B R I A N P H A N
D R . J A N E I S H M A E L
D E P A R T 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
S U M M E R 2 0 1 0

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.

LC8
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.

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.

Significance
 Understanding in the functional organization at pre- and post-synaptic sites.
 Insight into cellular mechanisms underlying neurological disorders.
 Importance in drug discovery.

NR1 Structure
NR1-1a

Techniques
• Use HEK293 cells as model system to transiently express NR1 and LC8.
• Co-immunoprecipitation reaction to identify protein complexes in cells.

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.
NH
2
Tetramerization with NR2 dimer?
C0 C1
… KDTST …
C2 COOH
LC8
Dimerization with another NR1.

Preliminary Results
 NR1-4a (NR1 without LC8 motif) still shows that
NR1 and LC8 are still associated together in a complex.
NH
2 C0
…
X
…
C2’
LC8
COOH

Immunoblot Analysis
 Transient expression of NMDAR1 splice variants and FLAGtagged LC8 in
HEK293 cells using
Calcium Phosphate transfection.
150
100
75
NR1-1a NR1-4a
 Western Blot analysis to identify expressed proteins X hr. later
15
10
FLAG-LC8

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.

Preliminary Results

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.

Methods
 Remove C1/C2 from NR1-1a (1-863)
NH
2
C0 C1 C2 COOH
NH
2
C0 COOH
 Does C0 contain another LC8 binding motif?

Methods
 Remove entire C-terminus from NR1-1a (1-834)
NH
2
C0 C1 C2 COOH
NH
2
COOH
 Is the LC8 binding domain within the C-terminus?

Methods


Mouse Anti-Glutamate Receptor Monoclonal
Antibody (Epitope 660-811)
Used to detect NR1 which migrates to ~120kDa on
SDS/PAGE.
NH
2 C-tail COOH

Construction of Eukaryotic Expression Vectors
 Design new primers and use PCR to amplify NR1 mutant inserts.
HindIII
XBa1

Ligate new NR1 insert into a cloning vector.

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

Transient expression in HEK293 cells


New DNA transfected into HEK293 cells.
Success in producing NR1 (1-863) construct and expression in HEK293.

Results of co-immunoprecipitation study
• 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.

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.

New Skills and Techniques




Producing and testing new DNA constructs.
Transient transfection into eukaryotic cells.
Cell culture and maintaining a cell line.
Co-immunoprecipitation.

Acknowledgements
 Dr. Jane Ishmael


Andrew Hau
Xiao Liu


HHMI
OSU Dept. of Pharmaceutical Sciences