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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

Functional expression of CBP/FLAG-GluR2 in heterologous cells and in transgenic mouse brain. (A) Schematic presentation of CBP/FLAG-GluR2. Calmodulin-binding peptide (CBP) and FLAG peptide sequences were inserted after the signal sequence of mouse GluR2 (flop form edited at position 586 (Q/R)). (B) In X. laevis oocytes expressing CBP/FLAG-GluR2, glutamate-evoked currents were vastly increased by coexpression of stargazin cRNA. (C) Surface receptors expressed in hippocampal neurons were live labeled with anti-FLAG M2 and total GluR2 was stained with anti-GluR2/3. Bar, 20 μm. (D) More efficient isolation of CBP/FLAG-GluR2 by immunoaffinity purification (IAP; anti-FLAG M2 agarose) than by conventional immunoprecipitation (anti-GluR2 antibody). Almost 100% of solubilized CBP/FLAG-GluR2 was isolated by IAP. FT, flow-through. (E) Two-step purification of CBP/FLAG-GluR2. The extracts of HEK cells transfected with mock vector or CBP/FLAG-GluR2 were subjected to sequential affinity chromatography: IAP and calmodulin-affinity chromatography (CaM). The arrow indicates the 110-kD protein (CBP/FLAG-GluR2). The arrowhead indicates the copurified 78-kD protein (identified as BiP/Grp78 by mass spectrometry). (F) Whole brain extracts (50 μg) from the indicated transgenic mouse lines and 100 fmol of CBP/FLAG-GluR2 purified from HEK cells were probed with the indicated antibodies. CBP/FLAG-GluR2 represents ∼50% of the endogenous protein in line 917. (G) Basal synaptic transmission is normal in transgenic GluR2 mice. (a) Input–output curve for basal synaptic transmission in hippocampal slices from wild type (Wt; n = 16) and transgenic (Tg; n = 18) mice. Each point represents the mean ± SEM for each bin. Sample fEPSPs at different stimulus intensities are shown on top. Bars: (y-axis) 0.5 mV; (x-axis) 20 ms. (b) AMPA/NMDA ratios were calculated by evoking dual-component EPSCs (Wt, n = 5; and Tg, n = 9). Histogram represents the AMPA/NMDA ratio mean ± SEM. Sample traces of the mixed and isolated AMPA- and NMDA-mediated EPSCs from wild-type and transgenic mice are shown on top. Bars: (y-axis) 100 pA; (x-axis) 20 ms.
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fig2: Functional expression of CBP/FLAG-GluR2 in heterologous cells and in transgenic mouse brain. (A) Schematic presentation of CBP/FLAG-GluR2. Calmodulin-binding peptide (CBP) and FLAG peptide sequences were inserted after the signal sequence of mouse GluR2 (flop form edited at position 586 (Q/R)). (B) In X. laevis oocytes expressing CBP/FLAG-GluR2, glutamate-evoked currents were vastly increased by coexpression of stargazin cRNA. (C) Surface receptors expressed in hippocampal neurons were live labeled with anti-FLAG M2 and total GluR2 was stained with anti-GluR2/3. Bar, 20 μm. (D) More efficient isolation of CBP/FLAG-GluR2 by immunoaffinity purification (IAP; anti-FLAG M2 agarose) than by conventional immunoprecipitation (anti-GluR2 antibody). Almost 100% of solubilized CBP/FLAG-GluR2 was isolated by IAP. FT, flow-through. (E) Two-step purification of CBP/FLAG-GluR2. The extracts of HEK cells transfected with mock vector or CBP/FLAG-GluR2 were subjected to sequential affinity chromatography: IAP and calmodulin-affinity chromatography (CaM). The arrow indicates the 110-kD protein (CBP/FLAG-GluR2). The arrowhead indicates the copurified 78-kD protein (identified as BiP/Grp78 by mass spectrometry). (F) Whole brain extracts (50 μg) from the indicated transgenic mouse lines and 100 fmol of CBP/FLAG-GluR2 purified from HEK cells were probed with the indicated antibodies. CBP/FLAG-GluR2 represents ∼50% of the endogenous protein in line 917. (G) Basal synaptic transmission is normal in transgenic GluR2 mice. (a) Input–output curve for basal synaptic transmission in hippocampal slices from wild type (Wt; n = 16) and transgenic (Tg; n = 18) mice. Each point represents the mean ± SEM for each bin. Sample fEPSPs at different stimulus intensities are shown on top. Bars: (y-axis) 0.5 mV; (x-axis) 20 ms. (b) AMPA/NMDA ratios were calculated by evoking dual-component EPSCs (Wt, n = 5; and Tg, n = 9). Histogram represents the AMPA/NMDA ratio mean ± SEM. Sample traces of the mixed and isolated AMPA- and NMDA-mediated EPSCs from wild-type and transgenic mice are shown on top. Bars: (y-axis) 100 pA; (x-axis) 20 ms.

Mentions: To permit more efficient isolation of GluR2 complexes, we designed an epitope-tagged GluR2 construct that has both a FLAG-recognition site and a calmodulin-binding peptide (CBP), which permit efficient and selective protein isolation from crude homogenates (Yang et al., 2002; Fig. 2 A). We first used this construct in heterologous expression systems and found that it expressed an appropriate and functional GluR2. As expected, the expressed protein migrated at a slightly larger monomeric molecular mass than did GluR2 without the engineered tags (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200501121/DC1). We found that glutamate-evoked currents from this GluR2 expressed in Xenopus laevis oocytes were vastly increased by coexpression of stargazin cRNA (Fig. 2 B). We also determined that this construct was expressed appropriately on the surface of transfected hippocampal neurons (Fig. 2 C).


Molecular constituents of neuronal AMPA receptors.

