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The ezrin protein family: membrane-cytoskeleton interactions and disease associations
659
The ezrin protein family: membrane-cytoskeleton interactions and disease associations Antti Vaheri*, Olli Carp6n, Leena Heiska, Tuula S Helander, Juha J .skel inen, P .ivi Majander-Nordenswan, Markku Sainio, Tuomo Timonen and Ossi Turunen Ezrin, radixin, moesin and merlin form a subfamily of conserved proteins in the band 4.1 superfamily. Ezrin protein subfamily members act as linkers between the plasma membrane and the cytoskeleton. Members of the subfamily have been shown to interact with each other, with cell adhesion molecules such as CD44 and with F-actin. Recent data indicate that intercellular adhesion molecules 1 and 2 also interact with ezrin. The proteins are also involved in the redistribution of intercellular adhesion molecules and the organization of cell membrane structures. Merlin is a tumor suppressor that is involved in tumorigenesis of schwannomas and meningiomas. Merlin has the same overall protein structure as the other proteins in the subfamily but may have partially distinct functions.
Addresses Haartman Institute, University of Helsinki, POB 21, FIN-O0014 Helsinki, Finland *e-mail: Antti.Vah eri@ helsinki.fi Correspondence: Antti Vaheri Current Opinion in Cell Biology 1997, 9:659-666
Although human ERM proteins share 75-80% identity with each other, human merlin shares about 45% sequence identity with ERM proteins. Although this suggests that merlin may have different functions to those of ezrin, radixin and moesin, the functional data obtained to date are sufficient to allow us to regard merlin as a functional member of the ERM family. In this review, we use the term ezrin protein family to include all the ERM proteins and merlin. T h e ERM proteins accumulate underneath the plasma membrane in various cell surface structures, such as microvilli, membrane ruffles and cell-cell contact sites. Ezrin has been localized by immunoelectron microscopy between the plasma membrane and the actin core structure in microvilli [3-5], suggesting a role as a linker between cell surface molecules and the actin cytoskeleton [6]. During recent years, information clarifying the biological functions of the ezrin protein family has rapidly accumulated. In this review, we focus on the membrane-cytoskeleton linker and tumor-suppressor roles of the family.
http://biomednet.com/elecref/0955067400900659 Actin-binding
© Current Biology Ltd ISSN 0955-0674 Abbreviations ERM ezrin, radixin, moesin ICAM intercellular adhesion molecule merlin moesin-ezrin-radixin-like protein NF2 neurofibromatosistype 2 NK natural killer PIP 2 phosphatidylinositol 4,5-bisphosphate
Introduction
to the ezrin
protein
family
Ezrin, the prototype member of the ezrin protein subfamily, was characterized as a component of brush-border and placental microvilli in the early 1980s [1,2]. T h e family now consists of four members in vertebrates, namely, ezrin, radixin, moesin (ERM proteins) and merlin (moesin-ezrin-radixin-like protein; also named schwannomin). Homologous proteins have been discovered in many invertebrate species, including Drosophila, Caenorhabditis elegans, sea urchin and parasites (Figure 1). T h e structure of all family members consists of an amino-terminal globular domain followed by an s-helical region and a carboxy-terminal domain. T h e family belongs to the band 4.1 superfamily on the basis of sequence homology of the amino-terminal domain with the erythrocyte membrane-cytoskeleton linker protein band 4.1.
ability
of ERM
proteins
Early attempts to demonstrate a direct interaction between ezrin and actin, using sedimentation assays for example, were disappointing. Weak binding was observed in nonphysiological but not in physiological salt concentrations [1,4]. However, it was observed that, following detergent extraction, ezrin remained partly in the cytoskeletal fraction [1,4]. Analysis of the ezrin cDNA sequence gave further support to an association of ezrin with the cytoskeieton. T h e amino-terminal domain of ezrin showed significant identity with band 4.1, the membrane-cytoskeleton linker in erythrocytes, and the a-helical domain was similar in sequence to domains of various cytoskeletal proteins [7,8]. T h e revelation of a masked actin-binding site in ezrin gave direct evidence that ERM proteins interact with the cytoskeleton. F-actin binding was detected in a truncated ezrin protein lacking the amino-terminal part [9]. In addition, full-length ERM proteins denaturated by SDS also showed F-actin binding [10]. These results suggest that the amino-terminal domain regulates actin binding. In line with this, head-to-tail binding in ERM proteins was reported, with the amino-terminal domain capable of binding the carboxy-terminal domain [11]. T h e interaction between the amino-terminal domain and the carboxyterminal domain has also been observed at another level,
660
Cell-to-cell contact and extracellular matrix
Figure 1
Amino-terminal domain I
~domain I
Carboxyterminal P domain II I
86%
69%
61%
86%
61%
62%
78%
26%
43%
76%
25%
57O/o
19O/o
61%
290/0
55%
20%
24%
47O/o
15%
15%
32O/o
80/0
Length (aa)
Protein
Overall sequence identity
585
Human ezrin
100%
583
Human radixin
77%
577
Human moesin
73%
578
Drosophilamoesin
55%
572
Sea urchin moesin
51%0
559
E, multilocularis EM 10
41%
595
Human merlin
43%
635
Drosophilamerlin
36%
654
C. elegans merlin
28O/o
587
Human band 4.1
22%
30%
37O/o
22%
150/o © 1997 Current Opinion in Cell Biology
The ezrin protein family. To date, a large number of genes encoding ezrin-like proteins have been cloned in a variety of species. The invertebrate ezrin-like proteins are Drosophila moesin and merlin, sea urchin moesin, the ezrin-like protein whose gene was cloned from the nematode C. elegans (this C. e/egans protein displayed properties typical of merlin, as shown by, for example, phytogenetic analysis and similarity in the carboxy-terminal 30 amino acids; O Turunen, A Vaheri, unpublished data) and E. mu/tilocularis EM10. All of these belong to the ERM family, with the exception of the merlins in Drosophila and C. elegans. Although the mammalian proteins are highly conserved, the ezrin-like proteins do not share high identity between mammals and invertebrates. The amino-terminal domain is very conserved across species, but the sequence identity between mammalian and invertebrate ezrin-like proteins can be very low in the (~ domain and carboxy-terminal domain, even as low as the homology of band 4.1 with ERM proteins. The amino-terminal domain is probably quite globular; the (x domain is probably helical according to computer analysis; the carboxy-terminal domain is also largely helical. The proline-rich region (P) interrupts the long c( helix in the middle of the protein in human ezrin and radixin, and in the merlins. In addition, mammalian moesin has a proline in the same position (not shown). Band 4.1 also has a group of prolines in the corresponding region and short amino acid motifs (not shown) in the carboxy-terminal domain are shared by ERM proteins and band 4.1. Percentage identities between different domains of the proteins are shown beneath the relevant domains; each percentage relates to similarity of the domain to human ezrin. Identities include gaps in the protein sequences that were introduced to maximize alignment. The black boxes indicate the actin-binding site of the ERM proteins. In the merlins, the role of the corresponding carboxy-terminal region (grey boxes) is unclear, aa, amino acids.
as the amino-terminal domain inhibits the morphogenic (cell extension) activity of the carboxy-terminal part [12]. Recently, it has also been shown that purified cellular ezrin interacts with actin, particularly the 13-actin isoform. Other studies demonstrated that ezrin could be directly co-sedimented with 13-actin, but only a weak association with ~-actin was observed. Furthermore, ezrin was shown to preferentially promote ~-actin polymerization [13"'].
It has also been reported that ezrin interacts indirectly with 13-actin filaments via the p73 protein, but not with a-actin filaments [14,15]. Although direct evidence is missing, a possible explanation for the conflicting binding results is that the isolated ezrin exists in different forms, depending on the cellular source. Yao etal. [13"'] isolated ezrin from gastric mucosa containing several ezrin isoforms, which were possibly the result of differences in phosphorylation. Furthermore, the indirect association of ezrin with l~-actin
The ezrin protein family Vaheri et a/.
was reported to occur at the barbed (fast-growing) end of actin [15], whereas the direct binding of ezrin to actin was observed at the filament sides [13°°]. These results suggest the existence of different physiologically significant modes of binding. Radixin was originally detected as a barbed end capping protein [16]. However, the barbed end binding was observed only in nonphysiological salt concentrations. The immunofluorescence data and binding experiments show that radixin also binds to the sides of actin filaments [16]. The data on radixin also showed that the barbed end capping activity inhibits actin polymerization [16], whereas ezrin appears to stimulate actin polymerization [13°°]. The role of the barbed end capping activity is not yet clear. The sequence similarity between the actin-binding sites of ERM proteins and of the barbed end capping protein CapZ 13 subunit may indicate that the barbed end capping activity is functional in the cells as well as in nonphysiological salt concentrations. Consequently, it is possible that ERM proteins regulate the length of actin filaments, by both inhibiting and stimulating actin polymerization. The actin-polymerizing activity probably accounts for the morphogenic effect of ERM proteins: both truncated and full-length ERM proteins induce cell surface extensions [12,17,18,19°']. Merlin also organizes cell surface structures, although we do not yet know how the protein affects actin polymerization [20]. The actin-binding site localizes to a few carboxy-terminal amino acids of ezrin and moesin [9,10]. In ezrin, the last 34 amino acids bind F-actin [9]. As there is high carboxy-terminal sequence identity (80%) between mammalian ERM proteins, moesin and radixin are also likely to have the same actin-binding site. The actin-binding site is highly conserved, even in invertebrate proteins. However, the corresponding site in merlin has a partially different sequence, also indicating a distinct function.
