Astrocytic and neuronal localization of the scaffold protein Na⁺/H⁺ exchanger regulatory factor 2 (NHERF‐2) in mouse brain

Abstract

The Na + /H + exchanger regulatory factor 2 (NHERF-2) is a scaffold protein that regulates cellular signaling by forming protein complexes. Several proteins known to interact with NHERF-2 are abundantly expressed in the central nervous system, but little is known about NHERF-2 localization in the brain. By using immunohistochemistry combined with light and electron microscopy, we found that many populations of astrocytes, as well as some populations of neurons, were immunopositive for NHERF-2 throughout the mouse brain. Quantitative analysis of the subcellular distribution of NHERF-2 immunostaining in four brain structures, cerebral cortex, hippocampus, striatum, and cerebellar cortex, showed that NHERF-2 was expressed mainly in astrocytic processes but was also sometimes observed in both pre-and postsynaptic neuronal elements. NHERF-2 immunostaining was associated mainly with the plasma membrane of neurons and astrocytes. However, NHERF-2 immunoreactivity was also observed in association with synaptic vesicles in putative glutamatergic axon terminals. The subcellular localization of NHERF-2 in brain is consistent with a role for NHERF-2 in forming complexes between cell surface and cytosolic proteins, and the preferential expression of NHERF-2 in astrocytes suggests that this scaffold protein may play an important role in astrocytic physiology. Indexing termsPDZ; E3KARP; NHERF2; scaffolding; receptor; signaling Scaffold proteins play an important role in regulating cellular signaling by physically tethering together key molecular components of signaling pathways. Many scaffold proteins consist of a series of modular domains that bind to specific motifs on target proteins. For example, NHERF-2 (also known as E3KARP, SIP-1, and TKA-1) is a scaffold protein that contains two PDZ domains and one ERM-binding domain (Yun et al., 1997). PDZ domains are named for the first three proteins in which they were described (postsynaptic density-95, discs large, zona occludens-1) and mediate protein-protein interactions by binding to the last few carboxyl- terminal residues of their target proteins (Hung and Sheng, 2002). ERM (ezrin, ra-dixin, moesin)-binding domains link NHERF-2 and the related scaffold NHERF-1 to the actin cytoskeleton by interacting with the ERM family of actin-binding proteins (Nguyen et al, 2001). Thus, via these multiple domains, NHERF-2 can link its interacting partners to each other and to the actin cytoskeleton.The PDZ domains of NHERF-2 are known to interact with a handful of specific G proteincoupled receptors (GPCRs), growth factor receptors, ion channels, transporters, and cellular signaling effectors (Shenolikar et al., 2004). By recruiting protein targets and forming protein complexes, NHERF-2 can regulate the targeting and/or trafficking of its binding partners as well as their physiological functions. For example, the simultaneous interaction of NHERF-2 with phospholipase Cβ (PLCβ) and either the purinergic P2Y 1 receptor or lysophosphatidic acid LPA 2 receptor results in enhanced rece...

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

“…Our observation that NHERF2 was present in endothelial cells of blood vessels and capillaries throughout the brain is consistent with recent data published in mouse brain using the same antibody (Paquet et al, 2006). In addition, however, the study in mouse brain showed labeling for NHERF2 in other cell types, including astrocytes and neurons.…”

“…We suggest these spatial disparities may reflect the probable role of NHERF1 not only in anchoring GLAST but probably in also complexing with other proteins that have PDZ binding motifs, such additional interactions precluding total colocalization at the molecular level and thus at the anatomic level. In light of the recent report that astrocytes in mouse brain show more restricted distribution of NHERF2 (Paquet et al, 2006), it is interesting to note that we find GLAST also binds NHERF2 in vitro (unpublished data); however, GLAST does not bind NHERF2 in vivo (as demonstrated by coimmunoprecipitation experiments).…”

“…5. Paquet et al (2006) used higher magnification electron microscopy to study subcellular localization of NHERF2 and were therefore able to detect NHERF2 in the synaptic elements. Interestingly, we found that NHERF2 staining of endothelial cells was prominent at the apical aspect of the cells, which may reflect a role for NHERF2 in the regulation of channels/transporters involved in the movement of ion/nutrients between the blood and brain.…”

The PDZ Scaffold NHERF-2 Interacts with mGluR5 and Regulates Receptor Activity

“…Brain sections were then prepared and processed for pre-embedding immunoperoxidase labeling as previously described (18). Briefly, 60-m thick sections were incubated with rabbit anti-mGluR5 (1:2,000) or rabbit anti-NHERF-2 (1:7,000) followed by biotinylated anti-rabbit (1:200, Vector Laboratories Burlingame, CA), rinsed and then incubated with avidin-biotin-peroxidase complex (1:200, Vector Laboratories).…”

Muscarinic-induced Recruitment of Plasma Membrane Ca2+-ATPase Involves PSD-95/Dlg/Zo-1-mediated Interactions

“…Our demonstration of rapid agonist-induced translocation of PMCA in a native cell type reveals a new mechanism for regulating Ca 2ϩ efflux. The recent identification of NHERFs in the brain and other excitable cells (66), and the fact that the brain specific PMCA 2b also binds NHERF-2 opens up the possibility that such mechanisms of regulation may be more widely operative.…”