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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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Related in: MedlinePlus

Two distinct AMPAR complexes. Immunoaffinity-purified brain AMPAR complexes (IAP) were reprecipitated (seqIP) with antibodies to either BiP or TARPs and analyzed by silver staining (top) and immunoblotting with the indicated antibodies (bottom). The BiP reprecipitation isolates 110- (arrow) and 78-kD (closed arrowhead; BiP) proteins; the TARP reprecipitation isolates 110- and 35-kD (open arrowhead; TARPs) proteins. The BiP reprecipitates include only GluR2, but not GluR1, TARPs, or PSD-95, whereas TARP reprecipitates include GluR1 and PSD-95 in addition to GluR2.
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fig4: Two distinct AMPAR complexes. Immunoaffinity-purified brain AMPAR complexes (IAP) were reprecipitated (seqIP) with antibodies to either BiP or TARPs and analyzed by silver staining (top) and immunoblotting with the indicated antibodies (bottom). The BiP reprecipitation isolates 110- (arrow) and 78-kD (closed arrowhead; BiP) proteins; the TARP reprecipitation isolates 110- and 35-kD (open arrowhead; TARPs) proteins. The BiP reprecipitates include only GluR2, but not GluR1, TARPs, or PSD-95, whereas TARP reprecipitates include GluR1 and PSD-95 in addition to GluR2.

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)

Two distinct AMPAR complexes. Immunoaffinity-purified brain AMPAR complexes (IAP) were reprecipitated (seqIP) with antibodies to either BiP or TARPs and analyzed by silver staining (top) and immunoblotting with the indicated antibodies (bottom). The BiP reprecipitation isolates 110- (arrow) and 78-kD (closed arrowhead; BiP) proteins; the TARP reprecipitation isolates 110- and 35-kD (open arrowhead; TARPs) proteins. The BiP reprecipitates include only GluR2, but not GluR1, TARPs, or PSD-95, whereas TARP reprecipitates include GluR1 and PSD-95 in addition to GluR2.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Two distinct AMPAR complexes. Immunoaffinity-purified brain AMPAR complexes (IAP) were reprecipitated (seqIP) with antibodies to either BiP or TARPs and analyzed by silver staining (top) and immunoblotting with the indicated antibodies (bottom). The BiP reprecipitation isolates 110- (arrow) and 78-kD (closed arrowhead; BiP) proteins; the TARP reprecipitation isolates 110- and 35-kD (open arrowhead; TARPs) proteins. The BiP reprecipitates include only GluR2, but not GluR1, TARPs, or PSD-95, whereas TARP reprecipitates include GluR1 and PSD-95 in addition to GluR2.
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