Regulation of ERM proteins Phosphorylation appears to regulate the binding of ERM proteins to actin, and is induced by several growth factors. The conformationally masked carboxy-terminal actin-binding site is thought to be opened by signals such as phosphorylation [11,21 ]. The regulation of actin-binding by phosphorylation appears to be mediated by several alternative mechanisms. Ezrin is phosphorylated at least at two tyrosine residues, Tyr145 and Tyr353, when A431 carcinoma cells are stimulated by epidermal growth factor (EGF), and ezrin is subsequently redistributed to microvilli [22,23]. The EGF-induced redistribution of ezrin involves the formation of ezrin dimers and oligomers [22,24]. In stimulated T lymphocytes, the tyrosine kinase p561ck is involved in the tyrosine phosphorylation of ezrin [25]. Serine/threonine phosphorylation regulates the distribution of ezrin in gastric parietal cells [26,27] and constitutive
661
serine/threonine phosphorylation of merlin has been observed [28]. cAMP is involved in the regulation of ezrin by serine/threonine phosphorylation in gastric parietal cells. A more general role for ezrin in cAMP-dependent cell regulation is revealed by the recent finding that ezrin functions as a protein that anchors the cAMP-dependent protein kinase [29°°]. It appears that ERM proteins can be regulated by phosphorylation of different protein domains. Tyr145 is located in the amino-terminal domain and Tyr353 in the a-helical domain, and phosphorylation of the carboxyterminal domain may also regulate the actin-binding ability of ERM proteins. Phosphorylation of Thr558 in only the carboxy-terminal actin-binding region of moesin occurs within minutes in thrombin activation of human platelets [30]. Activated platelets form extremely long filopodia and the phosphorylated moesin co-localizes with F-actin. Additional evidence for the tight association between ERM protein phosphorylation and their subcellular distribution can be seen following anoxic injury in the renal proximal tubule brush border, when rapid serine/threonine dephosphorylation of ezrin occurs and interaction of ezrin with the microvillar cytoskeleton is lost [31]. Inhibition of mast cell stimulation (which occurs by cross-linking receptor-bound IgE) by an antiallergic drug induces serine phosphorylation of moesin [32]. In macrophages, Thr558-phosphorylated moesin is found to be co-localized with F-actin in filopodial protrusions [33]. Treatment of H T l l 5 colon carcinoma cells with hepatocyte growth factor/scatter factor induced tyrosine phosphorylation of ezrin and its translocation from the cytosol to areas of ruffled membrane [34]. Recent evidence [35°,36,37,38 °°] suggests that Rhomediated signal transduction is involved in the activation of ERM proteins. The GTP-binding protein Rho regulates actin-dependent cellular events and organization of cell surface structures, and functions as an upstream factor in the regulation of ERM proteins. ERM proteins may also be regulated by phosphatidylinositol 4,5-bisphosphate (PIPz), which increases ERM protein binding to CD44, intercellular adhesion molecule (ICAM)-I and ICAM-2 ([38°°]; L Heiska et aL, unpublished data). The nature of the regulatory effect of PIP 2 is unclear but could involve conformational changes in ERM proteins in a manner analogous to PIP 2 regulation of vinculin activity [39].
Does merlin associate with the cytoskeleton? The mechanism of the cytoskeletal association of the fourth member of the ezrin protein family, merlin, has not yet been resolved. Merlin, like the ERM proteins, is localized to cortical cytoskeletal structures in cells and is also partly retained in the detergent-insoluble fraction in cell lysates [20,40,41]. The carboxy-terminal region corresponding to the ERM actin-binding site differs from
662
Cell-to-cell contact and extracellular matrix
that of ERM proteins, but still contains a number of basic amino acids that could, in principle, mediate binding to actin. Actin binding was not, however, observed in either full-length or truncated merlin ([28]; O Turunen, A Vaheri, unpublished data) in the chromatographic binding assay that revealed the actin-binding activity of ezrin [9]. Merlin's association with the cytoskeleton may differ from that of ERM proteins. Merlin may have a cytoskeleton-binding site in the middle of the protein, like band 4.1 [42]; the cytoskeleton association may be due to heterodimerization with ERM proteins; or other factors may be required for the cytoskeleton association. T h e overexpression of full-length merlin induces cell surface protrusions and elongation of cells [20]. Morphogenic effects at the cell surface, including blebbing, indicate extensive reorganization of the cortical cytoskeleton. This could occur as a result of distinct cytoskeleton-interacting properties of merlin.
Interactions family
between
members
of the ezrin
Ezrin can be isolated from cell extracts as monomers, dimers and oligomers [21,24,43]. However, attempts to generate dimers from monomers in solution have been unsuccessful. Monomers are very stable structures and dimerization apparently requires activation. However, ERM proteins readily associate when assayed by blot overlay. In these assays, ERM proteins displayed homotypic and heterotypic binding [AA n.6]. An important recent advance has been the demonstration [11] that ezrin self-association involves the binding of the amino-terminal domain (mapped to residues 1-296) to the carboxy-terminal domain (last 107 residues of the protein, 479-585). T h e self-association could also explain the inhibition of ezrin's morphogenic activity by its amino-terminal domain [12]. However, the in vivo role of heterodimerization in the regulation of ERM proteins and merlin is, as yet, unknown. The self-association may in principle occur in several ways, by the formation of head-to-tail dimers, parallel dimers and head-to-tail oligomers. Heterodimerization could modulate binding activity in the parallel dimers of ERM proteins, but in the oligomer-binding model (see Figure 2) the modulatory role of heterodimerization is not as evident. T h e interdomain interaction is not confined to the ERM family as the amino-terminal domain of radixin also binds to band 4.1 [46]. This raises the possibility that the activity of the whole band 4.1 protein family, including the tyrosine phosphatases, is regulated by similar mechanisms. In addition, ERM proteins may share other binding partners with band 4.1. Both band 4.1 and ezrin have been shown to interact in a co-precipitation complex with the murine form of hDlg, a homologue of the Drosophila Dlg tumor suppressor protein [47].
Figure 2
ICAM-1 ICAM-2 CD44 Cell membrane I ; ERM proteins PIP2
I PIP2
PIP2
Merlin.'? F-actin
ini n in
II
o ogy
i I
Model of the cell adhesion molecule-F-actin linker role of ERM proteins. The interaction of ERM proteins with actin filaments may occur such that the extended ERM molecule lies on the actin filament but only the carboxyl terminus (small shaded oval) actually binds actin. In this model, the self-association of the ERM proteins occurs in tandem with the binding of the amino-terminal domain (large shaded oval in ERM protein) of one protein to the carboxy-terminal domain of another [94]. Another possibility is that ERM proteins form parallel dimers that interact with actin filaments, such that each dimer would bind to the actin filament by a pair of actin-binding sites (not shown). In this alternative model, there is no place for an amino-terminal domain to carboxy-terminal domain interaction in the active molecule. Instead, the self-association could mainly be a way to inactivate the protein. On the other hand, the parallel dimer model may allow the modulation of the ERM protein activity by heterodimerization, which is not as evident in the head-to-tail oligomer-binding model. PIP2 is important in activating ERM protein interactions with adhesion molecules (ICAM-1, ICAM-2 and CD44) and is thus indicated in the figure. If merlin lacks the carboxy-terminal actin-binding site, it may not interact with actin in the way proposed by the tandem model.
