Synapse-Associated Protein 97 Selectively Associates with a Subset of AMPA Receptors Early in their Biosynthetic Pathway

SAP97 is associated with immature GluR1 and GluR2.A, Glycosylation state of SAP97-associated AMPARs.Left, Membrane homogenates (H) from hippocampus were solubilized with 0.5% SDS in the presence of 1% β-mercaptoethanol. After dilution with 1% NP-40, soluble extracts were incubated in the absence of enzyme (control samples) or in the presence of Endo-H or PNGaseF. GluR2 has a small, but distinct, population that is Endo-H-sensitive.Right, Triton-solubilized AMPARs were first immunoprecipitated with SAP97 and then deglycosylated with Endo-H or PNGaseF. Equal amounts of sample were treated and subjected to SDS-PAGE. Although SAP97 immunoprecipitated both Endo-H-sensitive and Endo-H-insensitive GluR2, there is a substantial relative increase in the Endo-H-sensitive component showing that SAP97 preferentially associates with immature AMPARs. B, Glycosylation state of the crude synaptic membrane (P2) and microsomal (P3) fraction. The P3 fraction is enriched in Endo-H-sensitive GluR1 and GluR2. P2 and P3 fractions of hippocampus were treated with Endo-H or PNGaseF and analyzed by SDS-PAGE and Western blotting. C, Glycosylation state of SAP97-associated AMPARs. Almost all SAP97-associated GluR1 and GluR2 in the P3 fraction is Endo-H-sensitive.

AMPARs associated with SAP97 are concentrated in intracellular membranes. The hippocampus was subfractionated into crude synaptic membrane (P2) and microsomal (P3) fraction and synaptic membrane (SM). Triton-solubilized subfractions were immunoprecipitated with anti-SAP97 antibodies. A, GluR1 and GluR2 remaining (unbound fraction) after IP. B,Quantitation of immunostaining remaining in the depleted fractions (mean ± SEM of three separate experiments) shows little SAP97-associated GluR1 and GluR2 in the SM fraction.

Isolation of PSD from the SM fraction using magnetic beads coated with antibodies to PSD-95. PSD were immunoisolated in the absence of detergent as described.A, Electron micrograph showing PSD (arrowheads) binding to magnetic beads (mb). Scale bar, 0.2 μm. B,Distribution of SAP97 and proteins associated with the PSD (PSD-95, SAP102, GluR1, GluR2–3, NR2B), synaptic vesicles (synaptophysin), and ER (calnexin). IgG refers to a control in which magnetic beads were coated with rabbit IgG. For P3, P2, and SM, 10 μg of protein was applied to each lane. Protein was not quantified in the immunoisolated fraction, but the same volume of sample was applied for each immunoblot.

GluRs from hippocampus and distribution of SAP97 in the CA1 region using light (A) and electron (B, C) microscopy. A, With light microscopy (immunofluorescence-FITC and immunoperoxidase-DAB), labeling is found throughout the cell body, apical dendrites, and neuropil in strata oriens (so), pyramidale, and radiatum (sr). B, EM (a–m), colocalization of SAP97 (10 nm gold) with GluR1 (5 nm gold;a–g, i) and GluR2–3 (5 nm gold:j–m) and immunolabeling for SAP97 (10 nm gold;h) in fixed (h) and unfixed (a–g, i–m) sections of the stratum pyramidale and radiatum of the CA1 region of the hippocampus, respectively.h, Arrowheads indicate gold particles decorating ER cisternae in the soma of a pyramidal neuron. a–g, i–m, Arrows indicate colocalization of SAP97 and AMPARs at synapses. Arrowheads indicate presumptive cytoplasmic vesicular or tubulovesicular structures that are double- or single-labeled. These labeled structures are common in dendrites (e–g, i, l, m) but also are seen in postsynaptic spines (j, k). Three labeled vesicle-like structures are seen in i, one double-labeled and one each labeled with either 5 or 10 nm gold. p, Presynaptic terminal. Scale bar: a–g, i–m, 0.2 μm; h, 0.4 μm.C, Histogram showing immunogold labeling for SAP97 (5 nm gold; stratum radiatum; fixed section), representing 28% of the total synapses. Although zero and one counts are dominant, there seems to be a great deal of unobserved heterogeneity. This leads to overdispersion (highly significant; p < 0.0001); that is, the actual variance exceeds the nominal Poisson variance. These results are consistent with there being a higher density of SAP97 in some synapses.

