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 (TARPs) are auxiliary subunits that regulate AMPA receptor trafficking to the plasma membrane and localization to postsynaptic sites. The classical TARP family consists of four members: stargazin/γ-2, γ-3, γ-4 and γ-8. The TARP 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 ß-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.