Membrane protein protein family
interactions
with
the ezrin
T h e homology between the amino-terminal domains of ezrin and members of the band 4.1 superfamily suggested that this domain may interact with membrane proteins, as the amino-terminal domain of band 4.1 interacts with glycophorin C, a major integral membrane protein in erythrocytes. This suggestion was supported by the observation that the amino-terminal domain of ezrin transfected into cultured cells associates with the plasma membrane [6]. By using antisense oligonucleotides, it was observed that the function of ERM proteins is critical to maintaining the cell adhesion [48]. CD44 was the first cell surface protein with which ERM proteins were demonstrated to interact [38**]. T h e binding of ERM proteins to CD44 is regulated by the GTP-binding protein Rho [35",38°°] and by phosphoinositides, particularly PIP 2 [38*']. Recombinant merlin also interacts with CD44 [20]. Our recent studies using ICAM cytoplasmic domain peptides and purified ezrin (L Heiska et al., unpublished data) demonstrate molecular interactions between ezrin and two members of the immunoglobulin superfamily, ICAM-1 and ICAM-2. These interactions are greatly promoted by the presence of
The ezrin protein family Vaheri et a/.
PIP z, which was earlier shown to bind the amino-terminal domain of ezrin [49]. ERM proteins may play an active role in targeting the cell adhesion molecules to specific cell surface regions. Transfected ezrin redistributes ICAM-2 to newly formed cellular protrusions (uropods) on the surface of tumor cells, which then become susceptible to lysis by natural killer (NK) cells [19"']. As ezrin interacts directly with the cytoplasmic tail of ICAM-2 (L Heiska et al., unpublished data), it is possible that direct interaction with ezrin drives ICAM-2 to the uropods, creating a surface structure that is recognized by NK cells (see Timonen and Helander, this issue, pp 667-673). In addition, ezrin may also be involved in other immunological events, such as stimulation of T lymphocytes [25]. T h e subcellular redistribution of adhesion molecules may also greatly facilitate cellular adhesion and other cell functions. T h e reorganization of cell adhesion molecules by ezrin could also be associated with NK cell killing in other pathological conditions, such as virus infections. Redistribution of ezrin has been observed to occur following infection of human fibroblasts with either herpes simplex virus or Semliki Forest virus [50]. Redistribution of uniformly located ezrin into newly formed microvilli occurs in a few hours, without any apparent change in ezrin quantity. Other cell adhesion molecules, like CD46, a complementbinding protein, and the highly glycosylated adhesion molecule CD43, may also interact with the ezrin protein family [37]. CD46 appears to interact with moesin in chemical cross-linking and co-immunoprecipitation experiments [51 ]. Merlin a s a t u m o r s u p p r e s s o r
Merlin is the product of the neurofibromatosis type 2 (NF2) gene [52,53]. It is a tumor suppressor protein that is defective or absent in NF2, a rare autosomal dominant disease (found in 1 in 35,000 people) that predisposes individuals to multiple nervous system tumors, vestibular and spinal schwannomas, meningiomas and ependymomas, and also to juvenile posterior lenticular opacities [54]. The NF2 gene is also somatically mutated in the majority of mesotheliomas [55]. The NF2 gene is located on human chromosome 22q12. In NF2, both germline and somatic mutations span the gene, with no clear 'hot spots' [54]. Like ERM proteins, merlins belong to the most conserved group of mammalian proteins. T h e mouse merlin shares 98% identity with human merlin, whereas the average identity of orthologous mouse and human proteins is 85% [56]. Mammalian merlin is expressed mainly in the nervous system and cells of the lens and is predominantly located in membrane ruffles and cellular protrusions. It is partially co-localized with ezrin, CD44 and F-actin [20,40,41,57,58].
663
In Drosophila, the distributions of the only Drosophila ERM proteins, moesin and merlin, are different, moesin being found on the plasma membrane and merlin on punctate structures in the membrane and cytoplasm [59"]. Like ERM proteins, merlin may also regulate cell adhesion. Antisense oligodeoxynucleotides to merlin inhibited cell adhesion [60] as has been observed for ezrin, radixin and moesin [48]. Transfection of the merlin gene suppresses a v-Ha-Ras-induced malignant phenotype [61] and inhibits the growth of NIH 3T3 cells [62]; on the other hand, transfection of ezrin appears to promote cell growth via loss of contact inhibition [63]. The growth-inhibiting activity of merlin is consistent with its role as a tumor suppressor. T h e inactivation of merlin is therefore probably associated with loss of cell growth control. T h e functional similarity between human merlin and ERM proteins raises the possibility that the whole protein family is associated with tumorigenesis. No cancer susceptibility syndromes have been linked as yet to the ezrin gene at chromosome position 6q25-26 [7,64], the radixin gene at 1lq12 [65] or the moesin gene at Xql 1.2-12 [66]. In vitro, however, high expression of ezrin in cultured cells causes cell transformation and is associated with cell proliferation [63,67]. Antisense oligonucleotides inhibit the ERM protein function and disrupt cell adhesion; lack of adhesion might contribute to metastasis [48]. Ezrin regulates ICAM-2-dependent tumor cell recognition and killing by NK cells [19°']. High levels of ezrin can be detected in the stromal cells of hemangioblastoma [68], a tumor associated with mutations in the von Hippel Lindau tumor suppressor gene. Also, moesin and radixin are upregulated by platelet-derived growth factor in experimental mesangial proliferative glomerulonephritis in rats [69]. These results suggest that disease associations for ERM proteins may exist. As the genomic structures of the human ezrin gene (it is 24 kilobases long and includes 13 exons) (P Majander-Nordenswan et al., unpublished data) and human moesin gene (which is >30 kilobases long and has 12 exons) [66] have now been revealed, their disease association can be studied more systematically by mutation screening. Conclusions
Ezrin, radixin, moesin and merlin appear to be proteins that are involved in the regulation of cell morphology, particularly of cell surface projections. Ezrin, radixin and moesin, and possibly also merlin, seem to function as linkers between cell adhesion molecules and the cytoskeleton, and they may organize the distribution of cell adhesion molecules. Furthermore, new findings demonstrate that all of these proteins may all play an active role in tumor biology. Particularly, it appears that killer cells may distinguish malignant cells through ezrin-controlled distribution of cell adhesion molecules. Protein phosphorylation appears to be critical to the
664
Cell-to-cell contact and extracellular matrix
activation of ERM proteins, resulting in the redistribution of ERM proteins and of intercellular adhesion molecules.
6.
Algrain M, Turunen O, Vaheri A, Louvard D, Arpin M: Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker. J Cell Bio/1993, 120:129-139.
T h e members of the ezrin protein family may interact with additional integral membrane proteins, and thus the important issue is how the interactions are regulated in each case. T h e search for cytoplasmic proteins that may also interact with and regulate the ERM proteins and merlin will be particularly interesting.
7.
TurunenO, Winqvist R, Grzeschik K-H, Pakkanen R, Wahlstr~m T, Vaheri A: Cytovillin, a microvillar Mr 75,000 protein, eDNA sequence, prokaryotic expression, and chromosomal localization. J Biol Chem 1989, 264:16727-16732.
8.
Gould KL, Bretscher A, Esch FS, Hunter T: cDNA cloning and sequencing of the protein-tyrosine kinase substrata, ezrin, reveals homology to band 4.1. EMBO J 1989, 8:4133-4142.
9.
TurunenO, Wahlstr6m T, Vaheri A: Ezrin has a COOH-tarminal actin-binding site that is conserved in the ezrin protein family. J Cell Biol 1994, 126:1445-1453.
10.
Pestonjamasp K, Amieva MR, Strassel CP, Nauseef WM, Furthmayr H, Luna EJ: Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes. Mo/ Biol Cell 1995, 6:247-259.
11.
Gary R, Bretscher A: Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site. Mol Biol Cell 1995, 6:1061-1075.
12.
Martin M, Andr6oli C, Sahuquet A, Montcourrier P, Algrain M, Mangeat P: Ezrin NH2-terminal domain inhibits the cell extension activity of the COOH-terminal domain. J Cell Bio/
As a structural protein, merlin is a novel type of tumor suppressor. We do not yet know how the tumor suppressor function is mediated and whether other members of the family share a similar function. Merlin-associated tumorigenesis occurs through inactivation of the protein, whereas the promotion of cell growth by ERM proteins is associated with increased expression levels in vitro. It may be that any association of ERM proteins with diseases differs from that of merlin. On the other hand, the inhibition of protein function of both ERM proteins and merlin disrupts cell adhesion. Future studies should clarify the differences and similarities between merlin and ERM proteins. T h e ezrin family proteins seem to form part of a network of protein interactions. An understanding of the structure-function relationships of this family is just emerging. There are many open questions about the interaction of ERM proteins with actin. T h e mechanisms underlying binding to cell adhesion molecules are even less well understood. Several modes of interaction could exist, as it is known that ERM proteins are located in different cellular structures, such as microvilli, ruffles and cell-cell contact sites. T h e cell type may also affect functional properties. A detailed structural analysis may reveal which structural properties are important in the specialized functions of each protein.
1995, 128:1081-1093.
