GLAST stability and activity are enhanced by interaction with the PDZ scaffold NHERF-2

Abstract

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...

Dynamic NHERF interaction with TRPC4/5 proteins is required for channel gating by diacylglycerol

“…Maintenance of the subcellular architecture by NHERF proteins is critical for salient cell functions, for example in the kidney, small intestine, and other organs, where they interact with transporters, ion channels, signaling proteins, transcription factors, enzymes, G protein-coupled receptors (GPCRs), and tyrosine kinase receptors (20)(21)(22)(23)(24). NHERF proteins regulate phosphate transport in proximal tubule cells (25), are involved in ion transport in the small intestine (26), and regulate the activity of the glutamate transporter GLAST and of the metabolic glutamate receptor mGlu5 in astrocytes (27,28). Moreover, they play a role in cell growth and cancer (29)(30)(31).…”

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

“…Moreover, the cellular distribution of NHERF1 and NHERF2 in polarized epithelial cells are mutually exclusive [26]. Conversely, both NHERF1 [5] and NHERF2 [6] are expressed in brain astrocytes, where GLAST is also highly expressed. Therefore, the serial interaction of NHERF proteins with GLAST during COPIImediated trafficking of GLAST would occur in brain astrocytes, but not in epithelial cells.…”

“…In this mechanism, GLAST, a vesicular cargo, transfers from NHERF1 to NHERF2 during COPII-mediated vesicular transport from the ER to the ERGIC. The sequential interaction of NHERF proteins with GLAST suggests that NHERF proteins not only stabilize GLAST at the plasma membrane [5,6], but also regulate the vesicular trafficking of the transporter, as evidenced by the finding that the interaction of GLAST with NHERF1 facilitated the export of GLAST from the ER (Fig. 2).…”

“…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].…”