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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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Stargazin enhances mature glycosylation and surface expression of an AMPA receptor subunit protein. (A) Cerebellar (Cb) cultures from +/stg(+/−) or stg/stg(−/−) were biotinylated, solubilized, and precipitated with streptavidin-agarose. Surface expression of the AMPA receptor subunit GluR2 is dramatically decreased in stg/stg cultures, whereas NMDAR subunits NR2A are not significantly altered. (B) In cerebellum of stargazer mouse (−/−), a large fraction of GluR2 remains immature and sensitive to EndoH glycosidase. By contrast, GluR2 in stargazer cerebral cortex (Cx) is mature and resistant to EndoH. In all cases, GluR2 glycosylation is removed by the nonspecific N-glycosidase, PNGaseF. Total levels of GluR2 were consistently decreased by ∼10–20% in cerebellum from stg/stg mice. Note that two independent examples from stargazer cerebellum are presented.
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fig8: Stargazin enhances mature glycosylation and surface expression of an AMPA receptor subunit protein. (A) Cerebellar (Cb) cultures from +/stg(+/−) or stg/stg(−/−) were biotinylated, solubilized, and precipitated with streptavidin-agarose. Surface expression of the AMPA receptor subunit GluR2 is dramatically decreased in stg/stg cultures, whereas NMDAR subunits NR2A are not significantly altered. (B) In cerebellum of stargazer mouse (−/−), a large fraction of GluR2 remains immature and sensitive to EndoH glycosidase. By contrast, GluR2 in stargazer cerebral cortex (Cx) is mature and resistant to EndoH. In all cases, GluR2 glycosylation is removed by the nonspecific N-glycosidase, PNGaseF. Total levels of GluR2 were consistently decreased by ∼10–20% in cerebellum from stg/stg mice. Note that two independent examples from stargazer cerebellum are presented.

Mentions: To determine directly whether stargazin is essential for AMPA receptor trafficking to the plasma membrane, we quantified the surface expression of glutamate receptor subunits in stargazer cerebellar granule cells, which lack expression of any TARP isoform. Cells were treated with a membrane-impermeable biotin reagent, and surface receptors were captured on avidin-linked agarose. These experiments revealed an ∼75% decrease in the surface expression of AMPA receptor subunit GluR2 in cerebellar cells from stargazer (Fig. 8 A), whereas total GluR2 levels were decreased by only 10–20% in stargazer cerebellum. As these cultures are a mixture of neurons, the remaining surface GluR2 in stargazer may represent receptors in Purkinje cells, which are enriched in GluR2 (Hampson et al., 1992; Lambolez et al., 1992) and are unaffected in stargazer mice (Chen et al., 1999; Hashimoto et al., 1999). This decrease in surface expression is selective for AMPA receptors, since NMDAR2A was unaffected (Fig. 8 A).


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)

Stargazin enhances mature glycosylation and surface expression of an AMPA receptor subunit protein. (A) Cerebellar (Cb) cultures from +/stg(+/−) or stg/stg(−/−) were biotinylated, solubilized, and precipitated with streptavidin-agarose. Surface expression of the AMPA receptor subunit GluR2 is dramatically decreased in stg/stg cultures, whereas NMDAR subunits NR2A are not significantly altered. (B) In cerebellum of stargazer mouse (−/−), a large fraction of GluR2 remains immature and sensitive to EndoH glycosidase. By contrast, GluR2 in stargazer cerebral cortex (Cx) is mature and resistant to EndoH. In all cases, GluR2 glycosylation is removed by the nonspecific N-glycosidase, PNGaseF. Total levels of GluR2 were consistently decreased by ∼10–20% in cerebellum from stg/stg mice. Note that two independent examples from stargazer cerebellum are presented.
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Related In: Results  -  Collection

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fig8: Stargazin enhances mature glycosylation and surface expression of an AMPA receptor subunit protein. (A) Cerebellar (Cb) cultures from +/stg(+/−) or stg/stg(−/−) were biotinylated, solubilized, and precipitated with streptavidin-agarose. Surface expression of the AMPA receptor subunit GluR2 is dramatically decreased in stg/stg cultures, whereas NMDAR subunits NR2A are not significantly altered. (B) In cerebellum of stargazer mouse (−/−), a large fraction of GluR2 remains immature and sensitive to EndoH glycosidase. By contrast, GluR2 in stargazer cerebral cortex (Cx) is mature and resistant to EndoH. In all cases, GluR2 glycosylation is removed by the nonspecific N-glycosidase, PNGaseF. Total levels of GluR2 were consistently decreased by ∼10–20% in cerebellum from stg/stg mice. Note that two independent examples from stargazer cerebellum are presented.
Mentions: To determine directly whether stargazin is essential for AMPA receptor trafficking to the plasma membrane, we quantified the surface expression of glutamate receptor subunits in stargazer cerebellar granule cells, which lack expression of any TARP isoform. Cells were treated with a membrane-impermeable biotin reagent, and surface receptors were captured on avidin-linked agarose. These experiments revealed an ∼75% decrease in the surface expression of AMPA receptor subunit GluR2 in cerebellar cells from stargazer (Fig. 8 A), whereas total GluR2 levels were decreased by only 10–20% in stargazer cerebellum. As these cultures are a mixture of neurons, the remaining surface GluR2 in stargazer may represent receptors in Purkinje cells, which are enriched in GluR2 (Hampson et al., 1992; Lambolez et al., 1992) and are unaffected in stargazer mice (Chen et al., 1999; Hashimoto et al., 1999). This decrease in surface expression is selective for AMPA receptors, since NMDAR2A was unaffected (Fig. 8 A).

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