Role of TARP interaction in S-SCAM-mediated regulation of AMPA receptors

Abstract

Scaffolding proteins are involved in the incorporation, anchoring, maintenance, and removal of AMPA receptors (AMPARs) at synapses, either through a direct interaction with AMPARs or via indirect association through auxiliary subunits of transmembrane AMPAR regulatory proteins (TARPs). Synaptic scaffolding molecule (S-SCAM) is a newly characterized member of the scaffolding proteins critical for the regulation and maintenance of AMPAR levels at synapses, and directly binds to TARPs through a PDZ interaction. However, the functional significance of S-SCAM-TARP interaction in the regulation of AMPARs has not been tested. Here we show that overexpression of the C-terminal peptide of TARP-γ2 fused to EGFP abolished the S-SCAM-mediated enhancement of surface GluA2 expression. Conversely, the deletion of the PDZ-5 domain of S-SCAM that binds TARPs greatly attenuated the S-SCAM-induced increase of surface GluA2 expression. In contrast, the deletion of the guanylate kinase domain of S-SCAM did not show a significant effect on the regulation of AMPARs. Together, these results suggest that S-SCAM is regulating AMPARs through TARPs.

Figure 1. Overexpression of Stargazin C-terminal peptides (GFP-StgC14) blocks the S-SCAM-induced increase of surface AMPA receptor levels in hippocampal neurons. (A) Sequence alignment of the last 14 amino acids of various TARPs involved in the PDZ interaction. (B) Representative images of surface GluA2 staining in dendritic segments of neurons transfected with GFP alone (control), GFP-StgC14 alone, GFP + myc-S-SCAM, or GFP-StgC14 + myc-S-SCAM. (C) Quantification of the effect of Stg-C14 and S-SCAM on surface GluA2. Scale bar represents 5 µm. ***p < 0.001; n.s, not significant; n = 30 per condition.

Figure 2. PDZ-5 domain of S-SCAM is required for the S-SCAM-mediated regulation of surface GluA2 levels in hippocampal neurons. (A) Representative images of surface GluA2 staining in neurons transfected with myc-S-SCAM (WT), myc-S-SCAM (ΔGK), or myc-S-SCAM (ΔPDZ-5). (B) Quantification of the effect of S-SCAM WT, ΔGK, and ΔPDZ5 mutant on surface GluA2 levels. ***p < 0.001, n = 30 per condition. Scale bar represents 5 µm.