NHE-RF, a Regulatory Cofactor for Na+-H+Exchange, Is a Common Interactor for Merlin and ERM (MERM) Proteins*

We have identified the human homologue of a regulatory cofactor of Na+-H+ exchanger (NHE-RF) as a novel interactor for merlin, the neurofibromatosis 2 tumor suppressor protein. NHE-RF mediates protein kinase A regulation of Na+-H+ exchanger NHE3 to which it is thought to bind via one of its two PDZ domains. The carboxyl-terminal region of NHE-RF, downstream of the PDZ domains, interacts with the amino-terminal protein 4.1 domain-containing segment of merlin in yeast two-hybrid assays. This interaction also occurs in affinity binding assays with full-length NHE-RF expressed in COS-7 cells. NHE-RF binds to the related ERM proteins, moesin and radixin. We have localized human NHE-RF to actin-rich structures such as membrane ruffles, microvilli, and filopodia in HeLa and COS-7 cells, where it co-localizes with merlin and moesin. These findings suggest that hNHE-RF and its binding partners may participate in a larger complex (one component of which might be a Na+-H+exchanger) that could be crucial for the actin filament assembly activated by the ERM proteins and for the tumor suppressor function of merlin.

Neurofibromatosis 2 (NF2), 1 is a dominantly inherited disorder characterized by bilateral occurrence of vestibular schwannomas and other brain tumors, especially meningiomas, and schwannomas of other cranial nerves and spinal nerve roots (1). The NF2 gene isolated by positional cloning encodes merlin, named for its striking similarity with moesin, ezrin, and radixin, three closely related proteins commonly referred to as the ERM family, a subclass of the protein 4.1 superfamily thought to link cytoskeletal components with proteins in the cell membrane (2,3). The ERM proteins share ϳ78% amino acid identity with each other, and all three are 45-47% identical to merlin (4).
In cultured cells, ERM proteins are highly concentrated in regions of contact between actin filaments and the plasma membrane, acting as possible linkers between integral membrane and cytoskeletal proteins (5)(6)(7)(8)(9). The carboxyl termini of both ezrin and moesin bind directly to actin in vitro (10,11) via a conserved actin binding site present in ezrin, moesin, and radixin but not in merlin. These findings suggest that the carboxyl terminus of the ERM proteins is responsible for their association with the actin-based cytoskeleton. Recently, however, another actin binding site in the amino-terminal domain of ezrin has been characterized in vitro and shown to be conserved in moesin, radixin, and merlin (12). The highly conserved amino-terminal half of the ERM proteins also contains the domain responsible for interaction with membrane proteins, particularly the glycoprotein CD44 (13). ERM-CD44 complexes are also associated with RhoGDI (RhoGDP dissociation inhibitor) (14), and the ERM proteins have been directly implicated in Rho-and Rac-dependent cytoskeletal reorganization in permeabilized cells (15).
We have reported that endogenous merlin localizes to the actin-rich motile regions (i.e. leading and ruffling edges) in human fibroblast and meningioma cells where it co-localizes with actin but is not associated with stress fibers (16). Merlin when overexpressed in cells, however, localizes to membrane ruffles as well as to other actin-rich structures such as microvilli and filopodia thus resembling the ERM proteins (17). Given the similarity between merlin and the ERM proteins, it is likely that merlin's normal function also involves interactions with membrane and cytoskeletal components. We have used the yeast two-hybrid interaction trap strategy to search for proteins that may associate with merlin. Here, we describe a human cDNA (merintc) that interacts not only with merlin, but also with the ERM proteins via their conserved aminoterminal domain. This cDNA encoded the carboxyl terminus of the human homologue of NHE-RF, which was originally identified as a cofactor that mediates protein kinase A inhibition of a renal brush-border membrane Na ϩ -H ϩ exchanger in rabbit (18). Exogenously expressed human NHE-RF also co-localizes with merlin and moesin. Our findings suggest that NHE-RF is a biologically significant interactor for the MERM (merlin and ERM) family of proteins and may be a participant in the activation of the Na ϩ -H ϩ exchange required for actin cytoskeleton reorganization. During the preparation of this manuscript a report appeared showing that NHE-RF binds to ezrin (19).

EXPERIMENTAL PROCEDURES
Molecular Cloning of Merintc Using the Yeast Two-hybrid System-DNA encoding the merlin sequence (aa 8 -595, isoform1) was polymerase chain reaction-amplified and cloned into the yeast LexA DNA binding vector pEG202 (20), and the plasmid was designated as pmerbait.
