Molecular NeurosciencePhospho-regulation of synaptic and extrasynaptic N-methyl-d-aspartate receptors in adult hippocampal slices
Section snippets
Antibodies and reagents
Rabbit polyclonal antibodies against NR2A and NR2B were generated previously in our laboratory and described elsewhere (Snell et al 1996, Grosshans and Browning 2001). The phospho-site specific antibodies against NR2B Tyr1252, Tyr1336, and Tyr1472 were obtained from PhosphoSolutions, Inc. (Aurora, CO, USA). Mouse monoclonal anti-phospho-tyrosine clone 4G10, anti–post-synaptic density (PSD) 95, anti-Src, and anti-Yes antibodies were obtained from Upstate Biotechnology (Lake Placid, NY, USA). The
Characterization of methods used to enrich synaptic and extrasynaptic membranes
A modified classical subcellular fractionation approach was utilized in order to separate plasma membrane fractions into synaptic and extrasynaptic membranes (Fig. 1a and see Carlin et al 1980, Cho et al 1992, Davies et al 2007). This method takes advantage of the fact that synaptic junctions and PSDs are insoluble in Triton-X 100 (Cotman and Taylor 1972, Cotman et al 1974, Matus and Taff-Jones 1978, Cohen 1997) and other membranes not tightly bound to the PSD, including membranes from the
Discussion
Accumulating evidence demonstrates that the tyrosine phosphorylation of NMDARs is positively associated with their surface expression (Dunah and Standaert 2001, Goebel et al 2005, Snyder et al 2005, Hallett et al 2006, Braithwaite et al 2006). Given that surface-bound NMDARs can be differentiated into synaptic and extrasynaptic receptors, it has remained unknown how tyrosine phosphatase and kinase inhibitors modulate NMDARs in these different compartments. Another unanswered question concerns
Acknowledgments
We thank Dr. Paul Lombroso for kindly providing the anti-STEP antibody and for helpful discussions. This research was supported by National Institutes of Health grants AA09675-10 and AG04418-19 and by an institutional National Institutes of Health training grant (HD41697-02). M.D.B. has a financial interest in PhosphoSolutions, Inc., a company which provided several of the antibodies used in this study.
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