Aim: The glutamate transporter EAAC1/EAAT3 mediates the uptake of glutamate from the synaptic cleft as well as the absorption of dicarboxylic amino acids in epithelial cells. Its cell-surface density is regulated by constitutive cycling, but the precise molecular mechanisms underlying this event are far from clear. Methods: We identified a conserved consensus sequence (-SQF) for interactions with class I PDZ domains and a non-conventional tyrosine-based internalisation signal (-YVNG-) in the C-terminus of EAAC1, and investigated their role in transporter localisation in epithelial cells. Results: Removal or manipulations of the PDZ-interacting sequence affected the cell surface expression of the transporter without altering its apical targeting or substrate affinity. Decreased cell surface expression was caused by faster internalisation and was prevented by hypertonic treatment or the overexpression of dominant-negative dynamine-K44A and the m2-W421A-subunit of AP-2 clathrin-adaptor. The endocytosis rate was dramatically attenuated following tyrosine mutagenesis in the internalisation signal, thus indicating that this motif can control the transporter's constitutive endocytosis. Conclusion: We suggest that EAAC1 density is controlled by balanced interactions with PDZ- and clathrin-adaptor proteins: the former retain the transporter at the cell surface, and the latter promote its constitutive endocytosis. Methods: We identified a conserved consensus sequence (-SQF) for interactions with class I PDZ domains and a non-conventional tyrosine-based internalisation signal (-YVNG-) in the C-terminus of EAAC1, and investigated their role in transporter localisation in epithelial cells. Results: Removal or manipulations of the PDZ-interacting sequence affected the cell surface expression of the transporter without altering its apical targeting or substrate affinity. Decreased cell surface expression was caused by faster internalisation and was prevented by hypertonic treatment or the overexpression of dominant-negative dynamine-K44A and the m2-W421A-subunit of AP-2 clathrin-adaptor. The endocytosis rate was dramatically attenuated following tyrosine mutagenesis in the internalisation signal, thus indicating that this motif can control the transporter's constitutive endocytosis. Conclusion: We suggest that EAAC1 density is controlled by balanced interactions with PDZ- and clathrin-adaptor proteins: the former retain the transporter at the cell surface, and the latter promote its constitutive endocytosis.

A PDZ protein target sequence and a clathrin-dependent endocytosis signal concur in regulating the surface expression of the glutamate transporter EAAC1 in epithelial cells / E.S. Di Cairano, G. Fantin, A. D'Amico, A. Soragna, N. Panzeri, F.V. Sacchi, C. Perego. - In: ACTA PHYSIOLOGICA. - ISSN 1748-1708. - 197:suppl. 672(2009), pp. P58-P58. ((Intervento presentato al 60. convegno Congresso Italiano di Fisiologia tenutosi a Siena nel 2009.

A PDZ protein target sequence and a clathrin-dependent endocytosis signal concur in regulating the surface expression of the glutamate transporter EAAC1 in epithelial cells

E.S. Di Cairano
Primo
;
A. D'Amico;A. Soragna;F.V. Sacchi
Penultimo
;
C. Perego
Ultimo
2009

Abstract

Aim: The glutamate transporter EAAC1/EAAT3 mediates the uptake of glutamate from the synaptic cleft as well as the absorption of dicarboxylic amino acids in epithelial cells. Its cell-surface density is regulated by constitutive cycling, but the precise molecular mechanisms underlying this event are far from clear. Methods: We identified a conserved consensus sequence (-SQF) for interactions with class I PDZ domains and a non-conventional tyrosine-based internalisation signal (-YVNG-) in the C-terminus of EAAC1, and investigated their role in transporter localisation in epithelial cells. Results: Removal or manipulations of the PDZ-interacting sequence affected the cell surface expression of the transporter without altering its apical targeting or substrate affinity. Decreased cell surface expression was caused by faster internalisation and was prevented by hypertonic treatment or the overexpression of dominant-negative dynamine-K44A and the m2-W421A-subunit of AP-2 clathrin-adaptor. The endocytosis rate was dramatically attenuated following tyrosine mutagenesis in the internalisation signal, thus indicating that this motif can control the transporter's constitutive endocytosis. Conclusion: We suggest that EAAC1 density is controlled by balanced interactions with PDZ- and clathrin-adaptor proteins: the former retain the transporter at the cell surface, and the latter promote its constitutive endocytosis. Methods: We identified a conserved consensus sequence (-SQF) for interactions with class I PDZ domains and a non-conventional tyrosine-based internalisation signal (-YVNG-) in the C-terminus of EAAC1, and investigated their role in transporter localisation in epithelial cells. Results: Removal or manipulations of the PDZ-interacting sequence affected the cell surface expression of the transporter without altering its apical targeting or substrate affinity. Decreased cell surface expression was caused by faster internalisation and was prevented by hypertonic treatment or the overexpression of dominant-negative dynamine-K44A and the m2-W421A-subunit of AP-2 clathrin-adaptor. The endocytosis rate was dramatically attenuated following tyrosine mutagenesis in the internalisation signal, thus indicating that this motif can control the transporter's constitutive endocytosis. Conclusion: We suggest that EAAC1 density is controlled by balanced interactions with PDZ- and clathrin-adaptor proteins: the former retain the transporter at the cell surface, and the latter promote its constitutive endocytosis.
glutamate transporter; endocytosis; PDZ protein; epithelial cells
Settore BIO/09 - Fisiologia
http://www.blackwellpublishing.com/aphmeeting/abstract.asp?MeetingID=760&id=79504
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/161248
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