To identify proteins interacting with merlin isoform 1, yeast strain EGY88 was sequentially transformed with the pmerbait and a human fetal frontal cortex interaction library fused to the activation domain of GAL4 in the plasmid pJG4-5 and selected on ura Ϫ , his Ϫ , trp Ϫ , leu Ϫ plates. A partial merintc clone was obtained (see "Results") and subsequently used as a probe to screen a human fetal brain library in ZAP (Stratagene) by standard techniques to obtain a full-length clone. These clones were sequenced, and sequences were analyzed using software * This work was supported in part by National Institutes of Health Grants NS24279 and NIDCD 03354 (to A. M.), the Deafness Foundation, and Neurofibromatosis Inc. (Massachusetts Chapter). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ The first two authors contributed equally to this work. § Supported in part by an young investigator award from the National Neurofibromatosis Foundation.
provided by the Genetics Computer Group (GCG) and the BLAST network service at the National Center for Biotechnology Information (NCBI) (21).
Protein Fusion Constructs-Full-length (aa 1-595) as well as the amino (aa 1-332) and carboxyl (aa 308 -595) portions of merlin were expressed as GST fusion proteins using the vector pGEX2T (16). Another full-length merlin construct encoding a naturally occurring NF2 missense mutation, Asn 220 3 Tyr (22) was generated by site-directed mutagenesis (Stratagene) and expressed as a GST fusion protein. Similarly full-length (aa 1-577), amino (aa 1-332), and carboxyl (aa 307-577) segments of human moesin were expressed as GST fusion proteins using the vector pGEX4T1. Expression and purification of the GST fusion proteins were performed as described previously for merlin (16) and using the standard method of Smith and Johnson (23) for moesin. For radixin, His 6 -tagged constructs were employed, and these have been described previously (24).
Mammalian Expression-The entire coding sequence of hNHE-RF-(1-358) engineered to have the influenza hemagglutinin (HA) epitope tag at the 5Ј end was cloned into the mammalian expression vector pcDNA3. The entire coding sequence of merlin was expressed as a GFP (green fluorescent protein) fusion protein employing the vector pEGFP-N1 (CLONTECH). These expression constructs were transfected in COS-7 cells and HeLa cells by the calcium phosphate method using the Cell Phect kit (Pharmacia Biotech Inc.) employing 10 -20 g of the plasmid DNA. Transient transfections were performed, and cells were harvested after 72 h.
Antibodies-An antipeptide rabbit polyclonal antibody was raised against the carboxyl-terminal amino acids SLAMAKERAHQKR (aa 328 -340) specific to human NHE-RF by Research Genetics, Inc., Huntsville, AL. The antiserum obtained (NP1) was further affinitypurified. The anti-merlin antibodies and the antibodies for moesin have been described previously (16,25). The hybridoma supernatant for the HA-tag was kindly provided by Dr. Ed Harlow (Massachusetts General Hospital, Boston, MA).
Affinity Precipitation of hNHE-RF from Cell Lysates-COS-7 cell lysates overexpressing hNHE-RF were incubated with 300 pmol of GST-merlin or GST-moesin immobilized on glutathione-Sepharose 4B (GSH) beads. The beads were washed with phosphate-buffered saline containing Pefabloc, resuspended in Laemmli loading buffer, subjected to 10% SDS-PAGE, and immunoblotted with anti-hNHE-RF (affinitypurified NP1). For radixin, COS-7 cell lysates overexpressing the interactor were incubated with 600 pmol of the His 6 -tagged full-length, amino-terminal, or carboxyl-terminal radixin polypeptides. The complexes were separated from the reaction mixture using Ni-NTA beads. The beads were washed (20 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, pH 8.0), and the specifically bound complexes were eluted with the same buffer containing 400 mM imidazole separated on SDS-PAGE gel and detected with NP1 antibody as described above.
Immunofluorescence-The immunofluorescence staining was performed as described previously (16). Anti-HA monoclonal antibody (1: 100) was used as a primary antibody to detect the localization of hNHE-RF. The affinity-purified moesin antibody was used as described earlier (9). Cells were examined on a Nikon microscope using a 40 ϫ 1.3 N.A. and 60 ϫ 1.4 N.A. objectives. For confocal microscopy, cells were examined with Leica TCS-NT 4D scanning laser confocal microscope.

RESULTS AND DISCUSSION
To identify proteins that associate with merlin, we screened a human fetal frontal cortex interaction library with pmerbait. About 10 6 primary transformants were pooled and replated (at a multiplicity of 20) onto galactose leu Ϫ selection plates. Thirtythree colonies, which showed galactose-dependent growth and blue color on leu Ϫ plates and on 5-bromo-4-chloro-3-indolyl ␤-D-galactopyranoside medium, respectively, were identified, and plasmids containing the cDNA clones were isolated. Restriction mapping and hybridization experiments revealed that these cDNAs were clustered into three different groups of overlapping clones, representing three novel cDNAs. In this study we report the characterization of one of the cDNAs, an 880-base pair fragment which we refer to as merintc. We tested merintc for associations in yeast strains containing either the amino-(aa 1-341) or the carboxyl-(aa 342-595) terminal portion of merlin as bait. Results of these tests indicated that merintc specifically associates with full-length and amino-terminal merlin constructs (data not shown).
