Indra A. Lim 1995-2005
Residency, Physical Medicine & Rehabilitation
U of Michigan Hospitals Ann Arbor, MI
University of Michigan 1995
Mollecular & Cellular Pharmacology PhD 2003
Johannes Hell
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
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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.
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| Thesis Publications |
- Lim IA, Merrill MA, Chen Y, Hell JW. Disruption of the NMDA receptor-PSD-95 interaction in hippocampal neurons with no obvious physiological short-term effect. Neuropharmacology 45:738-754, 2003.
- Seabold GK, Burette A, Lim IA, Weinberg RJ, Hell JW. 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, 2003.
- 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.
- Lim IA, Hall DD, Hell JW. Selectivity and promiscuity of the first and second PDZ domains of PSD-95 and SAP102. J Biol Chem 277:21697-21711, 2002.
- Leonard AS, Lim IA, Hemsworth DE, Horne MC, Hell JW. Calcium/calmodulin-dependent protein kinase II is associated with the N-methyl-D-aspartate receptor. Proc Natl Acad Sci USA 96:3239-44, 1999.
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