Direct interaction of post‐synaptic density‐95/Dlg/ZO‐1 domain‐containing synaptic molecule Shank3 with GluR1 α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid receptor

Abstract

A class of scaffolding protein containing the post-synaptic density-95/Dlg/ZO-1 (PDZ) domain is thought to be involved in synaptic trafficking of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors during development. To clarify the molecular mechanism of AMPA receptor trafficking, we performed a yeast two-hybrid screening system using the cytoplasmic tail of the GluR1 subunit of AMPA receptor as a bait and identified a synaptic molecule, Shank3/ProSAP2, as a GluR1 subunit-interacting molecule. Shank3 is a PDZ domain-containing multidomain protein and is predominantly expressed in developing neurons. Using the glutathione S-transferase pull-down assay and immunoprecipitation technique we demonstrated that the GluR1 subunit directly binds to the PDZ domain of Shank3 via its carboxyl terminal PDZ-binding motif. We raised anti-Shank3 antibody to investigate the expression of Shank3 in cortical neurons. The pattern of Shank3 immunoreactivity was strikingly punctate, mainly observed in the spines, and closely matched the pattern of post-synaptic density-95 immunoreactivity, indicating that Shank3 is colocalized with post-synaptic density-95 in the same spines. When Shank3 and the GluR1 subunit were overexpressed in primary cortical neurons, they were also colocalized in the spines. Taken together with the biochemical interaction of Shank3 with the GluR1 subunit, these results suggest that Shank3 is an important molecule that interacts with GluR1 AMPA receptor at synaptic sites of developing neurons. Keywords: a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor, development, GluR1 subunit, post-synaptic density-95/ Dlg/ ZO-1 domain, Shank3, synapse. Transmission at excitatory synapses is primarily mediated by glutamate acting on three classes of ligand-gated ion channels, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate and NMDA receptors (Wisden and Seeburg 1993;Hollmann and Heinemann 1994). In addition to their role in synaptic transmission, these glutamate receptors (GluRs) have been thought to play a crucial role in many brain functions, including activity-dependent synaptogenesis during development and synaptic plasticity (McDonald and Johnston 1990;Bliss and Collingridge 1993).Many excitatory synapses in young developing neurons have been found to express only NMDA receptors, which are continuously blocked by magnesium at resting membrane potentials. As no evoked transmission is observed even when glutamate is present, these synapses are referred to as 'silent synapses'. During later development, AMPA receptors are delivered and clustered on the synaptic membrane in an activity-dependent manner, and the synapses subsequently become functionally active (Durand et al. 1996;Wu et al. 1996;Pickard et al. 2000;Liao et al. 2001;Isaac 2003). Thus, the clustering of AMPA receptors on the synaptic membrane is an essential event during synaptogenesis. Address correspondence and reprint requests to S. Kohsaka, Department of Neurochemistry, National Institute of Neu...

Loss of Predominant Shank3 Isoforms Results in Hippocampus-Dependent Impairments in Behavior and Synaptic Transmission

“…The Morris water maze task was conducted essentially as described previously (Powell et al, 2004;Tabuchi et al, 2007). Briefly, a white, circular pool 1.2 m in diameter was filled with water (22 Ϯ 1 C) made opaque with nontoxic, "gothic white" liquid tempera paint, and a circular platform (10 cm in diameter) was submerged ϳ1 cm beneath the surface of the water.…”

“…Because long-term plasticity is dependent on proper function of glutamatergic AMPARs and NMDARs and because Shank3 is known to interact indirectly with both types of receptors (Naisbitt et al, 1999;Uchino et al, 2006;Arons et al, 2012), we hypothesized that the relative contributions of AMPARs and NMDARs to the EPSC would be altered in Shank3 ⌬C/⌬C mice. As predicted, there was a significant decrease in the NMDA/AMPA ratio in Shank3 ⌬C/⌬C compared with WT mice ( Fig.…”

“…Shank3 is a member of the Shank family of postsynaptic scaffolding proteins enriched in postsynaptic densities (PSDs) and was discovered in yeast two-hybrid screens as a binding partner of guanylate kinase-associated protein (GKAP) and postsynaptic density protein 95 (PSD-95;Naisbitt et al, 1999). Shank3 binds to the integral machinery of PSDs through its several functional domains.…”

“…The ankyrin repeat domain of Shank3 mainly interacts with cytoskeletal proteins (Böckers et al, 2001). Its PSD protein/Drosophila disc large tumor suppressor/zonula occludens-1 protein (PDZ) domain interacts with ionotropic glutamate receptors either directly or indirectly via GKAP and PSD-95 (Garner et al, 2000;Uchino et al, 2006). The Homer binding domain of Shank3 binds to Homer, which then binds to the group 1 metabotropic glutamate receptors, such as mGluR1/5 .…”

Phelan–McDermid Syndrome and SHANK3: Implications for Treatment

“…SH3 and multiple ankyrin repeat domains 3 (SHANK3/ Shank3) protein is expressed at low levels in all brain regions during early postnatal development; peak expression correlates with a significant increase in synaptogenesis and synaptic maturation [15][16][17]. Shank3 mRNA is localized to proximal and distal dendrites, and is highly expressed the hippocampus, cerebellar granular cells, caudate putamen, and thalamic nuclei [18].…”