We have identified the human homologue of a regulatory cofactor 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 twohybrid 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 (5-9). 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 ruf...

Neurofibromatosis 2 (NF2) is a dominantly inherited disorder characterized by bilateral vestibular schwannomas and meningiomas. Merlin, the neurofibromatosis 2 tumor suppressor protein, is related to the ERM (ezrin, radixin, moesin) proteins and, like its family members, is thought to play a role in plasma membrane-cytoskeletal interactions. We report a novel protein as a merlin-specific binding partner that we have named magicin (merlin and Grb2 interacting cytoskeletal protein) and show that the two proteins interact in vitro and in vivo as well as colocalize beneath the plasma membrane. Magicin is a 24 kDa protein that is expressed in many cell lines and tissues. Magicin, similar to merlin, associates with the actin cytoskeleton as determined by cofractionation, immunofluorescence and electron microscopy. Analysis of the magicin sequence reveals binding motifs for the adaptor protein Grb2. Employing affinity binding, blot overlay and co-immunoprecipitation assays, we demonstrate an interaction between Grb2 and magicin. In addition, merlin is capable of forming a ternary complex with magicin and Grb2. These results support a role for merlin in receptor-mediated signaling at the cell surface, and may have implications in the regulation of cytoskeletal reorganization.

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The introduction of human chromosome 17 suppresses the tumourigenicity of a neuroblastoma cell line in the absence of any effects on in vitro growth and the neurofibromatosis type 1 (NF1) gene may be responsible. Here we report that 4 out of 10 human neuroblastoma lines express little or no neurofibromin and that two of these lines show evidence of NF1 mutations, providing further proof that NF1 mutations occur in tumours that are not commonly found in NF1 patients. We also show that NF1 deficient neuroblastomas show only moderately elevated ras-GTP levels, in contrast to NF1 tumour cells, indicating that neurofibromin contributes differently to the negative regulation of ras in different cell types.