Functional regulation of gamma-aminobutyric acid transporters by direct tyrosine phosphorylation

Tyrosine phosphorylation regulates multiple cell signaling pathways and fun ctionally modulates a number of ion channels and receptors, Neurotransmitte r transporters, which act to clear transmitter from the synaptic cleft, are regulated by multiple second messenger pathways that exert their effects, at least in part, by causing a redistribution of the transporter protein to or from the cell surface. To test the hypothesis that tyrosine phosphoryla tion affects transporter function and to determine its mechanism of action, we examined the regulation of the rat brain gamma-aminobutyric acid (GABA) transporter GAT1 expressed endogenously in hippocampal neurons and express ed heterologously in Chinese hamster ovary cells. Inhibitors of tyrosine ki nases decreased GABA uptake; inhibitors of tyrosine phosphatases increased GABA uptake. The decrease in uptake seen with tyrosine kinase inhibitors wa s correlated with a decrease in tyrosine phosphorylation of GAT1 and result ed in a redistribution of the transporter from the cell surface to intracel lular locations. A mutant GAT1 construct that was refractory to tyrosine ph osphorylation could not be regulated by tyrosine kinase inhibitors. Activat ors of protein kinase C, which are known to cause a redistribution of GAT1 from the cell surface, were additive to the effects of tyrosine kinase inhi bitors suggesting that multiple signaling pathways control transporter redi stribution. Application of brain-derived neurotrophic factor, which activat es receptor tyrosine kinases, up-regulated GAT1 function suggesting one pot ential trigger for the cellular regulation of GAT1 signaling by tyrosine ph osphorylation, These data support the hypothesis that transporter expressio n and function is controlled by the interplay of multiple cell signaling ca scades.