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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 colocalize with AMPA receptors at excitatory synapses. (A) Western blotting shows that AMPA receptor trafficking proteins are all highly enriched in PSD fractions. By contrast, synaptophysin, which is also enriched in the crude synaptosomes is extracted with 0.5% Triton X-100 and does not occur in PSD fractions. (B) Pre-embedding DAB immunoperoxidase labeling for pan-TARP in the CA1 stratum radiatum of the hippocampus. Labeling is concentrated in dendritic shafts and associated postsynaptic spines. Note the dense concentration of labeling at postsynaptic densities (arrowheads). Patches of labeling also are evident in the spine and dendrite cytoplasm. (C–L) Double immunogold labeling for pan-TARP (5 nm gold) and GluR2/3 (10 nm gold) in the hippocampus. (C, D, and H) CA1 stratum radiatum. (F and G) Hilus. (E and I) CA3 stratum lucidum. (J) Molecular layer of the dentate gyrus. (K) CA1 stratum oriens. Black arrowheads indicate 5-nm gold particles that is associated closely with 10 nm gold particles. In C and I, one of the 5-nm gold particles overlaps a 10-nm gold particle. Close associations of 5- and 10-nm gold particles are more common at synapses (C–H) than in dendrite cytoplasm (I–K). H shows a very oblique synapse; 5 and 10-nm gold particles are associated in a presumptive vesicle fused to the lateral wall of the postsynaptic spine. Bars: (B) 500 nm; (in G, for C–K) 200 nm. (L) Distribution of gold particles (y axis) in the perpendicular (axodendritic) axis of synapses. Zero represents the postsynaptic membrane; + is toward presynaptic and – is toward postsynaptic. Note how most gold for TARPs and GluR2/3 is concentrated at the postsynaptic membrane and within the postsynaptic density.
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fig6: TARPs colocalize with AMPA receptors at excitatory synapses. (A) Western blotting shows that AMPA receptor trafficking proteins are all highly enriched in PSD fractions. By contrast, synaptophysin, which is also enriched in the crude synaptosomes is extracted with 0.5% Triton X-100 and does not occur in PSD fractions. (B) Pre-embedding DAB immunoperoxidase labeling for pan-TARP in the CA1 stratum radiatum of the hippocampus. Labeling is concentrated in dendritic shafts and associated postsynaptic spines. Note the dense concentration of labeling at postsynaptic densities (arrowheads). Patches of labeling also are evident in the spine and dendrite cytoplasm. (C–L) Double immunogold labeling for pan-TARP (5 nm gold) and GluR2/3 (10 nm gold) in the hippocampus. (C, D, and H) CA1 stratum radiatum. (F and G) Hilus. (E and I) CA3 stratum lucidum. (J) Molecular layer of the dentate gyrus. (K) CA1 stratum oriens. Black arrowheads indicate 5-nm gold particles that is associated closely with 10 nm gold particles. In C and I, one of the 5-nm gold particles overlaps a 10-nm gold particle. Close associations of 5- and 10-nm gold particles are more common at synapses (C–H) than in dendrite cytoplasm (I–K). H shows a very oblique synapse; 5 and 10-nm gold particles are associated in a presumptive vesicle fused to the lateral wall of the postsynaptic spine. Bars: (B) 500 nm; (in G, for C–K) 200 nm. (L) Distribution of gold particles (y axis) in the perpendicular (axodendritic) axis of synapses. Zero represents the postsynaptic membrane; + is toward presynaptic and – is toward postsynaptic. Note how most gold for TARPs and GluR2/3 is concentrated at the postsynaptic membrane and within the postsynaptic density.

Mentions: Subcellular fractionation studies showed that all four TARPs are enriched in synaptosomes and are resistant to extraction with 0.5% Triton X-100, suggesting association with the PSD (Fig. 6 A). To determine decisively whether TARPs localize to the PSD, we used immuno-EM labeling in adult hippocampus (Fig. 6). Preembedding immunocytochemistry using a pan-TARP antibody and 3′,3-diaminobenzidine tetrahydrochloride (DAB) produced dense labeling in the hippocampus (control sections were unlabeled). At the EM level, the neuropil showed dense labeling restricted almost entirely to the dendrites and postsynaptic spines (Fig. 6 B). Densest labeling was seen in some patches in the dendrites and in the postsynaptic densities. We used immunogold electron microscopic studies to assess possible colocalization of TARPs with AMPA receptors at the ultrastructural level (Fig. 6, C–L). Double labeling of hippocampal sections with antibodies to GluR2/3 (large particles) and a pan-TARP antibody (small particles) showed that both are concentrated on the postsynaptic side of excitatory synapses (Fig. 6, C–H and L). In addition, close association of gold particles corresponding to GluR2/3 and TARP-like proteins were also found, though less frequently, in dendritic cytoplasm (Fig. 6, I–K).


