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, 19 (1), 96-108

Citron Binds to PSD-95 at Glutamatergic Synapses on Inhibitory Neurons in the Hippocampus

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Citron Binds to PSD-95 at Glutamatergic Synapses on Inhibitory Neurons in the Hippocampus

W Zhang et al. J Neurosci.

Abstract

Synaptic NMDA-type glutamate receptors are anchored to the second of three PDZ (PSD-95/Discs large/ZO-1) domains in the postsynaptic density (PSD) protein PSD-95. Here, we report that citron, a protein target for the activated form of the small GTP-binding protein Rho, preferentially binds the third PDZ domain of PSD-95. In GABAergic neurons from the hippocampus, citron forms a complex with PSD-95 and is concentrated at the postsynaptic side of glutamatergic synapses. Citron is expressed only at low levels in glutamatergic neurons in the hippocampus and is not detectable at synapses onto these neurons. In contrast to citron, p135 SynGAP, an abundant synaptic Ras GTPase-activating protein that can bind to all three PDZ domains of PSD-95, and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) are concentrated postsynaptically at glutamatergic synapses on glutamatergic neurons. CaM kinase II is not expressed and p135 SynGAP is expressed in less than half of hippocampal GABAergic neurons. Segregation of citron into inhibitory neurons does not occur in other brain regions. For example, citron is expressed at high levels in most thalamic neurons, which are primarily glutamatergic and contain CaM kinase II. In several other brain regions, citron is present in a subset of neurons that can be either GABAergic or glutamatergic and can sometimes express CaM kinase II. Thus, in the hippocampus, signal transduction complexes associated with postsynaptic NMDA receptors are different in glutamatergic and GABAergic neurons and are specialized in a way that is specific to the hippocampus.

