The glutamate transporter excitatory amino acid carrier 1 associates with the actin-binding protein α-adducin

Abstract

Excitatory amino acid carrier 1 (EAAC1) belongs to the family of the Na +-dependent glutamate carriers. Although the association between defective EAAC1 function and neurologic disease has been repeatedly studied, EAAC1 regulation is not yet fully understood. We have reported that in C6 glioma cells both the activity and membrane targeting of EAAC1 require the integrity of actin cytoskeleton. Here we show that, in the same model, EAAC1 partially co-localizes with actin filaments at the level of cell processes. Moreover, perinuclear spots in which EAAC1 co-localizes with the actin binding protein α-adducin are observed in some cells and, consistently, faint co-immunoprecipitation bands between EAAC1 and α-adducin are detected. Co-localization and partial co-immunoprecipitation of EAAC1 and adducin are still detectable after cell treatment with phorbol esters, a condition that leads to a protein kinase C (PKC)-dependent increase of EAAC1 expression on the membrane and to the phosphorylation of adducin. A co-immunoprecipitation band was also detected in protein extracts of rat hippocampus. The amount of adducin co-immunoprecipitated with EAAC1 increases after the treatment of C6 cells with retinoic acid, a differentiating agent that induces EAAC1 overexpression in this cell model. Moreover, in clones of C6 cells transfected with a hemagglutinin (HA)-tagged adducin, the bands of EAAC1 immunoprecipitated by an anti-HA antiserum were proportional to EAAC1 expression. These results suggest the existence of a pool of EAAC1 transporters associated with the actin binding protein α-adducin in a PKC-insensitive manner.