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 colocalize with AMPA receptors at excitatory synapses. (A) Western blotting shows that AMPA receptor trafficking proteins are all highly enriched in PSD fractions. By contrast, synaptophysin, which is also enriched in the crude synaptosomes is extracted with 0.5% Triton X-100 and does not occur in PSD fractions. (B) Pre-embedding DAB immunoperoxidase labeling for pan-TARP in the CA1 stratum radiatum of the hippocampus. Labeling is concentrated in dendritic shafts and associated postsynaptic spines. Note the dense concentration of labeling at postsynaptic densities (arrowheads). Patches of labeling also are evident in the spine and dendrite cytoplasm. (C–L) Double immunogold labeling for pan-TARP (5 nm gold) and GluR2/3 (10 nm gold) in the hippocampus. (C, D, and H) CA1 stratum radiatum. (F and G) Hilus. (E and I) CA3 stratum lucidum. (J) Molecular layer of the dentate gyrus. (K) CA1 stratum oriens. Black arrowheads indicate 5-nm gold particles that is associated closely with 10 nm gold particles. In C and I, one of the 5-nm gold particles overlaps a 10-nm gold particle. Close associations of 5- and 10-nm gold particles are more common at synapses (C–H) than in dendrite cytoplasm (I–K). H shows a very oblique synapse; 5 and 10-nm gold particles are associated in a presumptive vesicle fused to the lateral wall of the postsynaptic spine. Bars: (B) 500 nm; (in G, for C–K) 200 nm. (L) Distribution of gold particles (y axis) in the perpendicular (axodendritic) axis of synapses. Zero represents the postsynaptic membrane; + is toward presynaptic and – is toward postsynaptic. Note how most gold for TARPs and GluR2/3 is concentrated at the postsynaptic membrane and within the postsynaptic density.
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Related In: Results  -  Collection

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fig6: TARPs colocalize with AMPA receptors at excitatory synapses. (A) Western blotting shows that AMPA receptor trafficking proteins are all highly enriched in PSD fractions. By contrast, synaptophysin, which is also enriched in the crude synaptosomes is extracted with 0.5% Triton X-100 and does not occur in PSD fractions. (B) Pre-embedding DAB immunoperoxidase labeling for pan-TARP in the CA1 stratum radiatum of the hippocampus. Labeling is concentrated in dendritic shafts and associated postsynaptic spines. Note the dense concentration of labeling at postsynaptic densities (arrowheads). Patches of labeling also are evident in the spine and dendrite cytoplasm. (C–L) Double immunogold labeling for pan-TARP (5 nm gold) and GluR2/3 (10 nm gold) in the hippocampus. (C, D, and H) CA1 stratum radiatum. (F and G) Hilus. (E and I) CA3 stratum lucidum. (J) Molecular layer of the dentate gyrus. (K) CA1 stratum oriens. Black arrowheads indicate 5-nm gold particles that is associated closely with 10 nm gold particles. In C and I, one of the 5-nm gold particles overlaps a 10-nm gold particle. Close associations of 5- and 10-nm gold particles are more common at synapses (C–H) than in dendrite cytoplasm (I–K). H shows a very oblique synapse; 5 and 10-nm gold particles are associated in a presumptive vesicle fused to the lateral wall of the postsynaptic spine. Bars: (B) 500 nm; (in G, for C–K) 200 nm. (L) Distribution of gold particles (y axis) in the perpendicular (axodendritic) axis of synapses. Zero represents the postsynaptic membrane; + is toward presynaptic and – is toward postsynaptic. Note how most gold for TARPs and GluR2/3 is concentrated at the postsynaptic membrane and within the postsynaptic density.
Mentions: Subcellular fractionation studies showed that all four TARPs are enriched in synaptosomes and are resistant to extraction with 0.5% Triton X-100, suggesting association with the PSD (Fig. 6 A). To determine decisively whether TARPs localize to the PSD, we used immuno-EM labeling in adult hippocampus (Fig. 6). Preembedding immunocytochemistry using a pan-TARP antibody and 3′,3-diaminobenzidine tetrahydrochloride (DAB) produced dense labeling in the hippocampus (control sections were unlabeled). At the EM level, the neuropil showed dense labeling restricted almost entirely to the dendrites and postsynaptic spines (Fig. 6 B). Densest labeling was seen in some patches in the dendrites and in the postsynaptic densities. We used immunogold electron microscopic studies to assess possible colocalization of TARPs with AMPA receptors at the ultrastructural level (Fig. 6, C–L). Double labeling of hippocampal sections with antibodies to GluR2/3 (large particles) and a pan-TARP antibody (small particles) showed that both are concentrated on the postsynaptic side of excitatory synapses (Fig. 6, C–H and L). In addition, close association of gold particles corresponding to GluR2/3 and TARP-like proteins were also found, though less frequently, in dendritic cytoplasm (Fig. 6, I–K).

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