Neto2 Interacts with the Scaffolding Protein GRIP and Regulates Synaptic Abundance of Kainate Receptors

Abstract

Kainate receptors (KARs) are a class of ionotropic glutamate receptors that are expressed throughout the central nervous system. The function and subcellular localization of KARs are tightly regulated by accessory proteins. We have previously identified the single-pass transmembrane proteins, Neto1 and Neto2, to be associated with native KARs. In the hippocampus, Neto1, but not Neto2, controls the abundance and modulates the kinetics of postsynaptic KARs. Here we evaluated whether Neto2 regulates synaptic KAR levels in the cerebellum where Neto1 expression is limited to the deep cerebellar nuclei. In the cerebellum, where Neto2 is present abundantly, we found a ∼40% decrease in GluK2-KARs at the postsynaptic density (PSD) of Neto2-null mice. No change, however, was observed in total level of GluK2-KARs, thereby suggesting a critical role of Neto2 on the synaptic localization of cerebellar KARs. The presence of a putative class II PDZ binding motif on Neto2 led us to also investigate whether it interacts with PDZ domain-containing proteins previously implicated in regulating synaptic abundance of KARs. We identified a PDZ-dependent interaction between Neto2 and the scaffolding protein GRIP. Furthermore, coexpression of Neto2 significantly increased the amount of GRIP associated with GluK2, suggesting that Neto2 may promote and/or stabilize GluK2:GRIP interactions. Our results demonstrate that Neto2, like Neto1, is an important auxiliary protein for modulating the synaptic levels of KARs. Moreover, we propose that the interactions of Neto1/2 with various scaffolding proteins is a critical mechanism by which KARs are stabilized at diverse synapses.

Neto2 is a KCC2 interacting protein required for neuronal Cl − regulation in hippocampal neurons

“…We elucidated the role of this association and characterized its importance in the normal neurophysiological function of KCC2. Because Neto proteins were previously identified as auxiliary subunits of ionotropic glutamate receptors, including kainate and NMDA receptors (17)(18)(19)(20)(21), this study further extends the role of the Neto proteins to inhibitory synapses.…”

“…Recent reports have demonstrated that Neto2 and its homologous protein Neto1 have important functions at excitatory synapses. Neto1 interacts with both the N-methyl D-aspartate receptor (NMDAR) and kainate receptor complexes (17,20,22), whereas Neto2 was shown to be an auxiliary subunit of the neuronal kainate receptor (18,(21)(22)(23). As the Neto proteins appear to have multiple functions in the nervous system, we chose to explore additional roles these proteins might have by conducting an unbiased screen to identify proteins that interact with the Neto proteins.…”

Distinct Subunit Domains Govern Synaptic Stability and Specificity of the Kainate Receptor

“…Several of the KAR subunits and auxiliary subunits have been implicated in the synaptic localization of KARs (Copits et al, 2011; Hirbec et al, 2003; Palacios-Filardo et al, 2014; Tang et al, 2012; Yan et al, 2004), but defining a specific domain involved in synaptic stabilization especially at anatomically defined synapses has not been possible. Generation of mice expressing a modified GluK2 subunit demonstrated that the carboxyl terminal cytoplasmic domain of GluK2 does not affect the surface (or total) expression of KARs and has no effect on the total current density of neuronal KARs.…”

Advances in the pharmacology of lGICs auxiliary subunits

“…kinetics. Both proteins also modulate synaptic targeting, but to a lesser extent than the GluK4 and GluK5 subunits, and also may interact with scaffolding proteins [120][121][122][123][124][125][126].…”