1 / 23

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)

blaise
Download Presentation

Characterization of LC8 Interaction with NR1 subunit of NMDA Receptors.

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Characterization of LC8 Interaction with NR1 subunit of NMDA Receptors. Brian Phan Dr. Jane Ishmael Department of Pharmaceutical Sciences Summer 2010

  2. 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.

  3. 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

  4. 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.

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

  6. NR1 Structure NR1-1a

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

  8. 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 COOH C0 C1 C2 LC8 Tetramerization with NR2 dimer? Dimerization with another NR1.

  9. Preliminary Results NR1-4a (NR1 without LC8 motif) still shows that NR1 and LC8 are still associated together in a complex. X …KDTST… NH2 COOH C0 C2’ LC8

  10. Immunoblot Analysis NR1-1a NR1-4a 150 100 75 15 FLAG-LC8 10 • 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

  11. Co-Immunoprecipitation Recognize FLAG-LC8 with antibody (m2 alpha-FLAG). Use protein G-sepharose beads to recognize and bind to the antibody-FLAG-LC8 complex. Elute FLAG-LC8 from lysate and determine if it is (or is not) associated with NR1. 2 1 3

  12. Preliminary Results IP: NR1-4a + FLAG-LC8 IP: NR1-1a + FLAG-LC8 NR1-4a + FLAG-LC8 IP: NR1-1a NR1-1a NR1-1a + FLAG-LC8

  13. 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.

  14. Methods Remove C1/C2 from NR1-1a (1-863) Does C0 contain another LC8 binding motif? NH2 COOH C0 C1 C2 NH2 COOH C0

  15. Methods Remove entire C-terminus from NR1-1a (1-834) Is the LC8 binding domain within the C-terminus? NH2 COOH C0 C1 C2 NH2 COOH

  16. 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 Epitope

  17. 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

  18. 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

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

  20. Results of co-immunoprecipitation study NR1 (1-863) + FLAG-LC8 IP: NR1-1a + FLAG-LC8 IP: NR1 (1-863) + FLAG-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. NR1-1a + FLAG-LC8

  21. 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.

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

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

More Related