13. ;e
Yao X, Chang L, Forte JG: Biochemical characterization of ezrin-actin interaction. J Biol Chem 1996, 271:7224-7229. authors of this paper studied the interaction of ezrin with actin by using purified proteins. The main results were that ezrin binds selectively to ~-actin, but only weakly to (~-actin, and that ezrin stimulates actin polymerization.The stronger interaction with 13*actinis significant as both ezrin and ~-actin have a similar location in the vicinity of the cell membrane. 14.
Shuster CB, Herman IM: Indirect association of ezrin with Factin: isoform specificity and calcium sensitivity. J Cell Biol 1995, 128:837-848.
15.
Shuster CB, Lin AY, Nayak R, Herman IM: bCAP73: a novel actin-specific binding protein. Cell Motil Cytoskeleton 1996, 354:1 75-187.
16.
TsukitaS, Hieda Y, Tsukita S: A new barbed end-capping protein localized in the cell-to-cell adherens junction: purification and characterization. J Cell Biol 1989, 108:2369-2382.
17.
Edwards KA, Montague RA, Shepard S, Edgar BA, Erikson RL, Kiehart DP: Identification of Drosophila cytoskeletal proteins by induction of abnormal cell shape in fission yeast. Proc Nat/ Acad Sci USA 1994, 91:4589-4593.
18.
Henry MD, Gonzalez-Agosti C, Solomon F: Molecular dissection of radixin: distinct and interdependent functions of the aminoand carboxy-terminal domains. J Cell Bio11995, 129:10071022.
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of special interest of outstanding interest Bretscher A: Purification of an 80,000-dalton protein that is a component of the isolated microvillus cytoskeleton, and its localization in nonmuscle cells. J Cell Bio/1983, 97:425-432. Suni J, N~.rv~nen A, WahlstrSm T, Pakkanen R, Aho M, Vaheri A, Copeland T, Cohen M, Oroszlan S: Human placental syncytiotrophoblastic Mr 75000 polypeptide defined by antibodies to a synthetic peptide based on cloned human endogenous retroviral DNA sequence. Proc Nat/Acad Sci USA 1984, 81:6197-6201.
3.
Pakkanen R, Hedman K, Turunen O, WahlstrSm T, Vaheri A: Microvillus-specific Mr75 000 surface protein of human choriocarcinoma cells. J Histochem Cytochem 1987, 35:809816.
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Bretscher A: Microfilament structure and function in the cortical cytoskeleton. Annu Rev Cell Biol 1991, 4:337-374.
5.
Berryman M, Franck Z, Bretscher A: Ezrin is concentrated in the apical microvilli of a wide variety of epithelial calls whereas moesin is found primarily in endothelial cells. J Cell Sci 1993, 105:1025-1043.
19. oo
Helander TS, Carp~n O, Turunen O, Kovanen PE, Vaheri A, Timonen T: ICAM-2 redistributed by ezrin as a target for killer cells. Nature 1996, 382:265-268. Ezrin was observed to organize cell surface structures and the localization of intercellular adhesion molecule-2 (ICAM-2). Transfection of ezrin cDNA to thymoma cells induced the formation of cell extensions (uropods) and ICAM-2 was redistributed to the newly formed uropods. The concentration of ICAM-2 in uropods sensitizes the ezrin-transfected thymoma cells to natural killer cell killing. 20.
Sainio M, Zhao F, Heiska L, Turunen O, den Bakker M, Zwarthoff E, Lutchman M, Rouleau G, J~i~,skel~iinenJ, Vaheri A, Carp~n O: Neurofibromatosis 2 tumor suppressor protein colocalizes with ezrin and CD44 and associates with actin-containing cytoskeleton. J Cell Sci 1997, in press.
21.
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22.
Bretscher A: Rapid phosphorylation end reorganization of ezrin and spectrin accompany morphological changes induced in A-431 cells by epidermal growth factor. J Cell Bio/1989, 108:921-930.
23.
Krieg J, Hunter T: Identification of two major epidermal growth factor-induced tyrosine phosphorylation sites in the microvillar core protein ezrin. J B/o/Chem 1992, 267:19258-19265.
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25.
Thuillier L, Hivroz C, Fagard R, Andreoli C, Mangeat P: Ligation of CD4 surface antigen induces rapid tyrosine phosphorylation of the cytoskeletsl protein ezrin. Cell Immunol 1994, 156:322-331.
41.
26.
Urushidani T, Hanzel DK, Forte JG: Characterization of an 80kDa phosphoprotein involved in parietal cell stimulation. Am J Physiol 1989, 256:G1070-G1081.
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42.
27.
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Schischrnanoff PO, Winardi R, Discher DE, Parra MK, Bicknese SE, Witkowska HE, Conboy JG, Mohandas N: Defining of the minimal domain in protein 4.1 involved in spectrin-actin binding. J Biol Chem 1995, 270:21243-21250.
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28.
Takeshima H, Izawa I, Lee PS, Safdar N, Levin VA, Saya H: Detection of cellular proteins that interact with the NF2 tumor suppressor gene product. Oncogene 1994, 9:2135-2144.
Pakkanen R, Vaheri A: Cytovillin and other microvillar proteins of human choriocarcinoma cells. J Cell Biochem 1989, 41:1-12.
44.
Gary R, Bretscher A: Heterotypic and homotypic associations between ezrin and moesin, two putative membranecytoskeletal linking proteins. Proc Nat/Acad Sci USA 1993, 90:10846-10850.
45.
Andreoli C, Martin M, Le-Borgne R, Reggio H, Mangeat P: Ezrin has properties to self-associate at the plasma membrane. J Cell Sci 1994, 107:2509-2521.
46.
Magendantz M, Henry MD, Lander A, Solomon F: Interdomain interactions of radixin in vitro. J Biol Chem 1995, 270:2532425327.
29. ,•
Dransfiald DT, Bradford A.I, Smith J, Martin M, Roy C, Mangeat PH, Goldenring JR: Ezrin is a cyclic AMP-dependent protein kinase anchoring protein. EMBO J 1997, 16:35-43. Ezrin was identified as a protein that sequestrates the cAMP-dependant protein kinase (A-kinase). A binding site for the type It A-kinase regulatory subunit (RII) was identified in the co-helical domain of ezrin. Radixin and moesin also had the same binding activity. This study shows that ezrin may redistribute proteins other than cell adhesion molecules in cell surface structures. 30.
Nakamura F, Amieva MR, Furthmayr H: Phosphorylation of threonine 558 in the carboxyl-terminal actin-binding domain of moesin by thrombin activation of human platelets. J Biol Chem 1995, 270:31377-31385.
47.
Lue RA, Brandin E, Chan EP, Branton D: Two independent domains of hDIg are sufficient for subcellular targeting: the PDZ1-2 conformational unit and an alternatively spliced domain. J Cell Biol 1996, 135:1125-1137.
31.
Chen J, Cohn JA, Mandel I1: Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury. Proc Nat/Acad Sci USA 1995, 92:7495-7499.
48.
32.
Correia I, Wang L, Pang X, Theoharides TC: Characterization of the 78 kDa mast cell protein phosphorylated by the antiallergic drug cromolyn and homology to moesin. Biochem Pharmacol 1996, 52:413-424.
Takeuchi K, Sato N, Kasahara H, Funayama N, Negafuchi A, Yonemura S, Tsukita Sa, Tsukita Sh: Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members. J Cell Biol 1994, 125:1371-1384.
49.
Niggli V, Andr6oli C, Roy C, Mangeat P: Identification of a phosphatidylinositol-4,5-biphosphate-binding domain in the N-terminal region of ezrin. FEBS Lett 1995, 376:172-176.
50.
Pakkanen R, yon Bonsdorff C-H, Turunen O, Wahlstr6m T, Vaheri A: Redistribution of Mr 75000 plasma membrane protein, cytovillin, into newly formed microvilli in herpes simplex and Semliki Forest virus infected human embryonal fibroblasts. Eur J Ceil Biol 1988, 46:435-443.
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Schneider-Schaulies J, Dunster LM, Schwarz-Albiez R, Krohne G, ter Meulen V: Physical association of moesin and CD46 as a receptor complex for measles virus. J Virol 1995, 69:22482256.
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Rouleau GA, Merel P, Lutchman M, Sanson M, Zucman J, Marineau C, Hoang-Xuan K, Demczuk S, Desmaze C, Plougastel B et al.: Alteration in a new gene encoding a putative membraneorganizing protein causes neurofibromatosis type 2. Nature 1993, 363:515-521.
53.
Trofatter JA, MacCollin MM, Rutter JL, Murrell JR, Duyso MP, Parry DM, Eldridge R, Kley N, Menon AG, Pulaski K et al.: A novel moesin-, ezrin-, radixin-like gene is a candidate for the neurofibromatosis 2 tumor suppressor. Cel/ 1993, 72:791-800.