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

Functional expression of CBP/FLAG-GluR2 in heterologous cells and in transgenic mouse brain. (A) Schematic presentation of CBP/FLAG-GluR2. Calmodulin-binding peptide (CBP) and FLAG peptide sequences were inserted after the signal sequence of mouse GluR2 (flop form edited at position 586 (Q/R)). (B) In X. laevis oocytes expressing CBP/FLAG-GluR2, glutamate-evoked currents were vastly increased by coexpression of stargazin cRNA. (C) Surface receptors expressed in hippocampal neurons were live labeled with anti-FLAG M2 and total GluR2 was stained with anti-GluR2/3. Bar, 20 μm. (D) More efficient isolation of CBP/FLAG-GluR2 by immunoaffinity purification (IAP; anti-FLAG M2 agarose) than by conventional immunoprecipitation (anti-GluR2 antibody). Almost 100% of solubilized CBP/FLAG-GluR2 was isolated by IAP. FT, flow-through. (E) Two-step purification of CBP/FLAG-GluR2. The extracts of HEK cells transfected with mock vector or CBP/FLAG-GluR2 were subjected to sequential affinity chromatography: IAP and calmodulin-affinity chromatography (CaM). The arrow indicates the 110-kD protein (CBP/FLAG-GluR2). The arrowhead indicates the copurified 78-kD protein (identified as BiP/Grp78 by mass spectrometry). (F) Whole brain extracts (50 μg) from the indicated transgenic mouse lines and 100 fmol of CBP/FLAG-GluR2 purified from HEK cells were probed with the indicated antibodies. CBP/FLAG-GluR2 represents ∼50% of the endogenous protein in line 917. (G) Basal synaptic transmission is normal in transgenic GluR2 mice. (a) Input–output curve for basal synaptic transmission in hippocampal slices from wild type (Wt; n = 16) and transgenic (Tg; n = 18) mice. Each point represents the mean ± SEM for each bin. Sample fEPSPs at different stimulus intensities are shown on top. Bars: (y-axis) 0.5 mV; (x-axis) 20 ms. (b) AMPA/NMDA ratios were calculated by evoking dual-component EPSCs (Wt, n = 5; and Tg, n = 9). Histogram represents the AMPA/NMDA ratio mean ± SEM. Sample traces of the mixed and isolated AMPA- and NMDA-mediated EPSCs from wild-type and transgenic mice are shown on top. Bars: (y-axis) 100 pA; (x-axis) 20 ms.
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Related In: Results  -  Collection

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fig2: Functional expression of CBP/FLAG-GluR2 in heterologous cells and in transgenic mouse brain. (A) Schematic presentation of CBP/FLAG-GluR2. Calmodulin-binding peptide (CBP) and FLAG peptide sequences were inserted after the signal sequence of mouse GluR2 (flop form edited at position 586 (Q/R)). (B) In X. laevis oocytes expressing CBP/FLAG-GluR2, glutamate-evoked currents were vastly increased by coexpression of stargazin cRNA. (C) Surface receptors expressed in hippocampal neurons were live labeled with anti-FLAG M2 and total GluR2 was stained with anti-GluR2/3. Bar, 20 μm. (D) More efficient isolation of CBP/FLAG-GluR2 by immunoaffinity purification (IAP; anti-FLAG M2 agarose) than by conventional immunoprecipitation (anti-GluR2 antibody). Almost 100% of solubilized CBP/FLAG-GluR2 was isolated by IAP. FT, flow-through. (E) Two-step purification of CBP/FLAG-GluR2. The extracts of HEK cells transfected with mock vector or CBP/FLAG-GluR2 were subjected to sequential affinity chromatography: IAP and calmodulin-affinity chromatography (CaM). The arrow indicates the 110-kD protein (CBP/FLAG-GluR2). The arrowhead indicates the copurified 78-kD protein (identified as BiP/Grp78 by mass spectrometry). (F) Whole brain extracts (50 μg) from the indicated transgenic mouse lines and 100 fmol of CBP/FLAG-GluR2 purified from HEK cells were probed with the indicated antibodies. CBP/FLAG-GluR2 represents ∼50% of the endogenous protein in line 917. (G) Basal synaptic transmission is normal in transgenic GluR2 mice. (a) Input–output curve for basal synaptic transmission in hippocampal slices from wild type (Wt; n = 16) and transgenic (Tg; n = 18) mice. Each point represents the mean ± SEM for each bin. Sample fEPSPs at different stimulus intensities are shown on top. Bars: (y-axis) 0.5 mV; (x-axis) 20 ms. (b) AMPA/NMDA ratios were calculated by evoking dual-component EPSCs (Wt, n = 5; and Tg, n = 9). Histogram represents the AMPA/NMDA ratio mean ± SEM. Sample traces of the mixed and isolated AMPA- and NMDA-mediated EPSCs from wild-type and transgenic mice are shown on top. Bars: (y-axis) 100 pA; (x-axis) 20 ms.
Mentions: To permit more efficient isolation of GluR2 complexes, we designed an epitope-tagged GluR2 construct that has both a FLAG-recognition site and a calmodulin-binding peptide (CBP), which permit efficient and selective protein isolation from crude homogenates (Yang et al., 2002; Fig. 2 A). We first used this construct in heterologous expression systems and found that it expressed an appropriate and functional GluR2. As expected, the expressed protein migrated at a slightly larger monomeric molecular mass than did GluR2 without the engineered tags (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200501121/DC1). We found that glutamate-evoked currents from this GluR2 expressed in Xenopus laevis oocytes were vastly increased by coexpression of stargazin cRNA (Fig. 2 B). We also determined that this construct was expressed appropriately on the surface of transfected hippocampal neurons (Fig. 2 C).

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