Indra A. Lim
| Years in MSTP | 1995-2005 |
| Current Position |
Molecular and Cellular Pharmacology, 2003 |
| Mentor | Johannes Hell |
| Thesis |
The Association of Post-Synaptic Density Protein 95 with the N-Methyl-D-Aspartate Type Glutamate Receptor: Specificity of PDZ Domain Interactions and Physiological Consequences of Binding |
| Residency | Rehabilitation medicine, University of Michigan |
Thesis Abstract
Post-synaptic Density protein 95 (PSD-95) is a structural protein that binds to NMDA-type glutamate receptors at the post-synaptic site. The essential role of the NMDA receptor in synaptic function, including learning and memory suggests that PSD-95 may be important for these functions. NMDA receptors bind protein-protein interaction domains on PSD-95 called PDZ domains.
In order to study how the primary structure of the NMDA-receptor affects affinity for PDZ domains, we constructed several libraries of peptides based on the PSD-95 binding domain of the NMDA receptor subunit NR2b, systematically substituting at each position archetypical amino acids.
Using fluorescence anisotropy, the affinity of the peptides for individual PDZ domains of PSD-95 and SAP 102, a related protein, were determined. From these data, an optimal sequence for binding to the PDZ1 and 2 and PSD-95 and SAP 102 was determined (E/Q-S/T D/E/Q/N-V). Searching the human genome for proteins ending with E/Q-S/T-X-V turned up several proteins. We tested and found several proteins for that had not previously been described to bind PSD-95 and SAP 102.
A peptide based on the C-terminus of NR2a (NR2aCT) could disrupt PSD-95/NMDA receptor interactions in vitro, and a membrane permeable form of NR2aCT could disrupt endogenous PSD-95/NMDA receptor in acute hippocampal slices. Whole cell recordings in acute hippocampal slices show that peptide can make it to post-synaptic sites in a relatively short time frame.
However, intracellular perfusion of peptide in this manner did not alter basal synaptic transmission or synaptic plasticity (long-term potentiation of evoked post-synaptic responses). This indicates that either the PSD-95 interaction with NMDA receptor (and other PDZ domain binding proteins) does not affect synaptic function, or the time scale of the recordings did not allow sufficient disruption of the interactions.
Publications
Lu Y, Zhang M, Lim IA, Hall DD, Allen M, Medvedeva Y, McKnight GS, Usachev YM, Hell JW. 2008. AKAP150-anchored PKA activity is important for LTD during its induction phase. J Physiol 586:4155-64. PMCID: 2652176
Lim IA, Merrill MA, Chen Y, Hell JW. 2003. Disruption of the NMDA receptor-PSD-95 interaction in hippocampal neurons with no obvious physiological short-term effect. Neuropharmacology 45:738-54.
Seabold GK, Burette A, Lim IA, Weinberg RJ, Hell JW. 2003. Interaction of the tyrosine kinase Pyk2 with the N-methyl-D-aspartate receptor complex via the Src homology 3 domains of PSD-95 and SAP102. J Biol Chem 278:15040-8.
Leonard AS, Bayer KU, Merrill MA, Lim IA, Shea MA, Schulman H, Hell JW. 2002. Regulation of calcium/calmodulin-dependent protein kinase II docking to N-methyl-D-aspartate receptors by calcium/calmodulin and alpha-actinin. J Biol Chem 277:48441-8.
Lim IA, Hall DD, Hell JW. 2002. Selectivity and promiscuity of the first and second PDZ domains of PSD-95 and synapse-associated protein 102. J Biol Chem 277:21697-711.
Leonard AS, Lim IA, Hemsworth DE, Horne MC, Hell JW. 1999. Calcium/calmodulin-dependent protein kinase II is associated with the N-methyl-D-aspartate receptor. Proc Natl Acad Sci USA 96:3239-44.
Leonard AS, Bayer KU, Merrill MA, Lim IA, Shea MA, Schulman H, Hell JW. Regulation of calcium/calmodulin-dependent protein kinase II docking to N-methyl-D-aspartate receptors by calcium/calmodulin and alpha-actinin. J Biol Chem 27:48441-48448, 2002.