Na⁺–H⁺ exchanger regulatory factor 1 is a PDZ scaffold for the astroglial glutamate transporter GLAST

Abstract

Glutamate is a key neurotransmitter and its levels in the synaptic cleft are tightly regulated by reuptake mechanisms that primarily involve transporters in astrocytes. This requires that the glutamate transporters be spatially constrained to effect maximum glutamate transport. GLAST (EAAT1) is the predominant astroglial transporter and contains a class I PDZ-binding consensus (ETKM) in its C-terminus. The epithelial Na + /H + exchanger regulatory factors NHERF1 and NHERF2 are PDZ proteins that contain two tandem PDZ domains and a C-terminal domain that binds members of the ERM (ezrin-radixin-moesin) family of membrane-cytoskeletal adaptors. NHERF proteins have been extensively characterized in renal epithelia and their expression in brain has recently been reported; however, their function in the brain remains unknown. The aims of the current study were to (1) determine the distribution of NHERF1/2 in the rodent brain and (2) investigate whether GLAST was a physiological ligand for NHERF1/2. Immunohistochemistry revealed that NHERF1 expression was widespread in rat brain (abundant in cerebellum, cerebral cortex, hippocampus, and thalamus) and primarily restricted to astrocytes whereas NHERF2 expression was primarily restricted to endothelial cells of blood vessels and capillaries. Importantly, NHERF1 distribution closely matched that of GLAST and confocal imaging demonstrated co-localization of the two proteins. Coimmunoprecipitation demonstrated that GLAST, NHERF1, and ezrin associate in vivo. In vitro binding assays showed that GLAST bound directly to the PDZ1 domain of NHERF1 via the Cterminal ETKM motif of GLAST. These findings implicate the GLAST-NHERF1 complex in the regulation of glutamate homeostasis in astrocytes.

Cytoskeletal Anchoring of GLAST Determines Susceptibility to Brain Damage

“…An in vivo association was previously shown between GLAST, NHERF1, and ezrin (17,21). In this study we have extended these findings; our D-aspartate uptake and co-immunoprecipitation experiments in cultured cells have highlighted the importance of the C-terminal PDZ binding motif (ETKM) of GLAST in both functional transport and in mediating the interaction with GFAP.…”

“…Mouse monoclonal anti-ezrin was purchased from Invitrogen. A rabbit polyclonal antibody against the C-terminal region of NHERF1 (a gift from Dr. C. Yun) was used as previously described (17). Monoclonal anti-green fluorescent protein antibody was purchased from BD Biosciences.…”

Na⁺-H⁺ Exchanger Regulatory Factor 1 (NHERF1) PDZ Scaffold Binds an Internal Binding Site in the Scavenger Receptor Megalin

“…Interestingly, only the PDZ2 domain of NHERF proteins is involved in binding to ClC-5 [9], NHE3 [12] and megalin (data shown here). This leaves the PDZ1 module free to potentially form homo-or heterodimers [26] thereby linking the different membrane proteins together in dynamic complexes in a spatio-temporal manner [11,27,28] (Fig. 7).…”

Modulatory roles of NHERF1 and NHERF2 in cell surface expression of the glutamate transporter GLAST

“…Glutamate transporters, such as GLAST [4], possess a type I PDZ domain-binding motif (S/T-X-U, where X is any residue, and U is a hydrophobic amino acid) at their C-terminus and bind to specific PDZ domaincontaining proteins. GLAST is known to bind to NHERF1 and NHERF2 [5,6], and interaction with these proteins has been shown to stabilize the surface expression of GLAST by linking it to the plasma membrane [5][6][7][8].…”

“…The proteins share similar domain structures composed of two tandem PDZ domains and protein 4.1-ezrin-radixin-moesin (FERM) binding-domains that recognize FERM family membrane anchor proteins and link them to the actin cytoskeleton [10]. Although NHERF1 and NHERF2 both function as scaffolding proteins for GLAST [5,6], PDZ domain-containing proteins in general are known to play multiple roles in the ER export, surface delivery, and endocytosis [11][12][13] of membrane proteins. Moreover, previous study has shown that NHERF1 and NHERF2 have different effects on trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) [14].…”