Functional Regulation of γ-Aminobutyric Acid Transporters by Direct Tyrosine Phosphorylation

116

Here we tested an hypothesis that PKC-mediated phosphorylation of NET is required for transporter internalization. Phosphoamino acid analysis of 32 Plabeled native NETs from rat placental trophoblasts and heterologously expressed wild type human NET (WT-hNET) from human placental trophoblast cells revealed that the phorbol ester (␤-PMA)-induced phosphorylation of NET occurs on serine and threonine residues. ␤-PMA treatment inhibited NE transport, reduced plasma membrane hNET levels, and stimulated hNET phosphorylation in human placental trophoblast cells expressing the WT-hNET. Substance P-mediated activation of the G␣ q -coupled human neurokinin 1 (hNK-1) receptor coexpressed with the WT-hNET produced effects similar to ␤-PMA via PKC stimulation. In striking contrast, an hNET double mutant harboring T258A and S259A failed to show NE uptake inhibition and plasma membrane redistribution by ␤-PMA or SP. Most interestingly, the plasma membrane insertion of the WT-hNET and hNET double mutant were not affected by ␤-PMA. Although the WT-hNET showed increased endocytosis and redistribution from caveolin-rich plasma membrane domains following ␤-PMA treatment, the hNET double mutant was completely resistant to these PKC-mediated effects. In addition, the PKC-induced phosphorylation of hNET double mutant was significantly reduced. In the absence of T258A and S259A mutations, alanine substitution of all other potential phosphosites within the hNET did not block PKC-induced phosphorylation and down-regulation. These results suggest that Thr-258 and Ser-259 serve as a PKC-specific phospho-acceptor site and that phosphorylation of this motif is linked to PKC-induced NET internalization. The norepinephrine (NE)2 transporter (NET) regulates noradrenergic signaling by mediating the clearance of NE and is an important target for antidepressants and psychostimulants (2-5). NE signaling is linked to behavioral arousal (6) and is affected in stress-related paradigms linked to depression (7,8). NE acutely inhibits nociceptive transmission, including that mediated by SP (NK1), potentiates opioid analgesia, and underlies part of antinociceptive effects of tricyclic antidepressants (9). Various biologic stimuli regulate NE signaling, and alterations in NE signaling, including NE clearance and NET density, are observed in cardiovascular diseases and brain disorders (10 -13). Recent studies provided evidence for protein kinase C (PKC)-mediated regulation of NET function attributed to alterations in NET surface redistribution (1, 14, 15). Signals mediated through G-protein-coupled receptors are a likely trigger for PKC-mediated regulation of NET, and such receptors are abundant on neuronal and non-neuronal cells.Human placenta expresses both SERT and NET (16 -18), and we have developed trophoblast cultures from the rat placenta that robustly express endogenous NET (19). Placenta also expresses several receptors, including receptors for peptides such as insulin, SP, and NKB, neurotransmitters,. Our recent study using rat placental trophobla...

Section: Discussionmentioning

confidence: 99%

Phosphorylation of the Norepinephrine Transporter at Threonine 258 and Serine 259 Is Linked to Protein Kinase C-mediated Transporter Internalization

Jayanthi

Annamalai

Samuvel

et al. 2006

116

“…To understand the extent of this influence, it is essential to understand the mechanisms underlying subcellular transporter trafficking. We and others have previously isolated transporter-containing synaptic-like vesicles that are potential mediators of transporter trafficking in axon terminals (10), and we have identified many signaling molecules that regulate subcellular transporter redistribution (12,14,15,22,24,28). In this report, by using a modified biotinylation assay to study GAT1 trafficking, (i) we defined an acutely recycling pool of GAT1 in cortical neurons that, in the basal state, comprises approximately one-third of total cellular GAT1; (ii) we measured the exocytosis rate (r exo ϭ 0.7 min Ϫ1 ) and the endocytosis rate (r endo ϭ 1.1 min Ϫ1 ) of acutely recycling GAT1 in the basal state; and (iii) we demonstrated that distinct transporter redistribution signals exert their effects by differentially regulating the recycling pool size or selectively uncoupling rates of exocytosis and endocytosis.…”

Section: Discussionmentioning

confidence: 99%

“…Multiple redistribution modulators have been identified, including second messengers (14 -18), transporter agonists and antagonists (19 -21), and interacting proteins (22)(23)(24)(25)(26). For example, the subcellular distribution of endogenous GAT1 in hippocampal neurons is under regulation by both protein kinase C and tyrosine kinase (13,27,28). Syntaxin 1A, a t-SNARE protein that interacts with GAT1, increases its plasma membrane expression (22,29).…”

