Skip to main content

Umbrella menu

  • SfN.org
  • eNeuro
  • The Journal of Neuroscience
  • Neuronline
  • BrainFacts.org

Main menu

  • HOME
  • CONTENT
    • Early Release
    • Featured
    • Current Issue
    • Issue Archive
    • Collections
  • ALERTS
  • FOR AUTHORS
    • Preparing a Manuscript
    • Submission Guidelines
    • Fees
    • Journal Club
    • eLetters
    • Submit
  • EDITORIAL BOARD
  • ABOUT
    • Overview
    • Advertise
    • For the Media
    • Rights and Permissions
    • Privacy Policy
    • Feedback
  • SUBSCRIBE
  • SfN.org
  • eNeuro
  • The Journal of Neuroscience
  • Neuronline
  • BrainFacts.org

User menu

  • Log in
  • Subscribe
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Neuroscience
  • Log in
  • Subscribe
  • My alerts
  • My Cart
Journal of Neuroscience

Advanced Search

Submit a Manuscript
  • HOME
  • CONTENT
    • Early Release
    • Featured
    • Current Issue
    • Issue Archive
    • Collections
  • ALERTS
  • FOR AUTHORS
    • Preparing a Manuscript
    • Submission Guidelines
    • Fees
    • Journal Club
    • eLetters
    • Submit
  • EDITORIAL BOARD
  • ABOUT
    • Overview
    • Advertise
    • For the Media
    • Rights and Permissions
    • Privacy Policy
    • Feedback
  • SUBSCRIBE
PreviousNext
ARTICLE, Cellular/Molecular

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

Nathalie Sans, Claudia Racca, Ronald S. Petralia, Ya-Xian Wang, Jennifer McCallum and Robert J. Wenthold
Journal of Neuroscience 1 October 2001, 21 (19) 7506-7516; DOI: https://doi.org/10.1523/JNEUROSCI.21-19-07506.2001
Nathalie Sans
1Laboratory of Neurochemistry, National Institute on Deafness and other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892-8027
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Claudia Racca
1Laboratory of Neurochemistry, National Institute on Deafness and other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892-8027
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ronald S. Petralia
1Laboratory of Neurochemistry, National Institute on Deafness and other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892-8027
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ya-Xian Wang
1Laboratory of Neurochemistry, National Institute on Deafness and other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892-8027
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jennifer McCallum
1Laboratory of Neurochemistry, National Institute on Deafness and other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892-8027
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Robert J. Wenthold
1Laboratory of Neurochemistry, National Institute on Deafness and other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892-8027
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF
Loading

Article Figures & Data

Figures

  • Fig. 1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 1.

    Co-immunoprecipitation of AMPAR subunits with SAP97 in rat hippocampus. Proteins were solubilized in 1% Triton X-100, and SAP97 was immunoprecipitated using anti-SAP97 antibodies. A, AMPAR subunits remaining (unbound fraction) after IP of SAP97 from hippocampus homogenate with SAP97 antibodies. The two left lanes of each panel show the non-immunoprecipitated Triton-solubilized fraction (Tx sol). The 100% lane represents 10 μl of sample applied, whereas the 5% lane represents 0.5 μl applied (after 1:10 dilution with sample buffer). These lanes represent the range of labeling for quantification of immunoreactivity in the depleted fractions. For each gel, standards of 75, 50, 25, and 10% of the solubilized fraction were also analyzed (data not shown). To determine the amount of immunoprecipitated proteins, 10 μl of the depleted fraction was analyzed, equivalent to an equal volume of the solubilized fraction (100%). B, IP of AMPAR subunits (bound fraction) using anti-SAP97 antibodies. Ten microliters of bound immunoprecipitate fractions were separated by SDS-PAGE, immunoblotted, and incubated with antibodies against SAP97, GluR1, GluR2, GluR3, and NR1. C, Percentage of immunostaining remaining in the depleted fractions is shown on the graph (mean ± SEM of three separate experiments).

  • Fig. 2.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 2.

    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.

  • Fig. 3.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 3.

    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.

  • Fig. 4.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 4.

    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.