33.
Nakamura F, Amieva MR, Hirota C, Mizuno Y, Furthmayr H: Phosphorylation of 5581" of moesin detected by site-specific antibodies in RAW264.7 macrophages. Biochem Biophys Res Commun 1996, 226:650-656.
34.
Jiang WG, Hiscox S, Singhrao SK, Puntis MCA, Nakamura T, Mansel RE, Hallett MB: Induction of tyrosine phosphorylation and translocation of ezrin by hepatocyta growth factor/scatter factor. Biochem Biophys Res Commun 1995, 217:1062-1069.
35. •
Kotani H, Takaishi K, Sasaki T, Takai Y: Rho regulates association of both ERM family and vinculin with the plasma membrane in MDCK cells. Oncogene 1997, 14:1705-1713. By microinjecting Rho guanine nucleotide dissociation inhibitor and C3, inhibitors of Rho, into cells, the authors showed that the inhibition of Rho inhibited the localization of ezrin, radixin, moesin (ERM) proteins to peripheral structures. On the other hand, it was observed that factors other than Rho may also be required to induce the redistribution of ERM proteins in the plasma membrane. 36.
Tsukita S, Yonemura S, Tsukita S: ERM (ezrin/redixin/moesin) family: from cytoskeleton to signal transduction. Curr Opin Cell Bio11997, 9:70-75.
54.
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Tsukita S, Yonemura S, Tsukita S: ERM proteins: head-to-tail regulation of actin-plasma membrane interaction. Trends Biochern Sci 1997, 22:53-58.
Gutmann DH: Molecular insights into neurofibromatosis 2. Neurobiol Disease 1997, 3:247-261.
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Sakido Y, Pass HI, Bader S, New DJY, Christman MF, Gazdar AF, Minna JD: Neurofibromatosis type 2 (NF2) gene is somatically mutated in mesothelloma but not in lung cancer. Cancer Res 1995, 55:1227-1231.
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Makalowski W, Zhang J, Boguski MS: Comparative analysis of 1196 orthologous mouse and human full-length mRNA and protein sequences. Genome Res 1996, 6:846-857.
57.
Claudio JO, Lutchman M, Rouleau GA: Widespread but cell typespecific expression of the mouse neurofibrometosis type 2 gene. Neuroreport 1995, 6:1942-1946.
58.
Scherer SS, Gutmann DH: Expression of the neurofibromatosis 2 tumor suppressor gene product, merlin, in Schwann cells. J Neurosci Res 1996, 46:595-605.
59. •
McCartney BM, Fehon RG: Distinct cellular and subcellular patterns of expression imply distinct functions for the Drosophila homologues of moesin and the neurofibromatosis 2 tumor suppressor, merlin. J Cell Bio11996, 133:843-852.
38. o-
Hirao M, Sato N, Kondo T, Yonemura S, Monden M, Sasaki T, Tsukita S, Tsukita S: Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway. J Cell Biol 1996, 135:37-51. The authors of this paper showed that the interaction of ezrin, radixin, moesin (ERM) proteins with cell adhesion molecule CD44 is direct and that no additional factors are needed to mediate the binding. However, the interaction of ERM proteins with CD44 was weak under physiological conditions. Factors were identified that may be required to activate the binding of ERM proteins to the cytoplasmic tail of CD44. The authors obtained evidence that phosphoinositidas, particularly phosphatidylinositol 4,5-biosphosphate, and Rho regulate the interaction of ERM proteins with CD44. The regulation by Rho possibly involves phosphatidylinositol turnover. 39.
Gilmore AP, Burridge K: Regulation of vinculin binding to talin and actin by phosphatidyl-inositol-4-5-bisphosphate. Nature 1996, 381:531-535.
666
Cell-to-cell contact and extracellular matrix
Ezrin, radixin, moesin (ERM) proteins and merlin are found in phylogenetically distant species, such as mammals and Drosophila. To date, the invertebrates have been shown to have only one ERM protein. A pair of an ERM protein and merlin may be a common pattern in invertebrates, showing that merlin has a significant general role. The authors of this paper showed that the Drosophila ERM protein and merlin had different locations in cells, the ERM protein being located in the membrane and merlin having a punctate distribution in the cytoplasm and plasma membrane. Thus, ERM proteins and merlin may have distinct roles. 60.
61.
Huynh QP, Pulst S-M: Neurofibromatosis 2 antisense oligodeoxynucleotides induce reversible inhibition of schwannomin synthesis and call adhesion in STS26T and T98G cells. Oncogene 1996, 13:73-84. Tikoo A, Varga M, Ramesh V, Gusella J, Maruta H: An antiras function of neurofibromatosis type 2 9ene product (NF2/merlin). J Biol Chem 1994, 269:23387-23390.
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Lutchman M, Rouleau GA: The neurofibromatosis type 2 gene product, schwannomin, suppresses growth of NIH 3T3 cells. Cancer Res 1995, 55:2270-2274.
63.
Kaul SC, Mitsui Y, Komatsu Y, Reddel RR, Wadhwa R: A highly expressed 81 kDa protein in immortalized mouse fibroblasts: its proliferative function and identity with ezrin. Oncogene 1996, 13:1231-1237. Ran PH, Murty VVVS, Gaidano G, Hauptschein R, Dalla-Favera R, Ch~,ganti RSK: Subregional mapping of 8 single copy loci to
64.
chromosome 6 by fluorescence in situ hybridization. Cytogenet Ceil Genet 1994, 66:272-273. 65.
Wilgenbus KK, Milatovich A, Francke U, Furthmayr H: Molecular cloning, cDNA sequence, and chromosomal assignment of the human radixin gene and two dispersed pseudogenes. Genomics 1993, 16:199-206.
66.
Wilgenbus KK, Hsieh C-L, Lankes WT, Milatovich A, Francke U, Furthmayr H: Structure and localization on the X chromosome of the gene coding for the human filopodial protein moesin (MSN). Genomics 1994, 19:326-333.
67.
Jooss KU, M011erR: Deregulation of genes encoding microfilament-associatad proteins during Fos-inducad morphological transformation. Oncogene 1995, 10:603-608.
68.
B~hling T, Turunen O, J~.skel~inan J, Carp6n O, Sainio M, Wahlstr6m T, Vaheri A, Haltia M: Ezrin expression in stromal cells of capillary hemangioblastoma. An immunohistochemical survey of brain tumors. Am J Pathol 1996, 148:367-373.
69.
Hugo C, Pichler R, Gordon K, Schmidt R, Amieva M, Couser WG, Furthmayr H, Johnson RJ: The cytoskeletal linking proteins, moesin and radixin, are upregulatad by platelet-derived growth factor, but not basic fibroblast growth factor in experimental mesanglal proliferative glomerulonephritis. J C/in Invest 1996, 97:2499-2508.
The ezrin protein family: membrane-cytoskeleton interactions and disease associations Antti Vaheri*, Olli Carp6n, Leena Heiska, Tuula S Helander, Juha J .skel inen, P .ivi Majander-Nordenswan, Markku Sainio, Tuomo Timonen and Ossi Turunen Ezrin, radixin, moesin and merlin form a subfamily of conserved proteins in the band 4.1 superfamily. Ezrin protein subfamily members act as linkers between the plasma membrane and the cytoskeleton. Members of the subfamily have been shown to interact with each other, with cell adhesion molecules such as CD44 and with F-actin. Recent data indicate that intercellular adhesion molecules 1 and 2 also interact with ezrin. The proteins are also involved in the redistribution of intercellular adhesion molecules and the organization of cell membrane structures. Merlin is a tumor suppressor that is involved in tumorigenesis of schwannomas and meningiomas. Merlin has the same overall protein structure as the other proteins in the subfamily but may have partially distinct functions.
