The neuronal glutamate transporter EAAC1/EAAT3(Excitatory Amino Acid Carrier-1) mediates the uptake of the excitatory neurotransmitter from the synaptic cleft. It is also expressed in epithelial cells where it provides the principal route of glutamate and aspartate absorption. The transporter activity and localization are modulated by auxiliary proteins that still have to be identified. In the C-terminus of the EAAC1/EAAT3 transporter, we observed a consensus sequence (-S-Q-F) for interaction with class I PDZ domains and we investigated the role of this motif in the transporter localization and activity. Mutant transporters were generated and overexpressed in the CV1, COS and MDCK (Madin Darby canine kidney) cell lines, and their localization and activity were tested by means of immunofluorescence, biotinylation, and uptake experiments. We found that removal of the PDZ-interacting sequence(T-S-Q-F) or substitution of the serine residue at -2 position with alanine or glutamate affected the cell surface stability of the transporter. Indeed, the steady state cell surface expression of mutant transporters was lower compared to wild type protein, but it was greatly increased by inhibition of the clathrindependent endocytosis (hyperosmotic stress). Double immunofluorescence experiments revealed that mutant transporters accumulated in an endocytic compartment which did not colocalize with transferrin, a marker of the recycling compartment. Instead, we found a partial colocalization with LAMP- 2, a marker of the lysosomal compartment, after inhibition of lysosomal degradation by means of leupeptine treatment. We suggest that the PDZ target sequence of EAAC1/EAAT3 transporter, and likely its interaction with PDZ proteins, may control the surface stability and/or recycling of the transporter. In the absence of this interaction, the transporter reaches the plasma membrane but instead of being retained in- or recycled to- the cell surface, it is internalized and degraded in a lysosomal compartment. We are now in the process to identify PDZ proteins interacting with the EAAC1/EAAT3 transporter in epithelial and neuronal cells.

A PDZ target sequence controls the surface expression and recycling of the EAAC1/EAAT3 glutamate transporter / A. D'Amico, A. Soragna, V.F. Sacchi, C. Perego. - In: ACTA BIO-MEDICA DE L'ATENEO PARMENSE. - ISSN 0392-4203. - 77:suppl. 3(2006), pp. 57-58. ((Intervento presentato al convegno Transporters 2006 tenutosi a Parma nel 2006.

A PDZ target sequence controls the surface expression and recycling of the EAAC1/EAAT3 glutamate transporter

A. D'Amico;A. Soragna;V.F. Sacchi;C. Perego
2006

Abstract

The neuronal glutamate transporter EAAC1/EAAT3(Excitatory Amino Acid Carrier-1) mediates the uptake of the excitatory neurotransmitter from the synaptic cleft. It is also expressed in epithelial cells where it provides the principal route of glutamate and aspartate absorption. The transporter activity and localization are modulated by auxiliary proteins that still have to be identified. In the C-terminus of the EAAC1/EAAT3 transporter, we observed a consensus sequence (-S-Q-F) for interaction with class I PDZ domains and we investigated the role of this motif in the transporter localization and activity. Mutant transporters were generated and overexpressed in the CV1, COS and MDCK (Madin Darby canine kidney) cell lines, and their localization and activity were tested by means of immunofluorescence, biotinylation, and uptake experiments. We found that removal of the PDZ-interacting sequence(T-S-Q-F) or substitution of the serine residue at -2 position with alanine or glutamate affected the cell surface stability of the transporter. Indeed, the steady state cell surface expression of mutant transporters was lower compared to wild type protein, but it was greatly increased by inhibition of the clathrindependent endocytosis (hyperosmotic stress). Double immunofluorescence experiments revealed that mutant transporters accumulated in an endocytic compartment which did not colocalize with transferrin, a marker of the recycling compartment. Instead, we found a partial colocalization with LAMP- 2, a marker of the lysosomal compartment, after inhibition of lysosomal degradation by means of leupeptine treatment. We suggest that the PDZ target sequence of EAAC1/EAAT3 transporter, and likely its interaction with PDZ proteins, may control the surface stability and/or recycling of the transporter. In the absence of this interaction, the transporter reaches the plasma membrane but instead of being retained in- or recycled to- the cell surface, it is internalized and degraded in a lysosomal compartment. We are now in the process to identify PDZ proteins interacting with the EAAC1/EAAT3 transporter in epithelial and neuronal cells.
Glutamate transporter ; PDZ protein ; epithelia ; neurons
Settore BIO/09 - Fisiologia
http://www.actabiomedica.it/data/2006/supp_3_2006/communications.pdf
Article (author)
File in questo prodotto:
Non ci sono file associati a questo prodotto.
Pubblicazioni consigliate

Caricamento pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/169596
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact