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Molecular constituents of neuronal AMPA receptors.

Fukata Y, Tzingounis AV, Trinidad JC, Fukata M, Burlingame AL, Nicoll RA, Bredt DS - J. Cell Biol. (2005)

Bottom Line: Although numerous AMPAR-interacting proteins have been identified, their quantitative and relative contributions to native AMPAR complexes remain unclear.We found that stargazin-like transmembrane AMPAR regulatory proteins (TARPs) copurified with neuronal AMPARs, but we found negligible binding to GRIP, PICK1, NSF, or SAP-97.To facilitate purification of neuronal AMPAR complexes, we generated a transgenic mouse expressing an epitope-tagged GluR2 subunit of AMPARs.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of California, San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
Dynamic regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) underlies aspects of synaptic plasticity. Although numerous AMPAR-interacting proteins have been identified, their quantitative and relative contributions to native AMPAR complexes remain unclear. Here, we quantitated protein interactions with neuronal AMPARs by immunoprecipitation from brain extracts. We found that stargazin-like transmembrane AMPAR regulatory proteins (TARPs) copurified with neuronal AMPARs, but we found negligible binding to GRIP, PICK1, NSF, or SAP-97. To facilitate purification of neuronal AMPAR complexes, we generated a transgenic mouse expressing an epitope-tagged GluR2 subunit of AMPARs. Taking advantage of this powerful new tool, we isolated two populations of GluR2 containing AMPARs: an immature complex with the endoplasmic reticulum chaperone immunoglobulin-binding protein and a mature complex containing GluR1, TARPs, and PSD-95. These studies establish TARPs as the auxiliary components of neuronal AMPARs.

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Immunoisolation of AMPARs from wild-type mice. (A and B) Brain extracts solubilized with 1% Triton X-100 were used for immunoprecipitation with anti-GluR2 antibody or nonimmune mouse IgG. AMPAR subunits and previously reported AMPAR binding proteins (see text) were examined by immunoblotting. Input lanes contain the indicated percentage of proteins used for immunoprecipitation (IP). (A) The percentages of binding (%) are indicated.
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fig1: Immunoisolation of AMPARs from wild-type mice. (A and B) Brain extracts solubilized with 1% Triton X-100 were used for immunoprecipitation with anti-GluR2 antibody or nonimmune mouse IgG. AMPAR subunits and previously reported AMPAR binding proteins (see text) were examined by immunoblotting. Input lanes contain the indicated percentage of proteins used for immunoprecipitation (IP). (A) The percentages of binding (%) are indicated.

Mentions: We solubilized whole brain extracts with 1% Triton X-100 and purified AMPAR complexes using a well-characterized antibody to GluR2. Under these conditions, ∼10% of GluR2 was isolated and along with it came ∼7.5% of total GluR1 (Fig. 1 A). Recovery of TARPs showed similar efficiency. In contrast, we were unable to detect any of the other reported AMPAR binding proteins including GRIP, NSF, PICK1, or SAP-97 (Fig. 1, A and B).


Molecular constituents of neuronal AMPA receptors.

Fukata Y, Tzingounis AV, Trinidad JC, Fukata M, Burlingame AL, Nicoll RA, Bredt DS - J. Cell Biol. (2005)

Immunoisolation of AMPARs from wild-type mice. (A and B) Brain extracts solubilized with 1% Triton X-100 were used for immunoprecipitation with anti-GluR2 antibody or nonimmune mouse IgG. AMPAR subunits and previously reported AMPAR binding proteins (see text) were examined by immunoblotting. Input lanes contain the indicated percentage of proteins used for immunoprecipitation (IP). (A) The percentages of binding (%) are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171936&req=5

fig1: Immunoisolation of AMPARs from wild-type mice. (A and B) Brain extracts solubilized with 1% Triton X-100 were used for immunoprecipitation with anti-GluR2 antibody or nonimmune mouse IgG. AMPAR subunits and previously reported AMPAR binding proteins (see text) were examined by immunoblotting. Input lanes contain the indicated percentage of proteins used for immunoprecipitation (IP). (A) The percentages of binding (%) are indicated.
Mentions: We solubilized whole brain extracts with 1% Triton X-100 and purified AMPAR complexes using a well-characterized antibody to GluR2. Under these conditions, ∼10% of GluR2 was isolated and along with it came ∼7.5% of total GluR1 (Fig. 1 A). Recovery of TARPs showed similar efficiency. In contrast, we were unable to detect any of the other reported AMPAR binding proteins including GRIP, NSF, PICK1, or SAP-97 (Fig. 1, A and B).

Bottom Line: Although numerous AMPAR-interacting proteins have been identified, their quantitative and relative contributions to native AMPAR complexes remain unclear.We found that stargazin-like transmembrane AMPAR regulatory proteins (TARPs) copurified with neuronal AMPARs, but we found negligible binding to GRIP, PICK1, NSF, or SAP-97.To facilitate purification of neuronal AMPAR complexes, we generated a transgenic mouse expressing an epitope-tagged GluR2 subunit of AMPARs.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of California, San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
Dynamic regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) underlies aspects of synaptic plasticity. Although numerous AMPAR-interacting proteins have been identified, their quantitative and relative contributions to native AMPAR complexes remain unclear. Here, we quantitated protein interactions with neuronal AMPARs by immunoprecipitation from brain extracts. We found that stargazin-like transmembrane AMPAR regulatory proteins (TARPs) copurified with neuronal AMPARs, but we found negligible binding to GRIP, PICK1, NSF, or SAP-97. To facilitate purification of neuronal AMPAR complexes, we generated a transgenic mouse expressing an epitope-tagged GluR2 subunit of AMPARs. Taking advantage of this powerful new tool, we isolated two populations of GluR2 containing AMPARs: an immature complex with the endoplasmic reticulum chaperone immunoglobulin-binding protein and a mature complex containing GluR1, TARPs, and PSD-95. These studies establish TARPs as the auxiliary components of neuronal AMPARs.

Show MeSH
Related in: MedlinePlus