Addresses Haartman Institute, University of Helsinki, POB 21, FIN-O0014 Helsinki, Finland *e-mail: Antti.Vah eri@ helsinki.fi Correspondence: Antti Vaheri Current Opinion in Cell Biology 1997, 9:659-666
Although human ERM proteins share 75-80% identity with each other, human merlin shares about 45% sequence identity with ERM proteins. Although this suggests that merlin may have different functions to those of ezrin, radixin and moesin, the functional data obtained to date are sufficient to allow us to regard merlin as a functional member of the ERM family. In this review, we use the term ezrin protein family to include all the ERM proteins and merlin. T h e ERM proteins accumulate underneath the plasma membrane in various cell surface structures, such as microvilli, membrane ruffles and cell-cell contact sites. Ezrin has been localized by immunoelectron microscopy between the plasma membrane and the actin core structure in microvilli [3-5], suggesting a role as a linker between cell surface molecules and the actin cytoskeleton [6]. During recent years, information clarifying the biological functions of the ezrin protein family has rapidly accumulated. In this review, we focus on the membrane-cytoskeleton linker and tumor-suppressor roles of the family.
http://biomednet.com/elecref/0955067400900659 Actin-binding
© Current Biology Ltd ISSN 0955-0674 Abbreviations ERM ezrin, radixin, moesin ICAM intercellular adhesion molecule merlin moesin-ezrin-radixin-like protein NF2 neurofibromatosistype 2 NK natural killer PIP 2 phosphatidylinositol 4,5-bisphosphate
Introduction
to the ezrin
protein
family
Ezrin, the prototype member of the ezrin protein subfamily, was characterized as a component of brush-border and placental microvilli in the early 1980s [1,2]. T h e family now consists of four members in vertebrates, namely, ezrin, radixin, moesin (ERM proteins) and merlin (moesin-ezrin-radixin-like protein; also named schwannomin). Homologous proteins have been discovered in many invertebrate species, including Drosophila, Caenorhabditis elegans, sea urchin and parasites (Figure 1). T h e structure of all family members consists of an amino-terminal globular domain followed by an s-helical region and a carboxy-terminal domain. T h e family belongs to the band 4.1 superfamily on the basis of sequence homology of the amino-terminal domain with the erythrocyte membrane-cytoskeleton linker protein band 4.1.
ability
of ERM
proteins
Early attempts to demonstrate a direct interaction between ezrin and actin, using sedimentation assays for example, were disappointing. Weak binding was observed in nonphysiological but not in physiological salt concentrations [1,4]. However, it was observed that, following detergent extraction, ezrin remained partly in the cytoskeletal fraction [1,4]. Analysis of the ezrin cDNA sequence gave further support to an association of ezrin with the cytoskeieton. T h e amino-terminal domain of ezrin showed significant identity with band 4.1, the membrane-cytoskeleton linker in erythrocytes, and the a-helical domain was similar in sequence to domains of various cytoskeletal proteins [7,8]. T h e revelation of a masked actin-binding site in ezrin gave direct evidence that ERM proteins interact with the cytoskeleton. F-actin binding was detected in a truncated ezrin protein lacking the amino-terminal part [9]. In addition, full-length ERM proteins denaturated by SDS also showed F-actin binding [10]. These results suggest that the amino-terminal domain regulates actin binding. In line with this, head-to-tail binding in ERM proteins was reported, with the amino-terminal domain capable of binding the carboxy-terminal domain [11]. T h e interaction between the amino-terminal domain and the carboxyterminal domain has also been observed at another level,
660
Cell-to-cell contact and extracellular matrix
Figure 1
Amino-terminal domain I
~domain I
Carboxyterminal P domain II I
86%
69%
61%
86%
61%
62%
78%
26%
43%
76%
25%
57O/o
19O/o
61%
290/0
55%
20%
24%
47O/o
15%
15%
32O/o
80/0
Length (aa)
Protein
Overall sequence identity
585
Human ezrin
100%
583
Human radixin
77%
577
Human moesin
73%
578
Drosophilamoesin
55%
572
Sea urchin moesin
51%0
559
E, multilocularis EM 10
41%
595
Human merlin
43%
635
Drosophilamerlin
36%
654
C. elegans merlin
28O/o
587
Human band 4.1
22%
30%
37O/o
22%
150/o © 1997 Current Opinion in Cell Biology
The ezrin protein family. To date, a large number of genes encoding ezrin-like proteins have been cloned in a variety of species. The invertebrate ezrin-like proteins are Drosophila moesin and merlin, sea urchin moesin, the ezrin-like protein whose gene was cloned from the nematode C. elegans (this C. e/egans protein displayed properties typical of merlin, as shown by, for example, phytogenetic analysis and similarity in the carboxy-terminal 30 amino acids; O Turunen, A Vaheri, unpublished data) and E. mu/tilocularis EM10. All of these belong to the ERM family, with the exception of the merlins in Drosophila and C. elegans. Although the mammalian proteins are highly conserved, the ezrin-like proteins do not share high identity between mammals and invertebrates. The amino-terminal domain is very conserved across species, but the sequence identity between mammalian and invertebrate ezrin-like proteins can be very low in the (~ domain and carboxy-terminal domain, even as low as the homology of band 4.1 with ERM proteins. The amino-terminal domain is probably quite globular; the (x domain is probably helical according to computer analysis; the carboxy-terminal domain is also largely helical. The proline-rich region (P) interrupts the long c( helix in the middle of the protein in human ezrin and radixin, and in the merlins. In addition, mammalian moesin has a proline in the same position (not shown). Band 4.1 also has a group of prolines in the corresponding region and short amino acid motifs (not shown) in the carboxy-terminal domain are shared by ERM proteins and band 4.1. Percentage identities between different domains of the proteins are shown beneath the relevant domains; each percentage relates to similarity of the domain to human ezrin. Identities include gaps in the protein sequences that were introduced to maximize alignment. The black boxes indicate the actin-binding site of the ERM proteins. In the merlins, the role of the corresponding carboxy-terminal region (grey boxes) is unclear, aa, amino acids.
as the amino-terminal domain inhibits the morphogenic (cell extension) activity of the carboxy-terminal part [12]. Recently, it has also been shown that purified cellular ezrin interacts with actin, particularly the 13-actin isoform. Other studies demonstrated that ezrin could be directly co-sedimented with 13-actin, but only a weak association with ~-actin was observed. Furthermore, ezrin was shown to preferentially promote ~-actin polymerization [13"'].
It has also been reported that ezrin interacts indirectly with 13-actin filaments via the p73 protein, but not with a-actin filaments [14,15]. Although direct evidence is missing, a possible explanation for the conflicting binding results is that the isolated ezrin exists in different forms, depending on the cellular source. Yao etal. [13"'] isolated ezrin from gastric mucosa containing several ezrin isoforms, which were possibly the result of differences in phosphorylation. Furthermore, the indirect association of ezrin with l~-actin
The ezrin protein family Vaheri et a/.
was reported to occur at the barbed (fast-growing) end of actin [15], whereas the direct binding of ezrin to actin was observed at the filament sides [13°°]. These results suggest the existence of different physiologically significant modes of binding. Radixin was originally detected as a barbed end capping protein [16]. However, the barbed end binding was observed only in nonphysiological salt concentrations. The immunofluorescence data and binding experiments show that radixin also binds to the sides of actin filaments [16]. The data on radixin also showed that the barbed end capping activity inhibits actin polymerization [16], whereas ezrin appears to stimulate actin polymerization [13°°]. The role of the barbed end capping activity is not yet clear. The sequence similarity between the actin-binding sites of ERM proteins and of the barbed end capping protein CapZ 13 subunit may indicate that the barbed end capping activity is functional in the cells as well as in nonphysiological salt concentrations. Consequently, it is possible that ERM proteins regulate the length of actin filaments, by both inhibiting and stimulating actin polymerization. The actin-polymerizing activity probably accounts for the morphogenic effect of ERM proteins: both truncated and full-length ERM proteins induce cell surface extensions [12,17,18,19°']. Merlin also organizes cell surface structures, although we do not yet know how the protein affects actin polymerization [20]. The actin-binding site localizes to a few carboxy-terminal amino acids of ezrin and moesin [9,10]. In ezrin, the last 34 amino acids bind F-actin [9]. As there is high carboxy-terminal sequence identity (80%) between mammalian ERM proteins, moesin and radixin are also likely to have the same actin-binding site. The actin-binding site is highly conserved, even in invertebrate proteins. However, the corresponding site in merlin has a partially different sequence, also indicating a distinct function.