mentioning

confidence: 99%

Trafficking of the Plasma Membrane γ-Aminobutyric Acid Transporter GAT1

Wang¹,

Quick

2005

Self Cite

Plasma membrane neurotransmitter transporters rapidly traffic to and from the cell surface in neurons. This trafficking may be important in regulating neuronal signaling. Such regulation will be subject to the number of trafficking transporters and their trafficking rates. In the present study, we define an acutely recycling pool of endogenous ␥-aminobutyric acid transporters (GAT1) in cortical neurons that comprises approximately one-third of total cellular GAT1. Kinetic analysis of this pool estimates exocytosis and endocytosis time constants of 1.6 and 0.9 min, respectively, and thus approximately one-third of the recycling pool is plasma membrane resident in the basal state. Recent evidence shows that GAT1 substrates, second messengers, and interacting proteins regulate GAT1 trafficking. These triggers could act by altering trafficking rates or by changing the recycling pool size. In the present study we examine three GAT1 modulators. Calcium depletion decreases GAT1 surface expression by diminishing the recycling pool size. Sucrose increases GAT1 surface expression by blocking clathrin-and dynamin-dependent endocytosis, but it does not change the recycling pool size. Protein kinase C decreases surface GAT1 expression by increasing the endocytosis rate, but it does not change the exocytosis rate or the recycling pool size. Based upon estimates of GAT1 molecules in cortical boutons, the present data suggest that ϳ1000 transporters comprise the acutely recycling pool, of which 300 are on the surface in the basal state, and five transporters insert into the plasma membrane every second. This insertion could represent the fusion of one transporter-containing vesicle.

“…In addition, a low level of phosphothreonine was consistently observed, and many threonines are found within PKC consensus sequences that are highly conserved throughout the monoamine transporter family (2). Recent studies (9,24,25) have reported the presence of phosphotyrosine on GAT1 and the functional regulation of DAT and GAT1 by tyrosine kinase inhibitors. We found no detectable phosphotyrosine from DATs prepared under the described conditions, but we did not employ treatments such as tyrosine kinase activators or phosphotyrosine phosphatase inhibitors specific for generation or preservation of phosphotyrosine.…”

Section: Fig 8 Tryptic and Cnbr Phosphopeptide Fragments Amentioning

confidence: 99%

“…Recent studies (5)(6)(7)(8)(9) have shown that DATs and other neurotransmitter transporters are phosphoproteins whose functions are acutely regulated by protein kinases, providing a mechanism for temporal and spatial control of synaptic neurotransmitter levels and neural signaling. Activators of protein kinase C (PKC) and inhibitors of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) cause an increase in DAT phosphorylation with a concomitant reduction in dopamine transport activity (5,10,11).…”

mentioning

confidence: 99%

Dopamine Transporters Are Phosphorylated on N-terminal Serines in Rat Striatum

Foster

Pananusorn²,

Vaughan

2002

134

171

Dopamine transporters (DATs) are neuronal phosphoproteins that clear dopamine from the synaptic cleft. Activation of protein kinase C (PKC) and inhibition of protein phosphatases by okadaic acid (OA) increase phosphorylation of DAT and lead to concomitant reduction in DAT activity and cell surface expression. Numerous potential sites for phosphorylation are present on DAT, but the sites utilized and their relationship to transport regulation are currently unknown. We used peptide mapping and epitope-specific immunoprecipitation to identify the region of DAT that undergoes phosphorylation in rat striatal tissue. Phosphoamino acid analysis revealed that basal and stimulated samples were phosphorylated primarily on serine. Digestion of 32 PO 4 -labeled DAT with trypsin and immunoprecipitation with N-or C-terminal specific antisera failed to isolate phosphopeptide fragments corresponding to photoaffinity-labeled fragments that contain all internal interhelical loops. However, digestion of 32 PO 4 -labeled DAT with endoproteinase asp-N and immunoprecipitation with an N-terminal antiserum extracted two phosphopeptide fragments from both basal and PKC/ OA-stimulated samples, demonstrating that the N-terminal cytoplasmic tail is a major site of phosphorylation. Aminopeptidase treatment of PKC-and/or OA-stimulated DAT cleaved essentially all 32 PO 4 label without proteolysis extending past transmembrane domains 1 and 2, providing further evidence that most phosphorylation sites are near the N terminus and not in intracellular loops or C-terminal domains. In situ proteolysis of the N-terminal tail indicates that the majority of stimulated phosphorylation sites are N-terminal to an antibody epitope at residues 42-59. Two-dimensional analysis of purified protein produced three tryptic phosphopeptides that may result from phosphorylation of multiple sites, but the fragments did not co-migrate with synthetic tryptic peptides phosphorylated at serines 2 and 4. These results indicate that most or all of the basal and stimulated phosphorylation of DAT in striatal tissue occurs on one or more residues in a group of six serines clustered near the distal end of the cytoplasmic N terminus.The concentration of dopamine in the synaptic and extrasynaptic space is controlled to a great extent by the action of dopamine transporters (DATs), 1 which utilize the energy of Na ϩ and Cl Ϫ ionic gradients to drive reuptake of the neurotransmitter into the presynaptic cell (1). DAT belongs to a family of transporters including those for norepinephrine and serotonin (SERT) that constitutes the major sites of action for tricyclic antidepressants and psychostimulants (2). Other neurotransmitter reuptake carriers include plasma membrane transporters for glycine (Glyt1) and ␥-aminobutyric acid (GAT1), and the synaptic vesicle monoamine and acetylcholine transporters, vesicular monoamine transporter and vesicular acetylcholine transporter (3, 4). These proteins are believed to have a similar structure of 12 transmembrane-spanning domains, cytoplasmica...