The two members of the group I metabotropic glutamate receptor family, mGluR1 and mGluR5, both couple to G q to mediate rises in intracellular calcium. The alternatively spliced C termini (CT) of mGluRs 1 & 5 are known to be critical for regulating receptor activity and to terminate in motifs suggestive of potential interactions with PDZ domains. We therefore screened the CTs of both mGluR1a and mGluR5 against a PDZ domain proteomic array. Out of 96 PDZ domains examined, the domain that bound most strongly to mGluR5-CT was the second PDZ domain of the Na ؉ /H ؉ exchanger regulatory factor 2 (NHERF-2). This interaction was confirmed by reverse overlay, and a single point mutation to the mGluR5-CT was found to completely disrupt the interaction. Full-length mGluR5 robustly associated with full-length NHERF-2 in cells, as assessed by co-immunoprecipitation and confocal microscopy experiments. In contrast, mGluR1a was found to bind NHERF-2 in vitro with a weaker affinity than mGluR5, and furthermore mGluR1a did not detectably associate with NHERF-2 in a cellular context. Immunohistochemical experiments revealed that NHERF-2 and mGluR5 exhibit overlapping patterns of expression in mouse brain, being found most abundantly in astrocytic processes and postsynaptic neuronal elements. In functional experiments, the interaction of NHERF-2 with mGluR5 in cells was found to prolong mGluR5-mediated calcium mobilization and to also potentiate mGluR5-mediated cell death, whereas coexpression of mGluR1a with NHERF-2 had no evident effects on mGluR1a functional activity. These observations reveal that NHERF-2 can selectively modulate mGluR5 signaling, which may contribute to cell-specific regulation of mGluR5 activity.Many of the physiological effects of L-glutamate as a neurotransmitter are mediated via stimulation of metabotropic glutamate receptors (mGluRs), 2 which are considered to be important therapeutic targets in the potential treatment of a number of different diseases (1-3). The mGluRs are G protein-coupled receptors that regulate the physiology of neurons and glia through activation of various secondary messenger systems (4). Eight subtypes of mGluRs have been identified and classified into three groups (I, II, III) based on sequence homology, signaling pathways and pharmacological selectivity. The various mGluR subtypes are known to display differential regional and cellular distributions throughout the mammalian brain (4). Activation of group I mGluRs (mGluR1 and mGluR5) leads to signal transduction primarily through coupling to G q and phospholipase C, which then hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to form the second messenger inositol 1,4,5-phosphate (IP 3 ). IP 3 binding to its receptor in the membrane of the endoplasmic reticulum causes release of free Ca 2ϩ in the cytosol, which generates a signaling cascade. Group I mGluRs can also couple to other signaling pathways in a cell type-specific manner (5). Despite many similarities in their signaling pathways, mGluR1 and mGluR5 expressed in th...
The astrocytic glutamate transporter GLAST (also known as EAAT1) is a key regulator of extracellular glutamate levels in many regions of vertebrate brains. To identify novel interacting partners that might regulate the localization and function of GLAST in astrocytes, we screened the transporter's C-terminus (GLAST-CT) against a proteomic array of 96 different PDZ domains. The GLAST-CT robustly and specifically interacted with PDZ domains from two related scaffolding proteins, the Na + /H + exchanger regulatory factors 1 and 2 (NHERF-1 and NHERF-2). Studies on cultured rat cortical astrocytes revealed that these cells are highly enriched in NHERF-2 relative to NHERF-1. Endogenous GLAST and NHERF-2 from cultured astrocytes were found to robustly co-immunoprecipitate, and further co-immunoprecipitation studies on mutant versions of GLAST expressed in transfected cells revealed the GLAST/NHERF-2 interaction to be dependent on the last amino acid of the GLAST-CT. Knockdown of endogenous NHERF-2 in astrocytes via siRNA treatment resulted in a significant reduction in GLAST activity, which corresponded to significantly reduced total expression of GLAST protein and reduced halflife of GLAST, as assessed in pulse-chase metabolic labeling studies. These findings reveal that NHERF-2 can interact with GLAST in astrocytes to enhance GLAST stability and activity.Glutamate is the most abundant neurotransmitter in the mammalian central nervous system and the mediator of excitatory neurotransmission at the majority of synapses in the brain. The extracellular concentration of glutamate must be tightly regulated, however, as excessive glutamate signaling can lead to excitotoxic cellular death [4]. Five transporters have been identified as the principal regulators of extracellular glutamate levels and therefore have been named excitatory amino acid transporters EAAT-1 (rat homologue known as GLAST), EAAT-2 (rat homologue known as GLT-1), EAAT-3, EAAT-4, and EAAT-5. Genetic studies have shed light on the functions of the individual transporter subtypes, revealing a dominant role for GLAST and GLT-1 in controlling synaptic glutamate levels [16,21]. Interestingly, GLAST and GLT-1 are exclusively expressed in astrocytes [3,12], thereby speaking to the importance of astrocytes in clearing synaptic glutamate. Moreover, mutations in GLAST are linked with a variety of disease states, © 2010 Elsevier Ireland Ltd. All rights reserved.Address for correspondence: Randy A. Hall, Rollins Research Center, room 5113, 1510 Clifton Rd., Emory University School of Medicine, Atlanta, GA, USA,. rhall@pharm.emory.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content...
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