Direct association of the unique C-terminal tail of transmembrane AMPA receptor regulatory protein γ-8 with calcineurin.

Transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor regulatory proteins are auxiliary subunits that regulate AMPA receptor trafficking to the plasma membrane and localization to postsynaptic sites. The classical family consists of four members: stargazin/γ-2, γ-3, γ-4 and γ-8. The Tduanyu37 γ-8 isoform, which is highly expressed in the hippocampus, has a unique, long C-terminal domain with five distinct regions: two glycine-rich regions, a serine/arginine-rich region, a proline/alanine (P/A) rich region, and a PSD-95/Dlg/ZO-1 (PDZ) binding motif. We performed mass spectrometry and immunoprecipitation assays to identify specific binding partners for the γ-8 C-terminal tail and found that γ-8, but not stargazin/γ-2, co-immunoprecipitated with calcineurin/PP2B, a Ca(2+) /calmodulin-dependent Ser/Thr phosphatase. Co-immunoprecipitation and immunoblot analyses of lysates from COS-7 cells co-transfected with calcineurin and either wild type or chimeric γ-8 revealed that a section of the C-terminal tail (residues 356-421) can bind calcineurin. Futhermore, γ-8 lacking the P/A-rich region (residues 383-399) did not bind to calcineurin. In addition, the GST-γ-8 C-terminal tail (residues 353-414) fusion protein containing the P/A-rich region bound to purified calcineurin in a Ca(2+) /calmodulin-dependent manner, whereas GST-γ-8 with a deletion of the P/A-rich region did not. Peptide competition assays demonstrated that γ-8 may interact with the hydrophobic pocket defined by β-sheet 14 and/or adjacent regions of the catalytic A subunit of calcineurin. These results indicate that the γ-8 P/A-rich region is essential for binding calcineurin, suggesting that the γ-8/calcineurin complex may regulate AMPA receptor phosphorylation and trafficking.

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