Transcript Characterization of LC8 Interaction with NR1 subunit of NMDA Receptors.

Characterization of LC8 Interaction
with NR1 subunit of NMDA Receptors.
BRIAN PHAN
DR. JANE ISHMAEL
DEPARTMENT OF
PHARMACEUTICAL SCIENCES
SUMMER 2010
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
 Conserved and dimeric
cargo binding subunit of
dynein motor complex.
 Increases ordered structure
of various proteins.
 LC8 can be found in
LC8
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.
…KDTST…
NH2
C0
Tetramerization
with NR2 dimer?
Dimerization with
another NR1.
C1
C2
COOH
LC8
Preliminary Results
 NR1-4a (NR1 without LC8 motif) still shows that
NR1 and LC8 are still associated together in a
complex.
X
…KDTST…
NH2
C0
COOH
C2’
LC8
Immunoblot Analysis
 Transient expression
of NMDAR1 splice
variants and FLAGtagged LC8 in
HEK293 cells using
Calcium Phosphate
transfection.
NR1-1a
NR1-4a
150
100
75
 Western Blot analysis
to identify expressed
proteins X hr. later
15
FLAG-LC8
10
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)
NH2
NH2
C0
C0
C1
C2
COOH
 Does C0 contain another LC8 binding motif?
COOH
Methods
 Remove entire C-terminus from NR1-1a (1-834)
NH2
C0
NH2
COOH
C1
C2
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.
NH2
C-tail
COOH
Construction of Eukaryotic Expression Vectors
 Design new primers and
use PCR to amplify NR1
mutant inserts.
 Ligate new NR1 insert
into a cloning vector.
XBa1
HindIII
Construct construction (cont.)
 Transform ligated insert
into bacterial cells (XLBlue10).
 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