Two phosphomimetic sites in the GABA transporter GAT1 C-terminus selectively regulate transporter interactions with distinct types of PDZ domains in vitro

Abstract

The GABA transporter GAT1 is a membrane protein that participates in GABA signalling in the brain and certain peripheral tissues. GAT1 contains a C-terminal PDZ motif (-A⁵⁹⁷Y⁵⁹⁸I⁵⁹⁹), previously classified as a type II (ΦXΦ) motif, where PDZ interaction should depend on non-phosphorylatable hydrophobic (Φ) residues at PDZ positions 0 (I⁵⁹⁹) and −2 (A⁵⁹⁷). We recently found that a GAT1 C-terminal peptide unconventionally binds to the PDZ1 domain of syntenin-1 and to the PDZ2 domain of PSD95 by using different PDZ interaction schemes, specifically 0,-1 and 0,-3, respectively. In this work, we used phosphomimetic amino acid mutagenesis to investigate the role of phosphorylatable amino acids in the GAT1 C-terminus in binding to these two domains. The mutagenesis and molecular docking experiments suggested that phosphorylation of tyrosine 598 at PDZ position −1 might affect the interaction with the PDZ1 domain of syntenin-1, but it did not affect the interaction with the PDZ2 domain of PSD95. On the other hand, a phosphomimetic but not a neutral mutation of the remote serine residues 592 and 594 at PDZ positions −7 and −5, respectively, affected the interaction of the GAT1 PDZ motif with the PDZ2 domain of PSD95, but not with the PDZ1 domain of syntenin-1. These findings suggest the existence of at least two domain-specific GAT1 PDZ interaction modes regulated by two distinct potentially phosphorylatable serine and tyrosine residues in the GAT1 C-terminus.