BRIAN PHANDR. JANE ISHMAEL

D EPA RT M EN T O F P H A R M A C E U T I C A L

S C I E N C ESSUMMER 2010

Characterization of LC8 Interaction with NR1 subunit of

NMDA Receptors.

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 postsynaptic sites in neurons.LC8

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.

COOH

NH2 C0 C2C1

…KDTST…

LC8Dimerization

with another NR1.

Tetramerization with NR2 dimer?

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

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

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

Preliminary Results

NR1

-4a

+ FL

AG-

LC8

NR1

-1a

+ FL

AG-

LC8NR1

-1a

IP: N

R1-1

a +

FLAG

-

LC8IP

: NR1

-1a

IP: N

R1-4

a +

FLAG

-

LC8

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)

Does C0 contain another LC8 binding motif?

C0 C1 C2NH2

NH2

COOH

COOH

C0

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

Methods

Mouse Anti-Glutamate Receptor Monoclonal Antibody (Epitope 660-811)

Used to detect NR1 which migrates to ~120kDa on SDS/PAGE.

NH2 COOH

C-tailEpitop

e

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

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.

NR1-1a

NR1 (1-8

63)

Results of co-immunoprecipitation study

NR1

(1-8

63) +

FLA

G-LC

8

NR1-1

a +

FLAG

-LC8

IP: N

R1-1

a +

FLAG

-LC8

IP: N

R1 (1

-863

) + F

LAG-

LC8

•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 HauXiao Liu

HHMIOSU Dept. of Pharmaceutical Sciences