BRIAN PHAN DR. JANE ISHMAEL DEPARTMENT OF PHARMACEUTICAL SCIENCES SUMMER 2010 Characterization of...
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BRIAN PHANDR. JANE ISHMAEL
D E PA RT M E N T O F P H A R M A C E U T I C A L
S C I E N C E S
SUMMER 2010
Characterization of LC8 Interaction with NR1 subunit of
NMDA Receptors.
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NMDA-type Glutamate Receptors
N-methyl-D-Aspartate receptors (NMDAR)
Glutamate receptors important for synaptic plasticity and memory function.
Tetramer made of 2 different subunits (NR1 and NR2)
Ligand and voltage-gated ion channel that facilitates movement of calcium across neural cell membranes.
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LC8
Conserved and dimeric cargo binding subunit of dynein motor complex.
Increases ordered structure of various proteins.
LC8 can be found in postsynaptic sites in neurons.
LC8
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Post-synaptic sites
Glutamate is the major excitatory amino acid neurotransmitter in mammalian CNS.
NMDAR regulates neurotransmission at postsynaptic sites.
Protein-protein interactions facilitate assembly of NMDA receptors.
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Significance
Understanding in the functional organization at pre- and post-synaptic sites.
Insight into cellular mechanisms underlying neurological disorders.
Importance in drug discovery.
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NR1 Structure
NR1-1a
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Techniques
•Use HEK293 cells as model system to transiently express NR1 and LC8.
•Co-immunoprecipitation reaction to identify protein complexes in cells.
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Working Hypothesis
Direct binding of LC8 onto the C-terminus of the NR1-1a subunit will facilitate NMDAR assembly and transport to the neural synapse.
COOH
NH2 C0 C2C1
…KDTST…
LC8Dimerization
with another NR1.
Tetramerization with NR2 dimer?
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Preliminary Results
NR1-4a (NR1 without LC8 motif) still shows that NR1 and LC8 are still associated together in a complex.
NH2 COOH
C0 C2’
…KDTST…X
LC8
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Immunoblot Analysis
Transient expression of NMDAR1 splice variants and FLAG-tagged LC8 in HEK293 cells using Calcium Phosphate transfection.
Western Blot analysis to identify expressed proteins X hr. later
15010075
15
10
NR1-1a
NR1-4a
FLAG-LC8
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Co-Immunoprecipitation
1. Recognize FLAG-LC8 with antibody (m2 alpha-FLAG).
2. Use protein G-sepharose beads to recognize and bind to the antibody-FLAG-LC8 complex.
3. Elute FLAG-LC8 from lysate and determine if it is (or is not) associated with NR1.
13
2
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Preliminary Results
NR
1-4a
+ F
LAG
-
LC8
NR
1-1a
+ F
LAG
-
LC8
NR
1-1a
IP: N
R1-
1a +
FLA
G-
LC8IP
: NR
1-1a
IP: N
R1-
4a +
FLA
G-
LC8
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Hypothesis
Another LC8 binding motif common in both NR1-1a and NR1-4a exists on the NR1 C-tail that interacts with LC8 to order and form the NR1 homodimer.
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Methods
Remove C1/C2 from NR1-1a (1-863)
Does C0 contain another LC8 binding motif?
C0 C1 C2NH2
NH2
COOH
COOH
C0
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Methods
Remove entire C-terminus from NR1-1a (1-834)
Is the LC8 binding domain within the C-terminus?
C0 C1 C2NH2
NH2
COOH
COOH
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Methods
Mouse Anti-Glutamate Receptor Monoclonal Antibody (Epitope 660-811)
Used to detect NR1 which migrates to ~120kDa on SDS/PAGE.
NH2 COOH
C-tailEpito
p
e
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Construction of Eukaryotic Expression Vectors
Design new primers and use PCR to amplify NR1 mutant inserts.
Ligate new NR1 insert into a cloning vector.
HindIII
XBa1
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Construct construction (cont.)
Transform ligated insert into bacterial cells (XL-Blue10).
Select single colonies
Mini-prep to extract DNA.
DNA digestion to confirm successful ligation and transformation.
Transfect new plasmid into HEK293 cells.
5.4 kb
2.5 kb
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Transient expression in HEK293 cells
New DNA transfected into HEK293 cells.Success in producing NR1 (1-863) construct
and expression in HEK293.
NR1-1a
NR1
(1-8
63)
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Results of co-immunoprecipitation study
NR1
(1-8
63) +
FLA
G-L
C8
NR1-
1a +
FLA
G-L
C8
IP: N
R1-
1a +
FLA
G-L
C8
IP: N
R1
(1-8
63) +
FLA
G-L
C8
•FLAG-LC8 appears to interact with NR1 (1-863) in HEK 293 cells
•Results need to be repeated for verification with NR1-1a without FLAG-LC8 as a control.
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Future Work
Sequence pCDNA3/ NR1 (1-863) DNA to verify that mutations were not introduced by PCR.
Continue constructing NR1 (1-834) and determine if an LC8 binding site exists on C-tail.
Biophysical approaches to further structurally characterize LC8-NR1 complex.
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New Skills and Techniques
Producing and testing new DNA constructs.Transient transfection into eukaryotic cells.Cell culture and maintaining a cell line.Co-immunoprecipitation.
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Acknowledgements
Dr. Jane Ishmael
Andrew HauXiao Liu
HHMIOSU Dept. of Pharmaceutical Sciences