Abstract
Addicsin (Arl6ip5) is a murine homologue of rat glutamate transporter-associated protein 3-18 (GTRAP3-18), a putative negative modulator of Na+-dependent neural glutamate transporter-excitatory amino acid carrier 1 (EAAC1). Here we report that ADP-ribosylation factor-like 6 interacting protein 1 (Arl6ip1) is a novel addicsin-associated partner that indirectly promotes EAAC1-mediated glutamate transport activity in a protein kinase C activity-dependent manner. Like addicsin, Arl6ip1 is expressed in numerous tissues and proved likely to be co-localized with addicsin in certain neurons in the matured brain. Arl6ip1 was not translocated from the subcellular compartments under any of the test conditions and had no association with any molecules on the plasma membrane. Immunoprecipitation assay demonstrated that Arl6ip1 bound directly to addicsin and that the hydrophobic region located at amino acids 103-117 of addicsin was crucial to the formation of the Arl6ip1-addicsin heterodimer and addicsin homodimer. Glutamate transport assay revealed that increasing the expression of Arl6ip1 in C6BU-1 cells markedly enhanced Na+-dependent EAAC1-mediated glutamate transport activity in the presence of 100 nm phorbol 12-myristate 13-acetate. Under these conditions, kinetic analyses demonstrated that EAAC1 altered glutamate transport activity by increasing its glutamate affinity but not its maximal velocity. Meanwhile, increasing expression of addicsin Y110A/L112A mutant lacking binding ability for Arl6ip1 showed no enhancement of EAAC1-mediated glutamate transport activity, regardless of phorbol 12-myristate 13-acetate activation, suggesting that association between addicsin and Arl6ip1 causes altered EAAC1-mediated glutamate transport activity. Our findings suggest that Arl6ip1 is a novel addicsin-associated partner that promotes EAAC1-mediated glutamate transport activity by decreasing the number of addicsin molecules available for interaction with EAAC1.
Highlights
Glutamate is the major excitatory neurotransmitter in excitatory synapses and the metabolic substrate for ␥-aminobutyric acid synthesis in the inhibitory neurons in the mammalian central nervous system
Our findings suggest that ADP-ribosylation factor-like 6 interacting protein 1 (Arl6ip1) is a novel addicsin-associated partner that promotes excitatory amino acid carrier 1 (EAAC1)-mediated glutamate transport activity by decreasing the number of addicsin molecules available for interaction with EAAC1
excitatory amino acid transporters (EAATs) can be classified into five different homologues, designated EAAT1 (glutamate/aspartate transporter (GLAST)), EAAT2 (glutamate trasnporter-2 (GLT-1)), EAAT3 (excitatory amino acid carrier 1 (EAAC1)), EAAT4, or EAAT5 [2]
Summary
Animals—Male ddY mice (6 weeks old; 25–30 g in body weight; Japan SLC, Inc., Shizuoka, Japan) were maintained in individual cages (12-hour light-dark cycle; 23–24 °C) and used in the experiments. Immmunoprecipitation Assay—For in vitro immunoprecipitation assay, at 48 h after transfection using Lipofectamine 2000, NG108-15 or COS7 cells were dissolved in the RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.0% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) containing 0.1% protease inhibitor mixture. Cells were rinsed three times with 500 l of sodium- or cholinecontaining buffer maintained at 37 °C (5 mM Tris-HCl, 10 mM HEPES, 2.5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 1.2 mM K2HPO4, 10 mM glucose, 140 mM NaCl, or choline chloride) before incubation with 0.5 M L-[3H]glutamate (2.5 Ci/ml) (GE Healthcare) and 30 M unlabeled glutamate for 5 min at 37 °C in a final volume of 400 l/well. The sections were fixed with acetone/methanol solution (w/w, 1:1) for 15 min and blocked with 1.5% normal goat serum for 20 min They were exposed to the rabbit antimouse Arl6ip IgG polyclonal antibody or rabbit anti-addicsin IgG overnight at 4 °C. After the cells had been coverslipped in Gel/Mount (Biomeda, Foster City, CA), images were acquired using a Fluoview FV1000 confocal laser-scanning microscope (Olympus, Tokyo, Japan)
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