Abstract
Neurotransmitter transporters regulate synaptic transmitter levels and are themselves functionally regulated by a number of different signal transduction cascades. A common theme in transporter regulation is redistribution of transporter protein between intracellular stores and the plasma membrane. The triggers and mechanisms underlying this regulation are important in the control of extracellular transmitter concentrations and hence synaptic signaling. Previously, we demonstrated that the gamma-aminobutyric acid transporter GAT1 is regulated by direct tyrosine phosphorylation, resulting in an up-regulation of transporter expression on the plasma membrane. In the present report, we show that two tyrosine residues on GAT1 contribute to the phosphorylation and transporter redistribution. Tyrosine phosphorylation is concomitant with a decrease in the rate of transporter internalization from the plasma membrane. A decrease in GAT internalization rates also occurs in the presence of GAT1 substrates, suggesting the hypothesis that tyrosine phosphorylation is required for the substrate-induced up-regulation of GAT1 surface expression. In support of this hypothesis, incubation of GAT1-expressing cells with transporter ligands alters the amount of GAT1 tyrosine phosphorylation, and substrate-induced surface expression is unchanged in a GAT1 mutant lacking tyrosine phosphorylation sites. These data suggest a model in which substrates permit the phosphorylation of GAT1 on tyrosine residues and that the phosphorylated state of the transporter is refractory for internalization.
Highlights
␥-Aminobutyric acid (GABA)1 is the major inhibitory neurotransmitter in the central nervous system
We showed that GAT1, endogenously expressed in hippocampal neurons and heterologously expressed in Chinese hamster ovary (CHO) cells, was a substrate for tyrosine-mediated phosphorylation, and that a GAT1 mutant lacking all five putative intracellular tyrosine residues was refractory for tyrosine phosphorylation [14]
Wild-type GAT1 and constructs containing tyrosine residues only at amino acid positions 107 or 317 [16] showed immunoreactive bands of the appropriate size; constructs containing individual tyrosine residues at amino acid positions 412, 481, and 598 and the GAT1 mutant lacking all five putative intracellular tyrosine residues (5YA) did not show immunoreactive bands. These results suggest that two tyrosine residues in GAT1, Tyr-107 in the putative first intracellular loop and Tyr-317 in the putative third intracellular loop, can be phosphorylated in vivo
Summary
␥-Aminobutyric acid (GABA)1 is the major inhibitory neurotransmitter in the central nervous system. A decrease in GAT internalization rates occurs in the presence of GAT1 substrates, suggesting the hypothesis that tyrosine phosphorylation is required for the substrate-induced up-regulation of GAT1 surface expression.
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