The Characterisation of NHERF-1 and NHERF-2 Scaffold Proteins in the Mammalian Brain

The Na+-H+ exchanger regulatory factors (NHERFs) are a family of well characterised PSD-95, Dlg, ZO-1 (PDZ) scaffold proteins that are predominantly expressed in epithelial cells (NHERF-1) or endothelial cells (NHERF-2) throughout the body. These proteins are able to bind a wide variety of proteins including membrane receptors, ion channels, transporters, cytoplasmic signalling molecules, and other scaffold proteins. NHERF-1 and NHERF-2 possess tandem PDZ domains and a carboxyl terminal domain that can bind members of the ezrin-radixin-moesin (ERM) family of actin-binding proteins. The multiple protein-binding domains allow NHERFs to form macromolecular complexes, while their ability to bind ERM proteins helps them to traffic and target protein-ligands to the plasma membrane, and to stabilise protein-ligands at sub-membranous sites in a spatially constrained manner. NHERFs can also regulate the function of protein-ligands by scaffolding effector and signalling molecules. While NHERF-1 and NHERF-2 have been extensively characterised in epithelial cells of the kidney and gut, very little information is known about their expression and distribution in the brain. Furthermore, NHERF-1 and NHERF-2 are known to interact with many protein ligands in peripheral organs, however very few have been characterised for either protein in the CNS. Therefore the aim of the current study was to characterise the expression and distribution of NHERF-1 and NHERF-2 in the adult rat brain as well as identify and fully characterise novel binding partners for these proteins in the adult rat brain. RT-PCR, Western blotting and immunohistochemistry identified the expression and distribution of NHERF-1 and NHERF-2 in the brain. NHERF-1 was expressed in astrocytes and Bergmann glia while NHERF-2 was primarily expressed in endothelial cells of the cerebral vasculature. In an attempt to identify novel binding partners for NHERF-1 and NHERF-2 in the brain, an analysis of all previously published NHERF-mediated interactions was performed, and a refined class I PDZ binding motif sequence, preferred by NHERF-1 and NHERF-2 was identified. This refined sequence was used to identify probable protein targets from a list of transmembrane proteins. Ultimately, the family of excitatory amino acid transporters (EAATs) were chosen for investigation. A combination of in vitro binding assays, co-immunoprecipitation experiments, and native tissue co-localisation studies fully characterised the interaction between NHERF-1 and the glutamateaspartate transporter (GLAST). NHERF-1 directly interacted with the C-terminal PDZ binding motif of GLAST via its amino-terminal PDZ binding domain. The binding of NHERF-1 to GLAST in the brain identifies a possible role for NHERF-1 in the regulation of glutamate up take by astrocytes. Further investigation revealed that NHERF-1 and GLAST associated with ezrin in vivo, however none of these proteins associated with the glutamate transporter GLT1b, presumably because it does not possess a refined class I PDZ binding motif preferred by NHERF-1 and NHERF-2. Since NHERF-1 is often identified in polarised epithelial cells an investigation into the distribution of NHERF-1 and NHERF-2 in the polarised epithelia of the brain was undertaken. NHERF-1 was identified at the apical surface of choroid plexus epithelial (CPE) cells while NHERF-2 was identified in fenestrated choroid plexus endothelial cells. The presence of NHERF-1 at the apical surface of CPE cells revealed a possible role for NHERF-1 in the scaffolding of proteins involved in the production or regulation of cerebrospinal fluid (CSF). It was hypothesised that GLAST might co-localise with NHERF-1 in CPE cells like it does in astrocytes. GLAST was identified in CPE cells using an antibody directed against an epitope at the carboxyl terminus of the GLAST protein, while an antibody raised against the amino terminus failed to label for GLAST in the choroid plexus. These data pointed to a probable post-translational modification of GLAST in the choroid plexus. The identification of GLAST in the choroid plexus prompted an investigation into the expression and distribution of all other known glutamate transporters as well as the cystineglutamate antiporter xCT in the choroid plexus and ependymal cells. GLT1a, GLT1b and xCT were localised to the apical surface of CPE cells, while the glutamate transporter EAAC1 was not observed in the choroid plexus. However, EAAC1 was identified in ependymal cells along with GLT1a, xCT and GLAST. The characterisation of the distribution of EAATs and xCT in the choroid plexus revealed a role for the choroid plexus in the uptake and removal of glutamate from the CSF. It is possible that the similar apical localisation of EAATs and xCT drives the uptake of cystine from the CSF into CPE cells, where it is available for use, along with glutamate in the production of glutathione, a major antioxidant in the brain. In summary, this study successfully characterised the distribution of NHERF-1 and NHERF-2 in the mammalian brain and identified GLAST as a binding partner for NHERF-1 in the brain. This association implicates NHERF-1 in the regulation of glutamate uptake in both astrocytes and choroid plexus epithelial cells