Figures

Fig. 1.
Fig. 1.
Sequences related to citron. A, Domain structure of citron. Ring-H2, Ring-H2 finger domain; PH, pleckstrin homology domain;CMG, citron/MRCK/Gek domain; tSXV, terminal S/TXV motif predicted to bind PDZ domains. The structures of cDNAs reveal two apparent alternative splice sites where two 15 residue sequences can be inserted (indicated byV). B, Proteins related to citron. A family of proteins that are effectors for the Rho/Rac/Cdc42 GTPases have domain structures similar to citron (see Results).MRCK, Myotonic dystrophy kinase-related kinase;GEK, Genghis Khan; ROK-α, Rho-associated kinase; RHO1 GEF, Rho-1 guanine nucleotide exchange factor; NIK, NCK-interacting kinase. C, Alignment of sequences in putative CMG domain. Sequences range from 26 to 43% identical to citron.Black boxes indicate four or more identities to citron.Open boxes indicate other identities.
Fig. 2.
Fig. 2.
Enrichment of citron in PSD fractions. Immunoblots of PSDs prepared by extraction with the indicated detergents were prepared and labeled with anti-citron antibody as described in Materials and Methods. Lane 1, Rat forebrain homogenate (50 μg); lane 2, synaptosomes (50 μg); lane 3, synaptosomes (7.5 μg); lane 4, PSD fraction extracted once with 0.5% Triton X-100 (7.5 μg); lane 5, PSD fraction extracted twice with 0.5% Triton X-100 (7.5 μg); lane 6, PSD fraction extracted once with 0.5% Triton X-100, followed by 3% sarcosyl (7.5 μg). Molecular weight markers are shown on the right.
Fig. 3.
Fig. 3.
Yeast two-hybrid assay for interaction of citron and PSD-95. A, Segments of cDNA encoding the portions of the citron C terminus indicated in the figure were inserted into the yeast pAS2–1 vector and tested in the two-hybrid assay for interaction with PSD-95. Full-length cDNA encoding PSD-95 was inserted in the pACT2 vector. Interaction and no interaction is indicated by + and −, respectively. B, A fragment mini-library of PSD-95 constructed in the prey vector pGADGH (Kornau et al., 1995) was screened with the bait vector pAS2–1/1 described in A. Clones encoding interacting segments of PSD-95 were isolated and sequenced as described in Materials and Methods. Nine positive clones encoded PDZ3, and three positive clones encoded PDZ2 of PSD-95.
Fig. 4.
Fig. 4.
Coimmunoprecipitation of citron and PSD-95 from rat brain. Aliquots of rat brain PSD fraction (150 μg in 600 μl), prepared as described in Materials and Methods, were mixed with 8 μl of antiserum against citron, 8 μl of antiserum against NR2B, or 8 μl of preimmune serum. Immune complexes were purified on agarose beads coupled to Protein A-Sepharose and fractionated by SDS-PAGE on 7.5% gels. Proteins were transferred to nitrocellulose and blotted with the indicated antisera as described in Materials and Methods. super., The supernatant (1/30 vol) from immunoprecipitation by antibodies against citron. (The supernatant from immunoprecipitation by antibodies against NR2B appeared identical.) No citron was detected in the supernatant from immunoprecipitation with anti-citron antibody. Positions of molecular weight markers are indicated on the left.
Fig. 5.
Fig. 5.
Immunofluorescent labeling of citron, GAD, NR2B, and PSD-95 in cultures of dissociated hippocampal neurons. Cultures were grown and labeled with antibodies against the indicated proteins as described in Materials and Methods. A,B, A neuron double stained for GAD (A) and citron (B). Citron stains dendrites and somas (arrow) but not axons (double arrowhead) of >90% of neurons that are stained with anti-GAD antibody. GAD/citron-positive neurons comprise 7–10% of neurons in the cultures. C, D, A neuron double stained for NR2B (C) and citron (D). Punctate labeling by antibody against citron along dendrites in this neuron (arrows) colocalizes with labeling by antibody against NR2B. E, F, A neuron double stained for PSD-95 (E) and citron (F). Punctate labeling by antibody against citron along dendrites of this neuron (arrows) colocalizes with labeling by antibody against PSD-95. Some anti-PSD-95-labeled puncta do not contain citron (arrowhead). Many of these appear to arise from a neighboring citron-negative neuron. Scale bars, 10 μm.
Fig. 6.
Fig. 6.
Immunofluorescent labeling of citron, the α subunit of CaM kinase II, and p135 SynGAP in cultures of dissociated hippocampal neurons. A, B, A field of three neurons double stained for αCaM kinase II (A) and citron (B). Two neurons (arrowheads) stain positively for αCaM kinase II, and one (arrow) stains positively for citron. The two proteins are present in distinct sets of neurons. The population of neurons that stain for citron represents ∼7–10% of the total. C, D, A field of neurons double stained for SynGAP (C) and citron (D). One neuron (arrows) is labeled by both antisera. Several dendrites in the field (arrowheads) are labeled only by antibody against SynGAP. Less than 20% of the citron-positive neurons also contain SynGAP. Essentially all of the citron-negative neurons are labeled by SynGAP. E, F, A field of dendrites double stained for SynGAP (E) and citron (F). Dendrites that contain only SynGAP are labeled with arrowheads. Dendrites that contain only citron are labeled with arrows. Scale bars, 10 μm.
Fig. 7.
Fig. 7.
Specificity of antibodies against citron used for immunocytochemistry. A, Aliquots of homogenate of rat forebrain (100 μg) were fractionated by SDS-PAGE and blotted to nitrocellulose as described in Materials and Methods. Thelanes were probed with either ascites 1B against citron (1) or the same amount of ascites 1B preabsorbed with antigen (2) as described in Materials and Methods. B, C, Hippocampal pyramidal layer in area CA1 labeled with anti-citron antisera (B) and a neighboring section labeled with anti-citron antiserum preadsorbed with antigen (C) as described in Materials and Methods (40× lens objective). The sections were photographed at identical microscope settings. D, E, Thalamic neurons labeled with anti-citron antisera (D) and a neighboring section labeled with anti-citron antiserum preadsorbed with antigen (E) as described in Materials and Methods (40× lens objective). The sections were photographed at identical microscope settings. Scale bars, 25 μm.
Fig. 8.
Fig. 8.
Immunocytochemical colocalization of citron, GAD, and CaM kinase II in hippocampal inhibitory interneurons. Coronal sections were cut from a rat brain fixed by perfusion as described in Materials and Methods. A, B, Colocalization of citron and GAD. Area CA1 of the rat hippocampus at approximately bregma −3.3 (Paxinos and Watson, 1998) was double labeled with anti-citron (A) and anti-GAD (B) as described in Materials and Methods (10× lens objective). Arrows, Neurons labeled with both antisera; white arrowhead, neuron labeled with anti-citron and not anti-GAD; black arrowhead, neuron labeled with anti-GAD and not anti-citron. sm, Stratum moleculare; sr, stratum radiatum; sp, stratum pyramidale; so, stratum oriens.C, D, Colocalization of citron and GAD. Area CA1 of the hippocampus in sections at approximately bregma −3 to −3.5 (Paxinos and Watson, 1998) was double-labeled with antibodies against citron (C) and GAD (D) as described in Materials and Methods (40× lens objective).Arrows, Neurons labeled with both antibodies.E, F, Colocalization of citron and CaM kinase II. Area CA1 of the rat hippocampus in sections at approximately bregma −3 to −3.5 (Paxinos and Watson, 1998) was double labeled with anti-citron (E) and anti-CaM kinase II (F) as described in Materials and Methods (40× lens). Arrowheads, Neurons labeled with anti-citron antibodies and devoid of labeling with anti-CaM kinase II antibodies. Scale bars, 25 μm.
Fig. 9.
Fig. 9.
Immunocytochemical localization of citron, GAD, and CaM kinase II in thalamus and neocortex. Coronal sections were cut from a rat brain fixed by perfusion as described in Materials and Methods. Sections at approximately bregma −3.0 to −3.5 (Paxinos and Watson, 1998) were double stained with antibodies against the pairs of proteins listed below. A, B, The rat thalamus double labeled with anti-citron (A) and anti-GAD (B) as described in Materials and Methods (20× lens objective). Arrowheads, Neurons labeled only with anti-citron antibodies and axons labeled only with anti-GAD antibodies. Very few GAD-positive neurons were seen in the central nuclei of the thalamus. C, D, Thalamus double labeled with anti-citron (C) and anti-CaM kinase II (D) as described in Materials and Methods (20× lens objective). Arrows, Several neurons labeled with both antibodies. E, F, Sensory neocortex double labeled with anti-citron (E) and anti-GAD (F) as described in Materials and Methods (63× lens objective).Arrows, A Cell labeled with anti-citron and anti-GAD antibodies; arrowheads, a cell and dendrites labeled with anti-citron antibodies but not anti-GAD antibodies.G, H, Sensory neocortex double labeled with anti-citron (G) and anti-CaM kinase II (H) as described in Materials and Methods (40× lens objective). Arrows, Neurons labeled with both antibodies; arrowheads, neurons labeled with anti-citron antibodies and unlabeled with anti-CaM kinase II antibodies. Scale bars, 25 μm.
Fig. 10.
Fig. 10.
Cartoon representing the partitioning of citron and CaM kinase II among GABAergic and glutamatergic neurons in the brain. The schematic neuron does not represent any particular neuronal type; the shapes of citron-positive neurons vary widely in different brain regions.

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