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Functional studies and distribution define a family of transmembrane AMPA receptor regulatory proteins.

Tomita S, Chen L, Kawasaki Y, Petralia RS, Wenthold RJ, Nicoll RA, Bredt DS - J. Cell Biol. (2003)

Bottom Line: Functional expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in cerebellar granule cells requires stargazin, a member of a large family of four-pass transmembrane proteins.Here, we define a family of transmembrane AMPA receptor regulatory proteins (TARPs), which comprise stargazin, gamma-3, gamma-4, and gamma-8, but not related proteins, that mediate surface expression of AMPA receptors.These studies indicate a general role for TARPs in controlling synaptic AMPA receptors throughout the central nervous system.

View Article: PubMed Central - PubMed

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

ABSTRACT
Functional expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in cerebellar granule cells requires stargazin, a member of a large family of four-pass transmembrane proteins. Here, we define a family of transmembrane AMPA receptor regulatory proteins (TARPs), which comprise stargazin, gamma-3, gamma-4, and gamma-8, but not related proteins, that mediate surface expression of AMPA receptors. TARPs exhibit discrete and complementary patterns of expression in both neurons and glia in the developing and mature central nervous system. In brain regions that express multiple isoforms, such as cerebral cortex, TARP-AMPA receptor complexes are strictly segregated, suggesting distinct roles for TARP isoforms. TARPs interact with AMPA receptors at the postsynaptic density, and surface expression of mature AMPA receptors requires a TARP. These studies indicate a general role for TARPs in controlling synaptic AMPA receptors throughout the central nervous system.

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TARPs interact specifically with AMPA receptors in brain extracts. (A) AMPA receptor subunits GluR1, GluR2, and GluR4 coimmunoprecipitate with stargazin (STG) in brain extracts from +/stg mice (+/−). In extracts from stg/stg mice (−/−), the antistargazin antibody does not immunoprecipitate GluR1, GluR2, or GluR4. (B–D) AMPA receptor subunits also coimmunoprecipitate with γ-3, γ-4, and γ-8 in extracts from adult cerebral cortex, postnatal day 7 cerebral cortex, and hippocampus, respectively. As control, other transmembrane proteins such as GABAARβ, N-cadherin, and Kv1.4 did not coimmunoprecipitate. (E) Subunit specificity for TARP interactions in cerebellum. Stargazin (STG) and γ-4 coimmunoprecipitate with GluR1 and GluR4, but only stargazin coimmunoprecipitates with GluR2/3 in cerebellum. (F) TARP isoforms are strictly segregated, since they do not coimmunoprecipitate with one another in cerebral cortex. (G) Surface GluR2 coimmunoprecipitates with γ-3. Primary cerebrocortical cultures were incubated with a membrane impermeable cross-linker (CL), and after SDS solubilization extracts were immunoprecipitated for γ-3; GluR2 coprecipitates with γ-3 in a cross-linker–dependent fashion, whereas another synaptic transmembrane protein, N-cadherin, does not. H, GluR2 coimmunoprecipitated with γ-3 in chemical cross-linked PSD fractions from brain, whereas another PSD protein, CaMKII, does not.
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fig7: TARPs interact specifically with AMPA receptors in brain extracts. (A) AMPA receptor subunits GluR1, GluR2, and GluR4 coimmunoprecipitate with stargazin (STG) in brain extracts from +/stg mice (+/−). In extracts from stg/stg mice (−/−), the antistargazin antibody does not immunoprecipitate GluR1, GluR2, or GluR4. (B–D) AMPA receptor subunits also coimmunoprecipitate with γ-3, γ-4, and γ-8 in extracts from adult cerebral cortex, postnatal day 7 cerebral cortex, and hippocampus, respectively. As control, other transmembrane proteins such as GABAARβ, N-cadherin, and Kv1.4 did not coimmunoprecipitate. (E) Subunit specificity for TARP interactions in cerebellum. Stargazin (STG) and γ-4 coimmunoprecipitate with GluR1 and GluR4, but only stargazin coimmunoprecipitates with GluR2/3 in cerebellum. (F) TARP isoforms are strictly segregated, since they do not coimmunoprecipitate with one another in cerebral cortex. (G) Surface GluR2 coimmunoprecipitates with γ-3. Primary cerebrocortical cultures were incubated with a membrane impermeable cross-linker (CL), and after SDS solubilization extracts were immunoprecipitated for γ-3; GluR2 coprecipitates with γ-3 in a cross-linker–dependent fashion, whereas another synaptic transmembrane protein, N-cadherin, does not. H, GluR2 coimmunoprecipitated with γ-3 in chemical cross-linked PSD fractions from brain, whereas another PSD protein, CaMKII, does not.

Mentions: We next performed immunoprecipitation experiments to determine whether TARPs associate with AMPA receptors in neurons. Cerebellar membranes were solubilized with Triton X-100 and were immunoprecipitated with an antibody to stargazin. AMPA receptor subunits GluR1, GluR2, and GluR4 coimmunoprecipitated with stargazin in extracts from wild-type (unpublished data) or heterozygous mice but not from stargazer mice (Fig. 7 A). Immunoprecipitation studies indicated that AMPA receptors also associate with the other TARP isoforms in brain tissues expressing high TARP levels. Thus, AMPA receptors coprecipitate with γ-3 in cerebral cortex, with γ-8 in hippocampus and with γ-4 in neonatal forebrain (Fig. 7, B–D). As controls, we found that K+ channel Kv 1.4, GABAA receptor subunit-β and neuronal cadherin did not associate with TARPs.


