Glutamate transporters play a fundamental role in maintaining extracellular glutamate below neurotoxic levels, and decreased glutamate transport activity is involved in neurodegenerative diseases [such as Alzheimer’s, Parkinson’s and Amyotrophic lateral sclerosis (ALS)]. The transport activity and surface expression of neurotransmitter transporters are often dynamically regulated through modulation of their intracellular trafficking. In the Madin-Darby canine kidney (MDCK) cell line, we have shown that PKC activation induces a time-dependent decrease in glutamate transport activity associated with clathrin dependent internalization of the transporter and relocation to recycling endosomes within 20 minutes. Using 32P-metabolic labeling experiments, we showed that PKC activation induces an early transitory peak of phosphorylation of a surface transporter otherwise dephosphorylated (5-10 minutes), and that accumulation in recycling compartments coincides with dephosphorylation of EAAC1 to its basal level. In addition, we found that PKC-induced decrease in transport activity and intracellular relocation of surface EAAC1 also depended on calcineurin phosphatase activity. In sharp contrast, we found that internalization and relocation of the GLT1 astroglial glutamate transporter to EAAC1 containing recycling compartments was dependent on PKC activation but independent of calcineurin activity. Moreover, an analysis of GLT1 and EAAC1 chimeras revealed that the cytosolic tail of EAAC1 (last 91 cytosolic C-terminal residues) containing sequences for regulated trafficking of this transporter (Cheng et al., 2002; Sheldon et al., 2006) was sufficient to confer calcineurin dependence to PKC induced internalization of GLT1. We are currently investigating the possibility that the tail of EAAC1 targets the protein to specific pathways of endocytosis sensitive to calcineurin activity. The existence of different mechanisms to modulate the trafficking of a specific glutamate transporter may explain the exclusive reduction of GLT1 in ALS.

Differential regulation of EAAC1 and GLT1 glutamate transporters by calcineurin / V. Padovano, S. Massari, S. Mazzucchelli, G. Pietrini. ((Intervento presentato al 5. convegno Molecular Mechanisms in Neuroscience tenutosi a Milano nel 2008.

Differential regulation of EAAC1 and GLT1 glutamate transporters by calcineurin

V. Padovano;S. Massari;G. Pietrini
2008

Abstract

Glutamate transporters play a fundamental role in maintaining extracellular glutamate below neurotoxic levels, and decreased glutamate transport activity is involved in neurodegenerative diseases [such as Alzheimer’s, Parkinson’s and Amyotrophic lateral sclerosis (ALS)]. The transport activity and surface expression of neurotransmitter transporters are often dynamically regulated through modulation of their intracellular trafficking. In the Madin-Darby canine kidney (MDCK) cell line, we have shown that PKC activation induces a time-dependent decrease in glutamate transport activity associated with clathrin dependent internalization of the transporter and relocation to recycling endosomes within 20 minutes. Using 32P-metabolic labeling experiments, we showed that PKC activation induces an early transitory peak of phosphorylation of a surface transporter otherwise dephosphorylated (5-10 minutes), and that accumulation in recycling compartments coincides with dephosphorylation of EAAC1 to its basal level. In addition, we found that PKC-induced decrease in transport activity and intracellular relocation of surface EAAC1 also depended on calcineurin phosphatase activity. In sharp contrast, we found that internalization and relocation of the GLT1 astroglial glutamate transporter to EAAC1 containing recycling compartments was dependent on PKC activation but independent of calcineurin activity. Moreover, an analysis of GLT1 and EAAC1 chimeras revealed that the cytosolic tail of EAAC1 (last 91 cytosolic C-terminal residues) containing sequences for regulated trafficking of this transporter (Cheng et al., 2002; Sheldon et al., 2006) was sufficient to confer calcineurin dependence to PKC induced internalization of GLT1. We are currently investigating the possibility that the tail of EAAC1 targets the protein to specific pathways of endocytosis sensitive to calcineurin activity. The existence of different mechanisms to modulate the trafficking of a specific glutamate transporter may explain the exclusive reduction of GLT1 in ALS.
Settore BIO/14 - Farmacologia
Società Italiana di Neuroscienze, Università degli Studi di Milano
Differential regulation of EAAC1 and GLT1 glutamate transporters by calcineurin / V. Padovano, S. Massari, S. Mazzucchelli, G. Pietrini. ((Intervento presentato al 5. convegno Molecular Mechanisms in Neuroscience tenutosi a Milano nel 2008.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/59196
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