Abstract
Synapse-associated protein 97 (SAP97) and postsynaptic density 95 (PSD-95) are closely related membrane-associated guanylate kinase homologs (Maguks) implicated in the synaptic targeting and anchoring of alpha-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid (AMPA)-selective glutamate receptors. Prompted by accumulating evidence for an oligomeric nature of Maguks, we examined the potential of SAP97 and PSD-95 to form heteromeric complexes. SAP97 and PSD-95 coimmunoprecipitated from rat brain detergent extracts and subsequent glutathione S-transferase pull-down and immunoprecipitation experiments showed that the interaction is mediated by binding of the N-terminal segment of SAP97 (SAP97(NTD)) to the Src homology 3 domain of PSD-95 (PSD-95(SH3)). In cultured hippocampal neurons, expression of green fluorescent protein-tagged PSD-95 triggered accumulation of SAP97 in synaptic spines, which was totally inhibited by coexpression of PSD-95(SH3). Furthermore, overexpression of green fluorescent protein-PSD-95 induced dendritic clustering of GluR-A subunit-containing AMPA receptors, which was strongly inhibited by cotransfection with SAP97(NTD) and PSD-95(SH3) constructs. Our results demonstrated a direct interaction between SAP97 and PSD-95 and suggested that this association may play a functional role in the trafficking and clustering of AMPA receptors.
Highlights
Trafficking of amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid (AMPA) receptors is under intensive study because of its believed central role in synaptic plasticity (9 –12)
The antibodies were specific for their respective Dlg proteins, as direct anti-postsynaptic density 95 (PSD-95) blot of rat brain did not detect any 120-kDa species, nor was the 95/80-kDa PSD-95 band labeled by direct anti-SAP97N blot (Fig. 1A, input lanes)
These findings indicate that Synapse-associated protein 97 (SAP97) and PSD-95 are present in the same molecular complexes in rat brain
Summary
Materials—N-terminally GFP-tagged SAP97 in pEGFP-C1 vector and C-terminally GFP-tagged PSD-95 in pGW1-CMV vector were generous gifts from Dr Craig Garner (Stanford University, Stanford, CA) and Dr David Bredt (University of California, San Francisco, CA), respectively. DNA fragments encoding the above described segments of SAP97 and PSD95 and PCR-generated SAP97 fragment coding for residues 101–223 (N2) were subcloned into pcDNA3.1(-) (Invitrogen) modified to carry sequences encoding an N-terminal c-Myc tag and/or a C-terminal His tag and into pEGFP-C1 (Clontech) for an N-terminal GFP tag. Proteins were extracted from adult rat brain homogenates or from transfected HEK293 cells in 50 mM Tris-HCl, pH 8.0, 140 mM NaCl, 1 mM EDTA, 1 mM Na3V04, 1 mM NaF, and 1 mM phenylmethylsulfonyl fluoride (TNE buffer) containing 1% Triton X-100, centrifuged for 20 min at 20,000 ϫ g, and incubated overnight at ϩ4 °C with 10 g of GST fusion protein bound to glutathione-Sepharose beads in a 1-ml total volume. Student’s unpaired t test was used for statistical analysis
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