A C-terminal motif found in the b2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the NayH exchanger regulatory factor family of PDZ proteins

The Na1yH1 exchanger regulatory factor (NHERF) binds to the tail of the b2-adrenergic receptor and plays a role in adrenergic regulation of Na1yH1 exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the b2 receptor. Mutagenesis studies of the b2 receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-SyT-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 b2 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 Na1yH1 exchanger regulatory factor (NHERF), has been recently characterized (8) and found to regulate the Na1yH1 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 b2-adrenergic receptor (10). The interaction of NHERF with the b2 receptor is mediated via binding of the first PDZ domain of NHERF to the last few amino acids of the b2 receptor tail (10). In the present study, we characterize this interaction via mutagenesis of the b2 receptor tail and saturation NHERF-binding studies. We furthermore demonstrate that NHERF-2, a close relative of NHERF, specifically binds to the b2 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 proteins account for previously reported interactions involving NHERF-2 and allow for predictions regarding other potential NHERFand NHERF-2-binding partners, such as the purinergic P2Y1 receptor and cystic fibrosis transmembrane conductance regulator (CFTR). MATERIALS AND METHODS NHERF PDZ1 Overlays. Hexahistidineand S-tagged NHERF domain 1 fusion protein (‘‘PDZ1’’; residues 1–151 of full-length NHERF) was created via insertion of rabbit NHERF cDNA (8) into pET-30A (Novagen) and expression according to manufacturer’s instructions. Wild-type or mutant human b2 receptor tails (10), as well as human P2Y1 and P2Y2 receptor tails, were expressed as glutathione S-transferase (GST) fusion proteins and examined for NHERF binding via a far Western blot overlay technique. Mutant b2 receptor tail GST constructs were created by PCR amplification from native receptor cDNA’s by using mutant sequence oligonucleotides and inserting the PCR products into the pGEX-2T vector (Pharmacia); all mutations were confirmed via sequencing with an Applied Biosystems 377 automated sequencer. The GST fusion proteins (25 mgylane) were run on 4–20% SDSy PAGE gels (NOVEX, San Diego), blotted, and overlaid with increasing concentrations of NHERF PDZ1 fusion protein in 2% milk and 0.1% Tween-20 in PBS (‘‘blot buffer’’) for 1 hr at room temperature. The blots were then washed three times with blot buffer, incubated for 1 hr at room temperature with an horseradish peroxidase-conjugated anti-S-tag antibody (Novagen) in blot buffer, washed three more times with blot buffer, and visualized via chemiluminescence. To perform saturation-binding curves, equal amounts of a given fusion protein were loaded into multiple lanes, and the resultant blots were cut into one-lane strips. The strips were incubated with increasing concentrations of NHERF PDZ1 fusion protein, and the amount of NHERF binding for each was quantified via The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y958496-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: NHERF, Na1yH1 exchanger regulatory factor; CFTR, cystic fibrosis transmembrane conductance regulator; GST, glutathione S-transferase. Data deposition: The sequence for NHERF-2 reported in this manuscript has been deposited in the GenBank database (accession no. AF035771). ‡To whom reprint requests should be addressed. e-mail: lefko001@ mc.duke.edu.