ABSTRACT
Phosphorylation of S880 within the GluR2 C-terminus has been reported to promote endocytosis of AMPA receptors (AMPARs) by preventing GluR2 interaction with the putative synaptic anchoring proteins GRIP and ABP. It is not yet established however, whether S880 phosphorylation induces removal of AMPARs from synaptic sites, and the trafficking of phosphorylated GluR2 subunits with surface and endocytosed GluR2 has not been directly compared within the same intact neurons. Here we show that phosphorylation of GluR2 subunits by PKC activated with phorbol esters is compartmentally restricted to receptors located at the cell surface. Endogenous AMPARs containing S880-phosphorylated GluR2 remained highly synaptic and colocalized with postsynaptic markers to the same extent as AMPARs which did not contain S880-phosphorylated GluR2. Moreover, following S880 phosphorylation, exogenous GluR2 homomers were found specifically at the cell surface and did not co-traffic with the internalized endosomal GluR2 population. We also show that GluR2 is endogenously phosphorylated by a constitutively active kinase pharmacologically related to PKC, and this phosphorylation is opposed by the protein phosphatase PP1. Our results demonstrate a population of hippocampal AMPARs which do not require interaction with GRIP/ABP for synaptic anchorage.
Subject(s)
Endocytosis/physiology , Hippocampus/cytology , Neurons/metabolism , Receptors, AMPA/genetics , Receptors, AMPA/metabolism , Synapses/metabolism , Animals , COS Cells , Chlorocebus aethiops , Dendrites/physiology , Mutagenesis , Nerve Tissue Proteins/metabolism , Neurons/cytology , Phosphorylation , Protein Kinase C/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , SAP90-PSD95 Associated Proteins , Serine/metabolism , Sindbis Virus , TransfectionABSTRACT
We have characterized a rodent-specific gene family designated alpha-takusan (meaning "many" in Japanese). We initially identified a member of the family whose expression is upregulated in mice lacking the NMDAR subunit NR3A. We then isolated cDNAs encoding 46 alpha-takusan variants from mouse brains. Most variants share an approximately 130 aa long sequence, which contains the previously identified domain of unknown function 622 (DUF622) and is predicted to form coiled-coil structures. Single-cell PCR analyses indicate that one neuron can express multiple alpha-takusan variants and particular variants may predominate in certain cell types. Forced expression in cultured hippocampal neurons of two variants, alpha1 or alpha2, which bind either directly or indirectly to PSD-95, leads to an increase in PSD-95 clustering, dendritic spine density, GluR1 surface expression, and AMPAR activity. Conversely, treating cultured neurons with RNAi targeting alpha-takusan variants resulted in the opposite phenotype. Hence, alpha-takusan represents a large gene family that regulates synaptic activity.
Subject(s)
Multigene Family/genetics , Synapses/physiology , Amino Acid Sequence , Animals , Brain Chemistry/physiology , COS Cells , Cells, Cultured , Chlorocebus aethiops , Dendrites/drug effects , Dendrites/metabolism , Disks Large Homolog 4 Protein , Electrophysiology , Green Fluorescent Proteins/metabolism , Guanylate Kinases , Hippocampus/cytology , Hippocampus/drug effects , Hippocampus/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Mice , Mice, Knockout , Molecular Sequence Data , Neurons/metabolism , Patch-Clamp Techniques , RNA, Messenger/biosynthesis , RNA, Small Interfering/genetics , Receptors, AMPA/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Receptors, N-Methyl-D-Aspartate/physiology , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Up-Regulation/physiologyABSTRACT
Long-term changes in excitatory synapse strength are thought to reflect changes in synaptic abundance of AMPA receptors mediated by receptor trafficking. AMPA receptor-binding protein (ABP) and glutamate receptor-interacting protein (GRIP) are two similar PDZ (postsynaptic density 95/Discs large/zona occludens 1) proteins that interact with glutamate receptors 2 and 3 (GluR2 and GluR3) subunits. Both proteins have proposed roles during long-term potentiation and long-term depression in the delivery and anchorage of AMPA receptors at synapses. Here we report a variant of ABP-L (seven PDZ form of ABP) called pABP-L that is palmitoylated at a cysteine residue at position 11 within a novel 18 amino acid N-terminal leader sequence encoded through differential splicing. In cultured hippocampal neurons, nonpalmitoylated ABP-L localizes with internal GluR2 pools expressed from a Sindbis virus vector, whereas pABP-L is membrane targeted and associates with surface-localized GluR2 receptors at the plasma membrane in spines. Mutation of Cys-11 to alanine blocks the palmitoylation of pABP-L and targets the protein to intracellular clusters, confirming that targeting the protein to spines is dependent on palmitoylation. Non-palmitoylated GRIP is primarily intracellular, but a chimera with the pABP-L N-terminal palmitoylation sequence linked to the body of the GRIP protein is targeted to spines. We suggest that pABP-L and ABP-L provide, respectively, synaptic and intracellular sites for the anchorage of AMPA receptors during receptor trafficking to and from the synapse.