Functional studies and distribution define a family of transmembrane AMPA receptor regulatory proteins.

Tomita S, Chen L, Kawasaki Y, Petralia RS, Wenthold RJ, Nicoll RA, Bredt DS - J. Cell Biol. (2003)

TARPs interact specifically with AMPA receptors in brain extracts. (A) AMPA receptor subunits GluR1, GluR2, and GluR4 coimmunoprecipitate with stargazin (STG) in brain extracts from +/stg mice (+/−). In extracts from stg/stg mice (−/−), the antistargazin antibody does not immunoprecipitate GluR1, GluR2, or GluR4. (B–D) AMPA receptor subunits also coimmunoprecipitate with γ-3, γ-4, and γ-8 in extracts from adult cerebral cortex, postnatal day 7 cerebral cortex, and hippocampus, respectively. As control, other transmembrane proteins such as GABAARβ, N-cadherin, and Kv1.4 did not coimmunoprecipitate. (E) Subunit specificity for TARP interactions in cerebellum. Stargazin (STG) and γ-4 coimmunoprecipitate with GluR1 and GluR4, but only stargazin coimmunoprecipitates with GluR2/3 in cerebellum. (F) TARP isoforms are strictly segregated, since they do not coimmunoprecipitate with one another in cerebral cortex. (G) Surface GluR2 coimmunoprecipitates with γ-3. Primary cerebrocortical cultures were incubated with a membrane impermeable cross-linker (CL), and after SDS solubilization extracts were immunoprecipitated for γ-3; GluR2 coprecipitates with γ-3 in a cross-linker–dependent fashion, whereas another synaptic transmembrane protein, N-cadherin, does not. H, GluR2 coimmunoprecipitated with γ-3 in chemical cross-linked PSD fractions from brain, whereas another PSD protein, CaMKII, does not.
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fig7: TARPs interact specifically with AMPA receptors in brain extracts. (A) AMPA receptor subunits GluR1, GluR2, and GluR4 coimmunoprecipitate with stargazin (STG) in brain extracts from +/stg mice (+/−). In extracts from stg/stg mice (−/−), the antistargazin antibody does not immunoprecipitate GluR1, GluR2, or GluR4. (B–D) AMPA receptor subunits also coimmunoprecipitate with γ-3, γ-4, and γ-8 in extracts from adult cerebral cortex, postnatal day 7 cerebral cortex, and hippocampus, respectively. As control, other transmembrane proteins such as GABAARβ, N-cadherin, and Kv1.4 did not coimmunoprecipitate. (E) Subunit specificity for TARP interactions in cerebellum. Stargazin (STG) and γ-4 coimmunoprecipitate with GluR1 and GluR4, but only stargazin coimmunoprecipitates with GluR2/3 in cerebellum. (F) TARP isoforms are strictly segregated, since they do not coimmunoprecipitate with one another in cerebral cortex. (G) Surface GluR2 coimmunoprecipitates with γ-3. Primary cerebrocortical cultures were incubated with a membrane impermeable cross-linker (CL), and after SDS solubilization extracts were immunoprecipitated for γ-3; GluR2 coprecipitates with γ-3 in a cross-linker–dependent fashion, whereas another synaptic transmembrane protein, N-cadherin, does not. H, GluR2 coimmunoprecipitated with γ-3 in chemical cross-linked PSD fractions from brain, whereas another PSD protein, CaMKII, does not.
Mentions: We next performed immunoprecipitation experiments to determine whether TARPs associate with AMPA receptors in neurons. Cerebellar membranes were solubilized with Triton X-100 and were immunoprecipitated with an antibody to stargazin. AMPA receptor subunits GluR1, GluR2, and GluR4 coimmunoprecipitated with stargazin in extracts from wild-type (unpublished data) or heterozygous mice but not from stargazer mice (Fig. 7 A). Immunoprecipitation studies indicated that AMPA receptors also associate with the other TARP isoforms in brain tissues expressing high TARP levels. Thus, AMPA receptors coprecipitate with γ-3 in cerebral cortex, with γ-8 in hippocampus and with γ-4 in neonatal forebrain (Fig. 7, B–D). As controls, we found that K+ channel Kv 1.4, GABAA receptor subunit-β and neuronal cadherin did not associate with TARPs.

Bottom Line: Functional expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in cerebellar granule cells requires stargazin, a member of a large family of four-pass transmembrane proteins.Here, we define a family of transmembrane AMPA receptor regulatory proteins (TARPs), which comprise stargazin, gamma-3, gamma-4, and gamma-8, but not related proteins, that mediate surface expression of AMPA receptors.These studies indicate a general role for TARPs in controlling synaptic AMPA receptors throughout the central nervous system.

View Article: PubMed Central - PubMed

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

ABSTRACT
Functional expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in cerebellar granule cells requires stargazin, a member of a large family of four-pass transmembrane proteins. Here, we define a family of transmembrane AMPA receptor regulatory proteins (TARPs), which comprise stargazin, gamma-3, gamma-4, and gamma-8, but not related proteins, that mediate surface expression of AMPA receptors. TARPs exhibit discrete and complementary patterns of expression in both neurons and glia in the developing and mature central nervous system. In brain regions that express multiple isoforms, such as cerebral cortex, TARP-AMPA receptor complexes are strictly segregated, suggesting distinct roles for TARP isoforms. TARPs interact with AMPA receptors at the postsynaptic density, and surface expression of mature AMPA receptors requires a TARP. These studies indicate a general role for TARPs in controlling synaptic AMPA receptors throughout the central nervous system.

Show MeSH
Related in: MedlinePlus