8.A.24 The Ezrin/Radixin/Moesin-binding Phosphoprotein 50 (EBP50) Family

EBP50 is a Na⁺/H⁺ exchange regulatory cofactor, called NHE-RF or NHERF-1, of 358 aas (Slc9 isoform A3, regulatory factor 3). It is an adaptor protein that organizes a number of cell receptors and channels (Li et al., 2005). It contains two PDZ domains that bind to the cytoplasmic domains of a number of membrane channels, carriers and receptors to coordinate the assembly and trafficking of these transmembrane proteins. Most target proteins harboring a C-terminus recognition motif bind more-or-less equivalently to either of the PDZ domains, which contain identical core-binding motifs. However some substrates such as the type II sodium-dependent phosphate co-transporter (NPT2A), uniquely bind only one PDZ domain (Mamonova et al. 2015). 

NHERF1 (TC# 8.A.24.1.1 and NHERF2 (E3KARP) (TC# 8.A.24.1.2) are homologous adapter proteins that play roles in membrane protein targeting, trafficking, and sorting (Park et al. 2005). Both NHERF1 and 2 interact with the Na⁺/H⁺ exchanger, NHE3, through their C-terminally extended second PDZ domain. The last 30 amino acids of these PDZ domain proteins interact with ezrin (TC# 8.A.25.1.1). Both soluble NHERF proteins have been purified (Park et al. 2005).

The carboxyl terminus of NHERF interacts with the FERM domain (a domain shared by protein 4.1, ezrin, radixin, and moesin) of a family of actin-binding proteins called the ezrin-radixin-moesin family (TC #8.A.25). NHERF enhances the channel activities of cystic fibrosis transmembrane conductance regulator (CFTR) (TC #3.A.1.202.1). Binding of the FERM domain of ezrin to NHERF regulates the cooperative binding of NHERF to bring two cytoplasmic tails of CFTR into spatial proximity to each other. Ezrin binding activates the second PDZ domain of NHERF to interact with the cytoplasmic tails of CFTR (C-CFTR), so as to form a specific 2:1:1 (C-CFTR)₂·NHERF·ezrin ternary complex. EPP50 is required both for plasma membrane localization and for maximal activation of CFTR (Broere et al., 2007). Without ezrin binding, the cytoplasmic tail of CFTR only interacts strongly with the first amino-terminal PDZ domain to form a 1:1 C-CFTR·NHERF complex. Because of the concentrated distribution of ezrin and NHERF in the apical membrane regions of epithelial cells and the diverse binding partners for the NHERF PDZ domains, the regulation of NHERF by ezrin may be employed as a general mechanism to assemble channels and receptors in the membrane cytoskeleton (Li et al., 2005).

The sodium-dependent glutamate transporter, glutamate transporter subtype 1 (GLT-1) is one of the main glutamate transporters in the brain. GLT-1 contains a COOH-terminal sequence similar to one in an isoform of Slo1 K⁺ channel protein previously shown to bind MAGI-1 (membrane-associated guanylate kinase with inverted orientation protein-1), a member of the EBP50 family (TC#8.A.24) (Zou et al., 2011). MAGI-1 is a scaffold protein which allows the formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. MAGI-1 is a binding partner of GLT-1. The interaction between MAGI-1 and GLT-1 was confirmed by co-immunoprecipitation. Immunofluorescence of MAGI-1 and GLT-1 demonstrated that the distribution of MAGI-1 and GLT-1 overlapped in astrocytes. Co-expression of MAGI-1 with GLT-1 in C6 Glioma cells resulted in a significant reduction in the surface expression of GLT-1, as assessed by cell-surface biotinylation. On the other hand, partial knockdown of endogenous MAGI-1 expression by small interfering RNA in differentiated cultured astrocytes increased glutamate uptake and the surface expression of endogenous GLT-1. Knockdown of MAGI-1 increased dihydrokainate-sensitive, Na⁺-dependent glutamate uptake, indicating that MAGI-1 regulates GLT-1-mediated glutamate uptake. These data suggest that MAGI-1 regulates surface expression of GLT-1 and the level of glutamate in the hippocampus (Zou et al., 2011). 

Many protein of TC families 8.A.22 and 8.A.24 and others contain PDZ, SH3 and kinase domains involved in signal transduction, often interacting with receptors and transporters. Therefore, these two families share about 400 aas in common.  PDZ proteins of the NHERF family act to stabilize and organize membrane targeting of multiple transmembrane proteins, including many clinically relevant drug transporters. These PDZ proteins are normally abundant at apical membranes, where they tether membrane-delimited transporters. NHERF expression is high at the apical membrane in polarized tissue such as intestinal, hepatic, and renal epithelia. NHERF proteins are determinants of drug transporter function in addition to their role in controlling membrane abundance and localization. They may have clinically significant roles in pharmacokinetics and pharmacodynamics of several pharmacologically active compounds and may affect drug action in cancer and chronic kidney disease. For these reasons, NHERF proteins represent a novel class of post-translational mediators of drug transport and novel targets for new drug development (Walsh et al. 2015).