A C-terminal motif found in the β ₂ -adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na ⁺ /H ⁺ exchanger regulatory factor family of PDZ proteins

Abstract

The Na ؉ ͞H ؉ exchanger regulatory factor (NHERF) binds to the tail of the ␤ 2 -adrenergic receptor and plays a role in adrenergic regulation of Na ؉ ͞H ؉ exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the ␤ 2 receptor. Mutagenesis studies of the ␤ 2 receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S͞T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the ␤ 2 receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling.PDZ domains are conserved protein modules that mediate protein-protein interactions (1-3). The term ''PDZ'' is derived from the first letters in the names of the three proteins in which these modules were originally characterized: PSD-95, Dlg, and ZO-1. PDZ domains bind to the C-terminal tails of target proteins, and the binding preferences of a number of PDZ domains have been characterized (4-7). Such characterization is useful because it allows for predictions regarding the set of proteins to which a given PDZ domain may potentially bind.A PDZ domain-containing protein, the Na ϩ ͞H ϩ exchanger regulatory factor (NHERF), has been recently characterized (8) and found to regulate the Na ϩ ͞H ϩ exchanger type 3 (NHE3) (8-9) via an interaction which does not involve binding of the NHE3 C terminus to the NHERF PDZ domains (9). The function and preferences of the two NHERF PDZ domains were unknown until NHERF was identified as a binding partner of the ␤ 2 -adrenergic receptor (10). The interaction of NHERF with the ␤ 2 receptor is mediated via binding of the first PDZ domain of NHERF to the last few amino acids of the ␤ 2 receptor tail (10). In the present study, we characterize this interaction via mutagenesis of the ␤ 2 receptor tail and saturation NHERF-binding studies. We furthermore demonstrate that NHERF-2, a close relative of NHERF, specifically binds to the ␤ 2 receptor tail and exhibits binding specificity similar to NHERF. Moreover, we demonstrate how the binding preferences of the first PDZ domain of the NHERF family of protein...

EBP50, a β-catenin-associating protein, enhances Wnt signaling and is over-expressed in hepatocellular carcinoma

“…Through PDZ domains, EBP50 scaffolds many transmembrane proteins (such as Na ϩ -H ϩ exchanger, ␤2-adrenergic receptor, CFTR, and PDGF receptor), cytoplasmic proteins (YAP, EPI64, and EBP50 itself), and the actin cytoskeleton. [32][33][34][35][36][37][38] Our in vitro binding assay demonstrated that the carboxyl (PDZ-2) and not the amino (PDZ-1) PDZ domain associates with ␤-catenin. PDZ domains associate with carboxyl-terminal sequences such as S/T-X-V/L.…”

Ezrin-radixin-moesin–binding phosphoprotein 50 is expressed at the apical membrane of rat liver epithelia

“…12,13 Their PDZ domains bind a number of distinct proteins, including Na ϩ /H ϩ exchanger 3 (NHE3), ␤2-adrenergic receptor, guanine nucleotide regulated kinase 6A, and CFTR. [14][15][16] Further, a non-PDZ domain at the COOH-terminus of EBP50 and E3KARP is capable of tethering to ERM proteins, a protein family with the capacity to bind both actin and protein kinase A. 17,18 In renal proximal tubule cells, EBP50 or E3KARP interaction with NHE3 is required for cAMP-dependent inhibition of NHE3 activity.…”

Postnatal developmental expression of the PDZ scaffolds Na+-H+ exchanger regulatory factors 1 and 2 in the rat cochlea

“…It is clear from the literature that NHERFs play key roles in mediating a number of transport and signaling phenomena by forming macromolecular complexes of transporters, ion channels, receptors, and structural proteins (Hall et al 1998;Fanning and Anderson 1999;Lederer et al 2003;Pushkin et al 2003;Yun 2003;Fam et al 2005;Weinman et al 2005). The rationale for the current study comes from the finding that the cochlea contains a number of proteins known to interact with NHERFs, plus numerous other proteins that have PDZ domains that could potentially interact with NHERFs.…”

“…Extracellular ATP affects cochlear function by influencing the endocochlear potential, micromechanics, and neurotransmission via P2X and P2Ypurinergic receptors (Housley et al 1999;Lee et al 2001;Housley et al 2002). The interaction of NHERF-2 with the PDZ-binding motif containing P2Y1 receptors (Hall et al 1998;Fam et al 2005) and the localization of P2Y1 receptors in sensory, supporting (e.g., Deiters cells and glia), and secretory cells of the cochlea (Teixeira et al 2000), in which we have shown NHERF-2 expression, indicate a potential role for NHERFs in the modulation of purinergic signaling.…”