  • Fig. 5.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 5.

    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.

  • Fig. 6.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 6.

    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.

  • Fig. 7.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 7.

    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.

  • Fig. 8.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 8.

    Model for the addition of AMPARs to the synaptic membrane. AMPARs with and without GluR1 are added to the SM following different pathways. The predominant AMPAR subunits in the adult hippocampus are GluR1 (shaded ovals), GluR2 (open ovals), and GluR3 (striped ovals). SAP97 is associated with GluR1 and plays a role only in initial delivery of receptors. After internalization, GluR1-containing complexes enter the constitutive receptor pool.

Back to top

In this issue

The Journal of Neuroscience: 21 (19)
Journal of Neuroscience
Vol. 21, Issue 19
1 Oct 2001
  • Table of Contents
  • Index by author
Email

Thank you for sharing this Journal of Neuroscience article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Synapse-Associated Protein 97 Selectively Associates with a Subset of AMPA Receptors Early in their Biosynthetic Pathway
(Your Name) has forwarded a page to you from Journal of Neuroscience
(Your Name) thought you would be interested in this article in Journal of Neuroscience.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Print
View Full Page PDF
Citation Tools
Synapse-Associated Protein 97 Selectively Associates with a Subset of AMPA Receptors Early in their Biosynthetic Pathway
Nathalie Sans, Claudia Racca, Ronald S. Petralia, Ya-Xian Wang, Jennifer McCallum, Robert J. Wenthold
Journal of Neuroscience 1 October 2001, 21 (19) 7506-7516; DOI: 10.1523/JNEUROSCI.21-19-07506.2001

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Respond to this article
Request Permissions
Share
Synapse-Associated Protein 97 Selectively Associates with a Subset of AMPA Receptors Early in their Biosynthetic Pathway
Nathalie Sans, Claudia Racca, Ronald S. Petralia, Ya-Xian Wang, Jennifer McCallum, Robert J. Wenthold
Journal of Neuroscience 1 October 2001, 21 (19) 7506-7516; DOI: 10.1523/JNEUROSCI.21-19-07506.2001
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • Footnotes
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF

Keywords

  • SAP97
  • GluR1
  • trafficking
  • ER–cis-Golgi
  • postsynaptic density
  • hippocampus

Responses to this article

Respond to this article

Jump to comment:

No eLetters have been published for this article.

Related Articles

Cited By...

More in this TOC Section

ARTICLE

  • Neural Correlates of Competing Fear Behaviors Evoked by an Innately Aversive Stimulus
  • Distinct Developmental Modes and Lesion-Induced Reactions of Dendrites of Two Classes of Drosophila Sensory Neurons
  • Functional Dissociation among Components of Remembering: Control, Perceived Oldness, and Content
Show more ARTICLE

Cellular/Molecular

  • Neural Correlates of Competing Fear Behaviors Evoked by an Innately Aversive Stimulus
  • Distinct Developmental Modes and Lesion-Induced Reactions of Dendrites of Two Classes of Drosophila Sensory Neurons
  • Functional Dissociation among Components of Remembering: Control, Perceived Oldness, and Content
Show more Cellular/Molecular
  • Home
  • Alerts
  • Visit Society for Neuroscience on Facebook
  • Follow Society for Neuroscience on Twitter
  • Follow Society for Neuroscience on LinkedIn
  • Visit Society for Neuroscience on Youtube
  • Follow our RSS feeds

Content

  • Early Release
  • Current Issue
  • Issue Archive
  • Collections

Information

  • For Authors
  • For Advertisers
  • For the Media
  • For Subscribers

About

  • About the Journal
  • Editorial Board
  • Privacy Policy
  • Contact
  • Feedback
(JNeurosci logo)
(SfN logo)

Copyright © 2021 by the Society for Neuroscience.
JNeurosci Online ISSN: 1529-2401

The ideas and opinions expressed in JNeurosci do not necessarily reflect those of SfN or the JNeurosci Editorial Board. Publication of an advertisement or other product mention in JNeurosci should not be construed as an endorsement of the manufacturer’s claims. SfN does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of any material contained in JNeurosci.