Abstract
The 14-3-3 family of proteins is widely distributed in the CNS where they are major regulators of essential neuronal functions. There are seven known mammalian 14-3-3 isoforms (ζ,, τ, ϵ, η, β, and σ), which generally function as adaptor proteins. Previously, we have demonstrated that 14-3-3ϵ isoform dynamically regulates forward trafficking of GluN2C-containing NMDA receptors (NMDARs) in cerebellar granule neurons, that when expressed on the surface, promotes neuronal survival following NMDA-induced excitotoxicity. Here, we report 14-3-3 isoform-specific binding and functional regulation of GluN2C. In particular, we show that GluN2C C-terminal domain (CTD) binds to all 14-3-3 isoforms except 14-3-3σ, and binding is dependent on GluN2C serine 1096 phosphorylation. Co-expression of 14-3-3 (ζ and ϵ) and GluN1/GluN2C promotes the forward delivery of receptors to the cell surface. We further identify novel residues serine 145, tyrosine 178, and cysteine 189 on α-helices 6, 7, and 8, respectively, within ζ-isoform as part of the GluN2C binding motif and independent of the canonical peptide binding groove. Mutation of these conserved residues abolishes GluN2C binding and has no functional effect on GluN2C trafficking. Reciprocal mutation of alanine 145, histidine 180, and isoleucine 191 on 14-3-3σ isoform promotes GluN2C binding and surface expression. Moreover, inhibiting endogenous 14-3-3 using a high-affinity peptide inhibitor, difopein, greatly diminishes GluN2C surface expression. Together, these findings highlight the isoform-specific structural and functional differences within the 14-3-3 family of proteins, which determine GluN2C binding and its essential role in targeting the receptor to the cell surface to facilitate glutamatergic neurotransmission.
Highlights
NMDA receptor trafficking to synapses is essential for excitatory neurotransmission
The last 175 amino acids within the C terminus of GluN2A, GluN2B, or GluN2C were fused to LexA DNA binding domain and co-transformed with the different 14-3-3 isoforms fused to Gal4 activation domain into yeast (Fig. 1A)
We observed no specific binding when the C terminus of GluN1, GluN2A, or GluN2B was co-expressed with each 14-3-3 isoform, demonstrating that the interaction between 14-3-3 and NMDA receptors is specific to GluN2C
Summary
NMDA receptor trafficking to synapses is essential for excitatory neurotransmission. Results: We have identified novel residues within 14-3-3 that determine isoform-specific interaction with GluN2C. We have demonstrated that 14-3-3⑀ isoform dynamically regulates forward trafficking of GluN2C-containing NMDA receptors (NMDARs) in cerebellar granule neurons, that when expressed on the surface, promotes neuronal survival following NMDA-induced excitotoxicity. Inhibiting endogenous 14-3-3 using a high-affinity peptide inhibitor, difopein, greatly diminishes GluN2C surface expression Together, these findings highlight the isoform-specific structural and functional differences within the 14-3-3 family of proteins, which determine GluN2C binding and its essential role in targeting the receptor to the cell surface to facilitate glutamatergic neurotransmission. While GluN1 is widely distributed, each GluN2 subunit displays unique spatiotemporal expression patterns throughout the brain and receptors assembled with different GluN2 subunits exhibit distinct pharmacological and functional properties. Our findings reveal a novel regulatory region in 14-3-3 that modulates isoform-specific interaction with GluN2C
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