Regulation of ERM proteins Phosphorylation appears to regulate the binding of ERM proteins to actin, and is induced by several growth factors. The conformationally masked carboxy-terminal actin-binding site is thought to be opened by signals such as phosphorylation [11,21 ]. The regulation of actin-binding by phosphorylation appears to be mediated by several alternative mechanisms. Ezrin is phosphorylated at least at two tyrosine residues, Tyr145 and Tyr353, when A431 carcinoma cells are stimulated by epidermal growth factor (EGF), and ezrin is subsequently redistributed to microvilli [22,23]. The EGF-induced redistribution of ezrin involves the formation of ezrin dimers and oligomers [22,24]. In stimulated T lymphocytes, the tyrosine kinase p561ck is involved in the tyrosine phosphorylation of ezrin [25]. Serine/threonine phosphorylation regulates the distribution of ezrin in gastric parietal cells [26,27] and constitutive
661
serine/threonine phosphorylation of merlin has been observed [28]. cAMP is involved in the regulation of ezrin by serine/threonine phosphorylation in gastric parietal cells. A more general role for ezrin in cAMP-dependent cell regulation is revealed by the recent finding that ezrin functions as a protein that anchors the cAMP-dependent protein kinase [29°°]. It appears that ERM proteins can be regulated by phosphorylation of different protein domains. Tyr145 is located in the amino-terminal domain and Tyr353 in the a-helical domain, and phosphorylation of the carboxyterminal domain may also regulate the actin-binding ability of ERM proteins. Phosphorylation of Thr558 in only the carboxy-terminal actin-binding region of moesin occurs within minutes in thrombin activation of human platelets [30]. Activated platelets form extremely long filopodia and the phosphorylated moesin co-localizes with F-actin. Additional evidence for the tight association between ERM protein phosphorylation and their subcellular distribution can be seen following anoxic injury in the renal proximal tubule brush border, when rapid serine/threonine dephosphorylation of ezrin occurs and interaction of ezrin with the microvillar cytoskeleton is lost [31]. Inhibition of mast cell stimulation (which occurs by cross-linking receptor-bound IgE) by an antiallergic drug induces serine phosphorylation of moesin [32]. In macrophages, Thr558-phosphorylated moesin is found to be co-localized with F-actin in filopodial protrusions [33]. Treatment of H T l l 5 colon carcinoma cells with hepatocyte growth factor/scatter factor induced tyrosine phosphorylation of ezrin and its translocation from the cytosol to areas of ruffled membrane [34]. Recent evidence [35°,36,37,38 °°] suggests that Rhomediated signal transduction is involved in the activation of ERM proteins. The GTP-binding protein Rho regulates actin-dependent cellular events and organization of cell surface structures, and functions as an upstream factor in the regulation of ERM proteins. ERM proteins may also be regulated by phosphatidylinositol 4,5-bisphosphate (PIPz), which increases ERM protein binding to CD44, intercellular adhesion molecule (ICAM)-I and ICAM-2 ([38°°]; L Heiska et aL, unpublished data). The nature of the regulatory effect of PIP 2 is unclear but could involve conformational changes in ERM proteins in a manner analogous to PIP 2 regulation of vinculin activity [39].
Does merlin associate with the cytoskeleton? The mechanism of the cytoskeletal association of the fourth member of the ezrin protein family, merlin, has not yet been resolved. Merlin, like the ERM proteins, is localized to cortical cytoskeletal structures in cells and is also partly retained in the detergent-insoluble fraction in cell lysates [20,40,41]. The carboxy-terminal region corresponding to the ERM actin-binding site differs from
662
Cell-to-cell contact and extracellular matrix
that of ERM proteins, but still contains a number of basic amino acids that could, in principle, mediate binding to actin. Actin binding was not, however, observed in either full-length or truncated merlin ([28]; O Turunen, A Vaheri, unpublished data) in the chromatographic binding assay that revealed the actin-binding activity of ezrin [9]. Merlin's association with the cytoskeleton may differ from that of ERM proteins. Merlin may have a cytoskeleton-binding site in the middle of the protein, like band 4.1 [42]; the cytoskeleton association may be due to heterodimerization with ERM proteins; or other factors may be required for the cytoskeleton association. T h e overexpression of full-length merlin induces cell surface protrusions and elongation of cells [20]. Morphogenic effects at the cell surface, including blebbing, indicate extensive reorganization of the cortical cytoskeleton. This could occur as a result of distinct cytoskeleton-interacting properties of merlin.
Interactions family
between
members
of the ezrin
Ezrin can be isolated from cell extracts as monomers, dimers and oligomers [21,24,43]. However, attempts to generate dimers from monomers in solution have been unsuccessful. Monomers are very stable structures and dimerization apparently requires activation. However, ERM proteins readily associate when assayed by blot overlay. In these assays, ERM proteins displayed homotypic and heterotypic binding [AA n.6]. An important recent advance has been the demonstration [11] that ezrin self-association involves the binding of the amino-terminal domain (mapped to residues 1-296) to the carboxy-terminal domain (last 107 residues of the protein, 479-585). T h e self-association could also explain the inhibition of ezrin's morphogenic activity by its amino-terminal domain [12]. However, the in vivo role of heterodimerization in the regulation of ERM proteins and merlin is, as yet, unknown. The self-association may in principle occur in several ways, by the formation of head-to-tail dimers, parallel dimers and head-to-tail oligomers. Heterodimerization could modulate binding activity in the parallel dimers of ERM proteins, but in the oligomer-binding model (see Figure 2) the modulatory role of heterodimerization is not as evident. T h e interdomain interaction is not confined to the ERM family as the amino-terminal domain of radixin also binds to band 4.1 [46]. This raises the possibility that the activity of the whole band 4.1 protein family, including the tyrosine phosphatases, is regulated by similar mechanisms. In addition, ERM proteins may share other binding partners with band 4.1. Both band 4.1 and ezrin have been shown to interact in a co-precipitation complex with the murine form of hDlg, a homologue of the Drosophila Dlg tumor suppressor protein [47].
Figure 2
ICAM-1 ICAM-2 CD44 Cell membrane I ; ERM proteins PIP2
I PIP2
PIP2
Merlin.'? F-actin
ini n in
II
o ogy
i I
Model of the cell adhesion molecule-F-actin linker role of ERM proteins. The interaction of ERM proteins with actin filaments may occur such that the extended ERM molecule lies on the actin filament but only the carboxyl terminus (small shaded oval) actually binds actin. In this model, the self-association of the ERM proteins occurs in tandem with the binding of the amino-terminal domain (large shaded oval in ERM protein) of one protein to the carboxy-terminal domain of another [94]. Another possibility is that ERM proteins form parallel dimers that interact with actin filaments, such that each dimer would bind to the actin filament by a pair of actin-binding sites (not shown). In this alternative model, there is no place for an amino-terminal domain to carboxy-terminal domain interaction in the active molecule. Instead, the self-association could mainly be a way to inactivate the protein. On the other hand, the parallel dimer model may allow the modulation of the ERM protein activity by heterodimerization, which is not as evident in the head-to-tail oligomer-binding model. PIP2 is important in activating ERM protein interactions with adhesion molecules (ICAM-1, ICAM-2 and CD44) and is thus indicated in the figure. If merlin lacks the carboxy-terminal actin-binding site, it may not interact with actin in the way proposed by the tandem model.
Membrane protein protein family
interactions
with
the ezrin
T h e homology between the amino-terminal domains of ezrin and members of the band 4.1 superfamily suggested that this domain may interact with membrane proteins, as the amino-terminal domain of band 4.1 interacts with glycophorin C, a major integral membrane protein in erythrocytes. This suggestion was supported by the observation that the amino-terminal domain of ezrin transfected into cultured cells associates with the plasma membrane [6]. By using antisense oligonucleotides, it was observed that the function of ERM proteins is critical to maintaining the cell adhesion [48]. CD44 was the first cell surface protein with which ERM proteins were demonstrated to interact [38**]. T h e binding of ERM proteins to CD44 is regulated by the GTP-binding protein Rho [35",38°°] and by phosphoinositides, particularly PIP 2 [38*']. Recombinant merlin also interacts with CD44 [20]. Our recent studies using ICAM cytoplasmic domain peptides and purified ezrin (L Heiska et al., unpublished data) demonstrate molecular interactions between ezrin and two members of the immunoglobulin superfamily, ICAM-1 and ICAM-2. These interactions are greatly promoted by the presence of
The ezrin protein family Vaheri et a/.
