Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, C128

Oral Communications

The adaptor protein PDZK1 interacts with the glutamate transporter EAAC1 and regulates its surface expression

E. S. Di Cairano1, G. Fantin1, A. D'Amico1, A. Soragna1, V. F. Sacchi1, C. Perego1

1. Dep. of Molecular Science applied to Biosystem, University of Milan, Milan, Italy.

  • Immunofluorescence. MDCK cells were stably transfected with the indicated GFP-EAAC1 mutant transporters, fixed and analyzed by confocal microscopy. WT EAAC1 is mainly expressed at the plasma membrane, whereas ΔTSQF mutant is expressed in an endocytotic compartment. Scale bar, 10 μm

  • Co-immunoprecipitation. Lysates of MDCK cells stably expressing EAAC1 wild type or ΔTSQF were immunoprecipitated with an anti-PDZK1 antibody; the immunocomplexes were resolved by 9% SDS-PAGE and immunostained with an anti-GFP antibody. 10% of the cell lysate used in the immunoprecipitation assay was probed (Lysate). PDZK1 antibody was able to co-immunoprecipitate only WT EAAC1 from cell lysates.

The glutamate transporter EAAC1/EAAT3 is expressed in the central nervous system (CNS), where it mediates the uptake of the neurotransmitter from the synaptic cleft. It is also expressed in non neuronal tissues, in particular intestine and kidney, where it represents the main pathway of amminoacid reabsorption [1]. Its surface expression is strictly regulated by interaction with accessory protein, e.g. PDZ proteins (from the name of the first proteins discovered PSD95, DLG and ZO1). These proteins contain multiple protein-protein interaction domains, that bring together and localize transporters, channels, enzymes and receptors in specific plasma membrane domains [2]. In the C-terminal of EAAC1 there is a class I PDZ proteins target sequence (-SQF), whose removal induced internalization of the transporter in endocytotic compartments (Fig.1). Therefore, PDZ proteins play a key role in regulating EAAC1 expression in the plasma membrane, but none of the EAAC1 interacting partners have been so far identified. Aim of this work was to identify the PDZ proteins interacting with EAAC1 and to verify whether they play a role in the EAAC1 localization at plasma membrane. In epithelia, possible candidate are members of the NHERF ( Na+/H+ Exchanger Regulatory Factor) family, PDZ proteins that organize the apical domain of the plasma membrane [3]. The interaction between PDZK1/NHERF3 and EAAC1 was proved by means of several experiments. By yeast two hybrid system assay [4], we found that the C-terminal tail of EAAC1 (last 40 or 26 aminoacids) directly interacted with PDZK1, and the interaction required the first and second PDZK1 PDZ domain (n=3). Affinity chromatography [5] confirmed that PDZK1 is retained by the C-terminal tail of wild type EAAC1 (WT) (n=4). Co-immunoprecipitation experiments demonstrated the binding of PDZK1 to wild type transporter (WT GFP-EAAC1), but not to a mutant transporter lacking the PDZ target sequence (ΔTSQF GFP-EAAC1) (n=3) (Fig.2). Similar experiments performed in intestinal tissues confirmed the EAAC1/PDZK1 interaction in vivo (n=3). In agreement with these results we found co-localization of PDZK1 with WT EAAC1 in over-expressing system (MDCK) as well as in native tissues (n=3). PDZK1 not only co-localized but also functionally interacted with the transporter, because the presence of mycPDZK1 caused a 1.34±0.13 fold increase (T-test, p≤0.05) in the WT EAAC1 surface activity, without affecting ΔTSQF activity. Thus, PDZK1 interacts with EAAC1 in vitro e in vivo, regulating its localization and function at plasma membrane. Further studies are needed to clarify the molecular mechanism by which PDZK1 controls the membrane localization of EAAC1.

Where applicable, experiments conform with Society ethical requirements