0 3 0. 03 Peptide dose (nmol/g) 0. 001

Summary NH 2 GAPDH Extracellular I II IV Extracellular AMPA activation Intracellular I II Intracellular COOH Nucleus p Siah 1 Glu. R 2 P 53 GAPDH III IV

Ultimate goal: Clinical Application PEPTIDE

Acknowledgement Dr. Shupeng Li Ms Ming Wang Dr. Lin Pei Dr. Dong. Xu Zhai Dr. Sheng Chen Dr. Yu-tian Wang Dr. Stephen Ferguson CAMH CIHR pop grant Bio. Discovery Ma. RS