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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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Quantitative association of TARPs with immunopurified AMPARs. (A) Gold colloidal total protein staining of immunoaffinity-purified CBP/FLAG-GluR2 (IAP) and immunoprecipitated TARPs (IP). The same preparations were also analyzed by Western blotting with anti-GluR1 or TARP antibody. The proteins with molecular masses of 110 (arrow), 78 (closed arrowhead), and 35 kD (open arrowhead) were detected in the transgenic mouse (Tg) but not in wild-type (Wt). 110- and 35-kD proteins were also detected in TARPs-IP, which correspond to AMPARs including GluR1 and TARPs, respectively. Asterisks denote the bands of IgG heavy and light chains. (B) IAP elution in A was immunoblotted with the indicated antibodies. The recovery (%) of each protein from input is indicated. (C) Quantitative binding of TARPs with AMPARs. For CBP/FLAG-GluR2, GluR1, and TARPs, input (1%) and purified AMPARs (IAP; 1%) from transgenic mouse brain were analyzed with the indicated amounts of purified CBP/FLAG-GluR2, HA-GluR1, and His6-stargazin COOH terminus. For PSD-95 and SAP-97, input (0.1%) and IAP elution (4%) were analyzed with purified PSD-95-GFP and SAP-97-GFP. These data are representative of five independent experiments.
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fig3: Quantitative association of TARPs with immunopurified AMPARs. (A) Gold colloidal total protein staining of immunoaffinity-purified CBP/FLAG-GluR2 (IAP) and immunoprecipitated TARPs (IP). The same preparations were also analyzed by Western blotting with anti-GluR1 or TARP antibody. The proteins with molecular masses of 110 (arrow), 78 (closed arrowhead), and 35 kD (open arrowhead) were detected in the transgenic mouse (Tg) but not in wild-type (Wt). 110- and 35-kD proteins were also detected in TARPs-IP, which correspond to AMPARs including GluR1 and TARPs, respectively. Asterisks denote the bands of IgG heavy and light chains. (B) IAP elution in A was immunoblotted with the indicated antibodies. The recovery (%) of each protein from input is indicated. (C) Quantitative binding of TARPs with AMPARs. For CBP/FLAG-GluR2, GluR1, and TARPs, input (1%) and purified AMPARs (IAP; 1%) from transgenic mouse brain were analyzed with the indicated amounts of purified CBP/FLAG-GluR2, HA-GluR1, and His6-stargazin COOH terminus. For PSD-95 and SAP-97, input (0.1%) and IAP elution (4%) were analyzed with purified PSD-95-GFP and SAP-97-GFP. These data are representative of five independent experiments.

Mentions: We used brains from these mice to isolate neuronal AMPAR complexes. Forebrain extracts were solubilized with Triton X-100. Purification from transgenic mouse brain extracts yielded protein bands of 110, 78, and 35 kD (Fig. 3 A). Quantitative Western blotting showed that the 110-, 78-, and 35-kD proteins corresponded to AMPAR subunits, BiP, and TARPs, respectively (Fig. 3, A–C; and see Fig. 4). We saw no other protein bands copurified specifically with AMPARs. As previously described (Tomita et al., 2004), when TARPs were immunoprecipitated from brain, a protein complex comprising 110- and 35-kD proteins, corresponding to AMPARs and TARPs respectively, was isolated (Fig. 3 A).


Molecular constituents of neuronal AMPA receptors.

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

Quantitative association of TARPs with immunopurified AMPARs. (A) Gold colloidal total protein staining of immunoaffinity-purified CBP/FLAG-GluR2 (IAP) and immunoprecipitated TARPs (IP). The same preparations were also analyzed by Western blotting with anti-GluR1 or TARP antibody. The proteins with molecular masses of 110 (arrow), 78 (closed arrowhead), and 35 kD (open arrowhead) were detected in the transgenic mouse (Tg) but not in wild-type (Wt). 110- and 35-kD proteins were also detected in TARPs-IP, which correspond to AMPARs including GluR1 and TARPs, respectively. Asterisks denote the bands of IgG heavy and light chains. (B) IAP elution in A was immunoblotted with the indicated antibodies. The recovery (%) of each protein from input is indicated. (C) Quantitative binding of TARPs with AMPARs. For CBP/FLAG-GluR2, GluR1, and TARPs, input (1%) and purified AMPARs (IAP; 1%) from transgenic mouse brain were analyzed with the indicated amounts of purified CBP/FLAG-GluR2, HA-GluR1, and His6-stargazin COOH terminus. For PSD-95 and SAP-97, input (0.1%) and IAP elution (4%) were analyzed with purified PSD-95-GFP and SAP-97-GFP. These data are representative of five independent experiments.
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Related In: Results  -  Collection

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fig3: Quantitative association of TARPs with immunopurified AMPARs. (A) Gold colloidal total protein staining of immunoaffinity-purified CBP/FLAG-GluR2 (IAP) and immunoprecipitated TARPs (IP). The same preparations were also analyzed by Western blotting with anti-GluR1 or TARP antibody. The proteins with molecular masses of 110 (arrow), 78 (closed arrowhead), and 35 kD (open arrowhead) were detected in the transgenic mouse (Tg) but not in wild-type (Wt). 110- and 35-kD proteins were also detected in TARPs-IP, which correspond to AMPARs including GluR1 and TARPs, respectively. Asterisks denote the bands of IgG heavy and light chains. (B) IAP elution in A was immunoblotted with the indicated antibodies. The recovery (%) of each protein from input is indicated. (C) Quantitative binding of TARPs with AMPARs. For CBP/FLAG-GluR2, GluR1, and TARPs, input (1%) and purified AMPARs (IAP; 1%) from transgenic mouse brain were analyzed with the indicated amounts of purified CBP/FLAG-GluR2, HA-GluR1, and His6-stargazin COOH terminus. For PSD-95 and SAP-97, input (0.1%) and IAP elution (4%) were analyzed with purified PSD-95-GFP and SAP-97-GFP. These data are representative of five independent experiments.
Mentions: We used brains from these mice to isolate neuronal AMPAR complexes. Forebrain extracts were solubilized with Triton X-100. Purification from transgenic mouse brain extracts yielded protein bands of 110, 78, and 35 kD (Fig. 3 A). Quantitative Western blotting showed that the 110-, 78-, and 35-kD proteins corresponded to AMPAR subunits, BiP, and TARPs, respectively (Fig. 3, A–C; and see Fig. 4). We saw no other protein bands copurified specifically with AMPARs. As previously described (Tomita et al., 2004), when TARPs were immunoprecipitated from brain, a protein complex comprising 110- and 35-kD proteins, corresponding to AMPARs and TARPs respectively, was isolated (Fig. 3 A).

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