The largest merintc fusion clone was sequenced, and a BLASTX search of GenBank revealed strong similarity with the carboxyl-terminal segment of rabbit NHE-RF, a protein that mediates protein kinase A inhibition of the renal brushborder membrane Na ϩ -H ϩ exchanger (18). A full-length cDNA clone was isolated from a human fetal brain library and was sequenced, revealing 88% identity (across the open reading frame) with rabbit NHE-RF. The predicted 358 amino acid protein shares 86% identity with both the rabbit protein and its mouse homologue and contains 2 PDZ repeats between amino FIG. 1. A, schematic representation of hNHE-RF. Lines represent the 5Ј-and 3Јuntranslated region. The coding region is indicated by the box. Shaded areas represent the two PDZ domains. The black area (aa 290 -358) represents the MERM binding domain. B, sequence comparison of human NHE-RF with rabbit and mouse NHE-RF and E3KARP. The amino acid identities between these sequences are boxed. The hNHE-RF sequence has been deposited in GenBank™, and the accession number is A5036241. acids 11-97 and 149 -236, with an expected molecular mass of 39 kDa (Fig. 1). Consequently, merintc appears to derive from the human homologue of NHE-RF which we refer to as hNHE-RF. Northern blot analysis of human tissues revealed that hNHE-RF is ubiquitously expressed with highest levels in kidney, liver, and pancreas (data not shown).
To confirm the interaction of full-length hNHE-RF with merlin, we expressed hNHE-RF with a 5ЈHA tag in COS-7 cells and tested its ability to bind with different segments of merlin expressed as bacterial GST fusion proteins and immobilized on GSH beads. We detected the bound protein in immunoblots, using the anti-hNHE-RF antibody (NP1). As shown in Fig. 2, hNHE-RF bound specifically to the amino-terminal and fulllength merlin fusion proteins, but did not bind to either the carboxyl terminus of merlin or to GST alone. Binding to a mutant merlin, encoding an NF2-associated missense mutation Asn 220 3 Tyr, is reduced compared with the wild type (Fig.  2, lane 4). Thus, as was predicted by the two-hybrid analysis, hNHE-RF associates with the amino-terminal but not with the carboxyl-terminal domain of merlin.
Because the amino-terminal domain of merlin shows significant similarity with ERM proteins, we also examined the interaction of NHE-RF with moesin and radixin. We separated GST-moesin and His-tagged radixin complexes from cell lysates expressing hNHE-RF by affinity chromatography. As shown in Fig. 2, hNHE-RF showed a strong association with the amino termini of both moesin and radixin but not with the carboxyl terminus of either protein. The interaction with fulllength radixin (Fig. 2B) is reduced compared with the aminoterminal domain alone. A similar result has been obtained with full-length moesin (data not shown) although no such difference was noted for merlin ( Fig. 2A). The immobilized amino termini of moesin and radixin completely bound and removed hNHE-RF from the lysate, whereas immobilized merlin, incubated with an equivalent amount of hNHE-RF, bound only a fraction of the protein (Fig. 2).
We have also determined the subcellular localization of exogenously expressed hNHE-RF in HeLa cells using immunocytochemistry. hNHE-RF localizes to the ruffling membrane, mi-crovilli, and filopodia reminiscent of the ERM proteins and of overexpressed merlin (Fig. 3). When we co-expressed both merlin and hNHE-RF in COS-7 and HeLa cells, we also observed the co-localization of these two proteins in membrane ruffles, microvilli, and filopodia (Fig. 3A). Double immunostaining of exogenous hNHE-RF and endogenous moesin in HeLa cells revealed co-localization in microvilli, ruffling membrane, and filopodia (Fig. 3B). However, hNHE-RF does not overlap with merlin and moesin in all microvilli and filopodia, and similarly merlin and moesin are not seen throughout the membrane ruffles where we observe hNHE-RF. The frequent co-localization of hNHE-RF with merlin and moesin is, however, consistent with the interaction of these proteins.
