The PDZ target motifs located in the C-terminal end of many receptors and ion channels mediate protein-protein interactions by binding to specific PDZ-containing proteins. These interactions are involved in the localization of surface proteins on specialized membrane domains of neuronal and epithelial cells. However, the molecular mechanism responsible for this PDZ protein-dependent polarized localization is still unclear. This study first demonstrated that the epithelial γ-aminobutyric acid (GABA) transporter (BGT-1) contains a PDZ target motif that mediates the interaction with the PDZ protein LIN-7 in Madin-Darby canine kidney (MDCK) cells, and then investigated the role of this interaction in the basolateral localization of the transporter. It was found that although the transporters from which the PDZ target motif was deleted were still targeted to the basolateral surface, they were not retained but internalized in an endosomal recycling compartment. Furthermore, an interfering BGT peptide determined the intracellular relocation of the native transporter. These data indicate that interactions with PDZ proteins determine the polarized surface localization of target proteins by means of retention and not targeting mechanisms. PDZ proteins may, therefore, act as a sort of membrane protein sorting machinery which, by recognizing retention signals (the PDZ target sequences), prevents protein internalization.

Abstract:The GLT-1 and GLAST astroglial transporters are the glutamate transporters mainly involved in maintaining physiological extracellular glutamate concentrations. Defects in neurotransmitter glutamate transport may represent an important component of glutamateinduced neurodegenerative disorders (such as amyotrophic lateral sclerosis) and CNS insults (ischemia and epilepsy). We characterized the protein expression of GLT-1 and GLAST in primary astrocyte-neuron cocultures derived from rat hippocampal tissues during neuron differentiation/maturation. GLT-1 and GLAST are expressed by morphologically distinct glial fibrillary acidic proteinpositive astrocytes, and their expression correlates with the status of neuron differentiation/maturation and activity. Up-regulation of the transporters paralleled the content of the synaptophysin synaptic vesicle marker p38, and down-regulation was a consequence of glutamateinduced neuronal death or the reduction of synaptic activity. Finally, soluble factors in neuronal-conditioned media prevented the down-regulation of the GLT-1 and GLAST proteins. Although other mechanisms may participate in regulating GLT-1 and GLAST in the CNS, our data indicate that soluble factors dependent on neuronal activity play a major regulating role in hippocampal cocultures.

The heterotrimeric PDZ complex containing LIN-2, LIN-7 and LIN-10 is known to be involved in the organization of epithelial and neuronal junctions in Caenorhabditis elegans and mammals. We report here that mammalian LIN-7 PDZ proteins form a complex with cadherin and b-catenin in epithelia and neurons. The association of LIN-7 with cadherin and b-catenin is Ca 2+ dependent and is mediated by the direct binding of LIN-7 to the C-terminal PDZ target sequence of b-catenin, as demonstrated by means of co-immunoprecipitation experiments and in vitro binding assays with the recombinant glutathione S-transferase:LIN-7A. The presence of b-catenin at the junction is required in order to relocate LIN-7 from the cytosol to cadherin-mediated adhesions, thus indicating that LIN-7 junctional recruitment is b-catenin dependent and that one functional role of the binding is to localize LIN-7. Moreover, when LIN-7 is present at the b-catenin-containing junctions, it determines the accumulation of binding partners, thus suggesting the mechanism by which b-catenin mediates the organization of the junctional domain.

It has been suggested that glutamate-induced excitotoxicity plays a central role in the development of motor neuron diseases such as amyotrophic lateral sclerosis (ALS). The GLT-1 isoform of the glutamate transporter gene family is the most important transporter involved in keeping extracellular glutamate concentration below neurotoxic levels. Its loss and an increase in extracellular glutamate has been documented in cases of sporadic and familial ALS, as well as in animal models expressing ALS-linked Cu2+-Zn2+ superoxide dismutase (SOD1) mutations, but the underlying molecular mechanisms are still unclear. We developed and characterised a cell model consisting of polarised epithelial Madin-Darby Canine Kidney (MDCK) cell lines stably expressing wild-type SOD1 or the ALS-linked SOD1 G93A mutant, and analysed the expression of glutamate transporters after transient transfection of the corresponding cDNAs. Like ALS patients and animal models of ALS, the G93A-expressing MDCK cell system showed reduced total glial GLT-1 expression, with no change in the expression of the neuronal EAAC1 glutamate transporter isoform. Morphological analysis revealed the intracellular redistribution of GLT-1 to acidic compartments, whereas the surface distribution of other glutamate transporters (neuronal EAAC1 and glial GLAST) was not affected. Moreover, mutant SOD1 affected the cytosolic tail of GLT-1 because reduced protein expression of EAAC-GLT but not GLT-EAAC chimeras was found in G93A-expressing cell lines. GLT-1 downregulation was greatly induced by inhibition of protein synthesis, and prevented by treatment with chloroquine aimed at inhibiting the activity of acidic degradative compartments. Negligible effect on the protein level or distribution of GLT-1 was observed in cells overexpressing wild-type SOD1. The specific decrease in the GLT-1 isoform of glutamate transporters is therefore recapitulated in G93A-expressing MDCK cell lines, thus suggesting an autonomous cell mechanism underlying the loss of GLT-1 in ALS. Our data indicate that the continuous expression of mutant SOD1 causes the downregulation of GLT-1 by increasing the internalisation and degradation of the surface transporter, and suggest that the cytosolic tail of GLT-1 is required to target the transporter to degradation.

The Na/Cl-dependent BGT1 transporter has osmoprotective functions by importing the small osmolyte betaine into the cytosol of renal medullary epithelial cells. We have demonstrated previously that the surface localization of the transporter in Madin-Darby canine kidney cells depends on its association with the LIN7 PDZ protein through a PDZ target sequence in the last 5 residues of the transporter (-KETHL). Here we describe a protein kinase C (PKC)-mediated mechanism regulating the association between BGT1 and LIN7. Reduced transport activity paralleled by the intracellular relocalization of the transporter was observed in response to the PKC activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. This activation caused clathrindependent internalization of the transporter and its targeting to a recycling compartment that contains the truncated transporter lacking the LIN7 binding motif (BGT⌬5) but not the LIN7 partner of BGT1. The decreased association between BGT1 and LIN7 was demonstrated further by coimmunoprecipitation studies and in vitro binding to recombinant LIN7 fusion protein.The TPA treatment induced phosphorylation of surface BGT1 on serine and threonine residues. However, a greater increase in phosphothreonines than phosphoserines was measured in the wild type transporter, whereas the opposite was true in the BGTSer mutant in which a serine replaced the threonine 612 in the LIN7 association motif (-KESHL). No similar increase in relative phosphoserines or phosphothreonines was found in the BGT⌬5 transporter. Moreover, phosphorylation of threonine 612 in a BGT COOH-terminal peptide impaired its association with recombinant LIN7. Taken together, these data demonstrate that the post-translational regulation of BGT1 surface density is a result of transporter phosphorylation and that threonine 612 is an essential residue in this PKC-mediated regulation.