PIP z, which was earlier shown to bind the amino-terminal domain of ezrin [49]. ERM proteins may play an active role in targeting the cell adhesion molecules to specific cell surface regions. Transfected ezrin redistributes ICAM-2 to newly formed cellular protrusions (uropods) on the surface of tumor cells, which then become susceptible to lysis by natural killer (NK) cells [19"']. As ezrin interacts directly with the cytoplasmic tail of ICAM-2 (L Heiska et al., unpublished data), it is possible that direct interaction with ezrin drives ICAM-2 to the uropods, creating a surface structure that is recognized by NK cells (see Timonen and Helander, this issue, pp 667-673). In addition, ezrin may also be involved in other immunological events, such as stimulation of T lymphocytes [25]. T h e subcellular redistribution of adhesion molecules may also greatly facilitate cellular adhesion and other cell functions. T h e reorganization of cell adhesion molecules by ezrin could also be associated with NK cell killing in other pathological conditions, such as virus infections. Redistribution of ezrin has been observed to occur following infection of human fibroblasts with either herpes simplex virus or Semliki Forest virus [50]. Redistribution of uniformly located ezrin into newly formed microvilli occurs in a few hours, without any apparent change in ezrin quantity. Other cell adhesion molecules, like CD46, a complementbinding protein, and the highly glycosylated adhesion molecule CD43, may also interact with the ezrin protein family [37]. CD46 appears to interact with moesin in chemical cross-linking and co-immunoprecipitation experiments [51 ]. Merlin a s a t u m o r s u p p r e s s o r
Merlin is the product of the neurofibromatosis type 2 (NF2) gene [52,53]. It is a tumor suppressor protein that is defective or absent in NF2, a rare autosomal dominant disease (found in 1 in 35,000 people) that predisposes individuals to multiple nervous system tumors, vestibular and spinal schwannomas, meningiomas and ependymomas, and also to juvenile posterior lenticular opacities [54]. The NF2 gene is also somatically mutated in the majority of mesotheliomas [55]. The NF2 gene is located on human chromosome 22q12. In NF2, both germline and somatic mutations span the gene, with no clear 'hot spots' [54]. Like ERM proteins, merlins belong to the most conserved group of mammalian proteins. T h e mouse merlin shares 98% identity with human merlin, whereas the average identity of orthologous mouse and human proteins is 85% [56]. Mammalian merlin is expressed mainly in the nervous system and cells of the lens and is predominantly located in membrane ruffles and cellular protrusions. It is partially co-localized with ezrin, CD44 and F-actin [20,40,41,57,58].
663
In Drosophila, the distributions of the only Drosophila ERM proteins, moesin and merlin, are different, moesin being found on the plasma membrane and merlin on punctate structures in the membrane and cytoplasm [59"]. Like ERM proteins, merlin may also regulate cell adhesion. Antisense oligodeoxynucleotides to merlin inhibited cell adhesion [60] as has been observed for ezrin, radixin and moesin [48]. Transfection of the merlin gene suppresses a v-Ha-Ras-induced malignant phenotype [61] and inhibits the growth of NIH 3T3 cells [62]; on the other hand, transfection of ezrin appears to promote cell growth via loss of contact inhibition [63]. The growth-inhibiting activity of merlin is consistent with its role as a tumor suppressor. T h e inactivation of merlin is therefore probably associated with loss of cell growth control. T h e functional similarity between human merlin and ERM proteins raises the possibility that the whole protein family is associated with tumorigenesis. No cancer susceptibility syndromes have been linked as yet to the ezrin gene at chromosome position 6q25-26 [7,64], the radixin gene at 1lq12 [65] or the moesin gene at Xql 1.2-12 [66]. In vitro, however, high expression of ezrin in cultured cells causes cell transformation and is associated with cell proliferation [63,67]. Antisense oligonucleotides inhibit the ERM protein function and disrupt cell adhesion; lack of adhesion might contribute to metastasis [48]. Ezrin regulates ICAM-2-dependent tumor cell recognition and killing by NK cells [19°']. High levels of ezrin can be detected in the stromal cells of hemangioblastoma [68], a tumor associated with mutations in the von Hippel Lindau tumor suppressor gene. Also, moesin and radixin are upregulated by platelet-derived growth factor in experimental mesangial proliferative glomerulonephritis in rats [69]. These results suggest that disease associations for ERM proteins may exist. As the genomic structures of the human ezrin gene (it is 24 kilobases long and includes 13 exons) (P Majander-Nordenswan et al., unpublished data) and human moesin gene (which is >30 kilobases long and has 12 exons) [66] have now been revealed, their disease association can be studied more systematically by mutation screening. Conclusions
Ezrin, radixin, moesin and merlin appear to be proteins that are involved in the regulation of cell morphology, particularly of cell surface projections. Ezrin, radixin and moesin, and possibly also merlin, seem to function as linkers between cell adhesion molecules and the cytoskeleton, and they may organize the distribution of cell adhesion molecules. Furthermore, new findings demonstrate that all of these proteins may all play an active role in tumor biology. Particularly, it appears that killer cells may distinguish malignant cells through ezrin-controlled distribution of cell adhesion molecules. Protein phosphorylation appears to be critical to the
664
Cell-to-cell contact and extracellular matrix
activation of ERM proteins, resulting in the redistribution of ERM proteins and of intercellular adhesion molecules.
6.
Algrain M, Turunen O, Vaheri A, Louvard D, Arpin M: Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker. J Cell Bio/1993, 120:129-139.
T h e members of the ezrin protein family may interact with additional integral membrane proteins, and thus the important issue is how the interactions are regulated in each case. T h e search for cytoplasmic proteins that may also interact with and regulate the ERM proteins and merlin will be particularly interesting.
7.
TurunenO, Winqvist R, Grzeschik K-H, Pakkanen R, Wahlstr~m T, Vaheri A: Cytovillin, a microvillar Mr 75,000 protein, eDNA sequence, prokaryotic expression, and chromosomal localization. J Biol Chem 1989, 264:16727-16732.
8.
Gould KL, Bretscher A, Esch FS, Hunter T: cDNA cloning and sequencing of the protein-tyrosine kinase substrata, ezrin, reveals homology to band 4.1. EMBO J 1989, 8:4133-4142.
9.
TurunenO, Wahlstr6m T, Vaheri A: Ezrin has a COOH-tarminal actin-binding site that is conserved in the ezrin protein family. J Cell Biol 1994, 126:1445-1453.
10.
Pestonjamasp K, Amieva MR, Strassel CP, Nauseef WM, Furthmayr H, Luna EJ: Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes. Mo/ Biol Cell 1995, 6:247-259.
11.
Gary R, Bretscher A: Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site. Mol Biol Cell 1995, 6:1061-1075.
12.
Martin M, Andr6oli C, Sahuquet A, Montcourrier P, Algrain M, Mangeat P: Ezrin NH2-terminal domain inhibits the cell extension activity of the COOH-terminal domain. J Cell Bio/
As a structural protein, merlin is a novel type of tumor suppressor. We do not yet know how the tumor suppressor function is mediated and whether other members of the family share a similar function. Merlin-associated tumorigenesis occurs through inactivation of the protein, whereas the promotion of cell growth by ERM proteins is associated with increased expression levels in vitro. It may be that any association of ERM proteins with diseases differs from that of merlin. On the other hand, the inhibition of protein function of both ERM proteins and merlin disrupts cell adhesion. Future studies should clarify the differences and similarities between merlin and ERM proteins. T h e ezrin family proteins seem to form part of a network of protein interactions. An understanding of the structure-function relationships of this family is just emerging. There are many open questions about the interaction of ERM proteins with actin. T h e mechanisms underlying binding to cell adhesion molecules are even less well understood. Several modes of interaction could exist, as it is known that ERM proteins are located in different cellular structures, such as microvilli, ruffles and cell-cell contact sites. T h e cell type may also affect functional properties. A detailed structural analysis may reveal which structural properties are important in the specialized functions of each protein.
1995, 128:1081-1093.
13. ;e
Yao X, Chang L, Forte JG: Biochemical characterization of ezrin-actin interaction. J Biol Chem 1996, 271:7224-7229. authors of this paper studied the interaction of ezrin with actin by using purified proteins. The main results were that ezrin binds selectively to ~-actin, but only weakly to (~-actin, and that ezrin stimulates actin polymerization.The stronger interaction with 13*actinis significant as both ezrin and ~-actin have a similar location in the vicinity of the cell membrane. 14.
Shuster CB, Herman IM: Indirect association of ezrin with Factin: isoform specificity and calcium sensitivity. J Cell Biol 1995, 128:837-848.
15.
Shuster CB, Lin AY, Nayak R, Herman IM: bCAP73: a novel actin-specific binding protein. Cell Motil Cytoskeleton 1996, 354:1 75-187.
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Edwards KA, Montague RA, Shepard S, Edgar BA, Erikson RL, Kiehart DP: Identification of Drosophila cytoskeletal proteins by induction of abnormal cell shape in fission yeast. Proc Nat/ Acad Sci USA 1994, 91:4589-4593.
18.
Henry MD, Gonzalez-Agosti C, Solomon F: Molecular dissection of radixin: distinct and interdependent functions of the aminoand carboxy-terminal domains. J Cell Bio11995, 129:10071022.
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666
Cell-to-cell contact and extracellular matrix
Ezrin, radixin, moesin (ERM) proteins and merlin are found in phylogenetically distant species, such as mammals and Drosophila. To date, the invertebrates have been shown to have only one ERM protein. A pair of an ERM protein and merlin may be a common pattern in invertebrates, showing that merlin has a significant general role. The authors of this paper showed that the Drosophila ERM protein and merlin had different locations in cells, the ERM protein being located in the membrane and merlin having a punctate distribution in the cytoplasm and plasma membrane. Thus, ERM proteins and merlin may have distinct roles. 60.
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