We demonstrate here that hNHE-RF, a widely expressed protein whose rabbit homologue was first identified by its involvement in mediating protein kinase A regulation of the renal brush-border Na ϩ -H ϩ exchanger (18) interacts both with merlin and with its close relatives, moesin and radixin from the ERM family. Interestingly, while this manuscript was in preparation, Reczek et al. (19) identified hNHE-RF as a physiologically relevant interactor (which they named EBP50) for ezrin, the third ERM protein.
hNHE-RF possesses two PDZ domains, motifs thought to mediate protein-protein interactions, particularly at the plasma membrane during assembly of components involved in FIG. 2. Affinity precipitation of hNHE-RF from cell lysates. RIPA (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS containing a 1ϫ protease inhibitor mixture) lysates from COS-7 cells overexpressing hNHE-RF were incubated with merlin or moesin expressed as GST fusion proteins and immobilized on GSH beads (A), and radixin expressed with a His-tag and eluted from Ni-NTA beads (B). The beads were extensively washed, separated on 10% SDS-PAGE, and immunoblotted with affinity-purified anti hNHE-RF antibody (NP1). N and C represent the amino and carboxyl regions of merlin, moesin, and radixin. FL represents full-length merlin and radixin, and Mut-Mer is an NF2-associated missense mutation of merlin. GST protein expressed alone is shown as a control (GST). Total cell extracts from either COS-7 cells overexpressing full-length hNHE-RF or wild type COS-7 cells are also shown as controls. The supernatants that were not bound to the beads are shown to point out that the amino portion of moesin is completely bound by hNHE-RF (A). The arrow shows the hNHE-RF at 50 kDa.

FIG. 3. Co-localization of hNHE-RF with merlin and moesin by double immunocytochemistry.
A, merlin is visualized as GFPtagged protein (a, d). Rhodamine-conjugated secondary anti-mouse antibody was used to detect hNHE-RF (b, e). The partial co-localization of these two proteins can be visualized in COS-7 cells at the ruffling membrane (small arrows), microvilli and filopodia (large arrow, c), and similar co-localization in microvilli is observed in HeLa cells (f). Bar, 10 m. B, fluorescein isothiocyanate-conjugated secondary anti-mouse antibody was used to detect hNHE-RFh (a, d), and rhodamine-conjugated secondary anti-mouse antibody was used to detect endogenous moesin (b, e) in HeLa cells. At the top of the cells (a-c), there is strong overlap of these two proteins in the microvilli (c). In the lower part of the cells close to substrate (d-f), the two proteins co-localize very clearly at ruffling membrane and filopodia. Bar, 20 m. cellular signaling (26,27). E3KARP (also called TKA-1), a protein recently isolated as an interactor for the small intestinal and renal proximal tubule brush-border Na ϩ -H ϩ exchanger NHE3, shares 51% identity with hNHE-RF primarily in the PDZ domains (28). However, the interaction with the aminoterminal domain of merlin and the ERM proteins occurs outside of the hNHE-RF PDZ domains, via the carboxyl-terminal region spanning amino acids 290 -358. This suggests that the hNHE-RF and its MERM binding partners might participate in a larger complex, one component of which might be a Na ϩ -H ϩ exchanger.
Multiple forms of Na ϩ -H ϩ exchangers exist in mammalian cells, and at least five of these, designated as NHE1-5, sharing an overall 34 -60% amino acid identity have been cloned and characterized (29). Whereas NHE3 expression is limited to kidney and intestine, NHE1 is a housekeeping protein that is ubiquitously expressed (30). NHE1 has been shown to accumulate in membrane ruffles where vinculin, talin, and F-actin are concentrated suggesting that Na ϩ -H ϩ exchangers are sequestered to these regions by interacting with the cytoskeletal network (31). NHE1 is necessary for Rho-induced stress fiber formation (32).
Our demonstration of NHE-RF as a binding partner for MERM family members suggests a potential functional connection with the involvement of Na ϩ -H ϩ exchange in cytoskeletal rearrangement (Fig. 4). Indeed, a recent study demonstrates that ERM proteins are also essential for Rho-and Rac-induced cytoskeletal changes such as stress fiber assembly, formation of focal adhesions, elaboration of lamellipodia, and membrane ruffling (15). The prospect that MERM-NHE-RF interaction plays a role in these changes and the fact that NHE-RF is expressed in tissues not containing NHE3 raise the question of whether NHE-RF is also capable of regulating other Na ϩ -H ϩ exchangers, particularly NHE1. Interestingly, increased Na ϩ -H ϩ exchange has long been known to be associated with cell proliferation, differentiation, and neoplastic transformation (33). Thus, an intriguing possibility that merits investigation is that merlin-NHE-RF interaction is the basis of merlin's tumor suppressor function since failure of the interaction might abrogate regulation of a Na ϩ -H ϩ exchanger in NF2 target cells and lead to formation of meningiomas and schwannomas.