SAP97 is colocalized with a subpopulation of surface GluR1 in cultured hippocampal neurons. A, Live cultured hippocampal neurons were surface-labeled with antibodies to the GluR1-N and subsequently double-labeled for SAP97. Surface GluR1 forms clusters distributed throughout the dendritic tree (red). SAP97 immunofluorescence (green) was observed along dendrites and in the soma and showed a more diffuse pattern of distribution than GluR1 along dendrites. SAP97 labeling rarely overlapped with AMPAR labeling (arrows). B, Dendritic distribution of GluR1 (green) and BiP (red) (right panel) and SAP97 (green) and BiP (left panel). Both GluR1 and SAP97 showed partial colocalization (yellow) with BiP, an ER marker, within dendrites. C, Live neurons were prelabeled with antibodies to GluR1-N, stimulated with 100 μm AMPA and 50 μm APV, or 20 μm NMDA, returned to growth medium for 10–15 min at 37°C, and internalized GluR1 (red) was detected together with SAP97 (green). Internalized GluR1 accumulated in puncta along dendrites. SAP97 labeling is also punctate, but SAP97 and internalized GluR1 very rarely colocalize. D, Stimulated neurons were double-immunolabeled for SAP97 (green) and early/recycling endosomes (EEA1 and Rab4) and late endosomes (Lamp1) (red). Under all stimulation conditions (control shown), labeling for endosomes was punctiform and observed within dendritic and somatic cytoplasm. The endosomal compartments showed no SAP97 labeling. Scale bar, 2 μm.

SAP97 is associated with some surface receptors in cultured cortical neurons. To determine if surface AMPARs are associated with SAP97, cortical cultures were biotinylated with NHS–SS–biotin, and presence of biotinylated AMPARs associated with SAP97 was assayed. Three experimental approaches were used to assess the relationship between SAP97 and biotinylated receptors.A, Two-week-old cortical cultures were biotinylated, and membranes were solubilized with 1% SDS (with boiling) or with 1% Triton X-100. The detergent-soluble fraction was added to streptavidin-conjugated beads and incubated at 4°C. Solubilized membranes (S), IgG-precipitated (control), and streptavidin-precipitated proteins, were loaded so that each lane represents 1% of the material from the plate. The blots were probed with antibodies to GluR1, GluR2, SAP97, and PSD-95. BiP and tubulin antibodies were used as controls. The presence of SAP97 in the streptavidin fraction shows that some SAP97 is associated with biotinylated surface receptors. Note that this is seen only with Triton solubilization but not SDS solubilization, which disrupts protein–protein interactions. The absence of SAP97 in the SDS solubilized material is a control for biotinylation of intracellular proteins. Tubulin or BiP, an ER protein, are not biotinylated.B, Cultures of cortical neurons were biotinylated and membranes were solubilized with 1% SDS or with 1% Triton X-100. The detergent-soluble fraction was added to SAP97-conjugated protein A and incubated at 4°C. Solubilized membranes (S), IgG-precipitated (control), and SAP97-precipitated proteins were loaded so that each lane represents 1% of the material from the plate. The blots were probed with GluR1, GluR2, SAP97, and NR1 antibodies, and with streptavidin-HRP. SAP97 immunoprecipitated unbiotinylated subunits along with biotinylated ones. In the streptavidin–HRP-stained panel, a band that co-migrates with GluR1 and GluR2 is seen (arrow). SAP97 did not associate with NMDARs.C, Co-migration of streptavidin–HRP with GluR1 and GluR2, as shown in B, does not prove that the biotinylated band is indeed GluR1–GluR2. To establish this, double affinity purification of biotinylated AMPARs by sequential anti-SAP97 and streptavidin IP was done. Cortical neurons were exposed to 1 mg/ml NHS–SS–biotin or to PBS–Ca²⁺–Mg²⁺ (control) and processed as described. Lanes 1 and 2, Anti-SAP97 immunoprecipitated GluRs; lanes 3 and4, anti-SAP97 and streptavidin-precipitated GluRs.D, To show that Endo-H-sensitive receptors are not present on the cell surface, surface receptors immunoprecipitated with streptavidin-conjugated beads (IP), were treated with Endo-H or PNGaseF and analyzed by SDS-PAGE and Western blotting. Treatment of surface-biotinylated protein with Endo-H has no effect on GluR1 or GluR2, showing that surface GluR2 are Endo-H-resistant. Total homogenates (H) contain an Endo-H-sensitive component.