Download Ezrin: a protein requiring conformational activation to link

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Journal of Cell Science 110, 3011-3018 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
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COMMENTARY
Ezrin: a protein requiring conformational activation to link microfilaments to
the plasma membrane in the assembly of cell surface structures
Anthony Bretscher, David Reczek and Mark Berryman
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca NY 14853, USA
*Author for correspondence (e-mail: [email protected])
SUMMARY
The cortical cytoskeleton of eucaryotic cells provides structural support to the plasma membrane and also contributes
to dynamic processes such as endocytosis, exocytosis, and
transmembrane signaling pathways. The ERM (ezrinradixin-moesin) family of proteins, of which ezrin is the best
studied member, play structural and regulatory roles in the
assembly and stabilization of specialized plasma membrane
domains. Ezrin and related molecules are concentrated in
surface projections such as microvilli and membrane ruffles
where they link the microfilaments to the membrane.
The present knowledge about ezrin is discussed from an
historical perspective. Both biochemical and cell biological
studies have revealed that ezrin can exist in a dormant conformation that requires activation to expose otherwise
masked association sites. Current results indicate that
activated ezrin monomers or head-to-tail oligomers
associate directly with F-actin through a domain in its C
terminus, and with the membrane through its N-terminal
domain. The association of ezrin with transmembrane
proteins can be direct, as in the case of CD44, or indirect
through EBP50. Other binding partners, including the
regulatory subunit of protein kinase A and rho-GDI,
suggest that ezrin is an integral component of these
signaling pathways. Although the membrane-cytoskeletal
linking function is clear, further studies are necessary to
reveal how the activation of ezrin and its association with
different binding partners is regulated.
INTRODUCTION
Ezrin is the best studied member of the ERM family,
proteins that share approximately 75% primary sequence
identity (Funayama et al., 1991; Gould et al., 1989; Lankes
and Furthmayr, 1991; Sato et al., 1992; Turunen et al., 1989).
ERM proteins belong to the band 4.1 superfamily, because
they show homology to the first ~300 residues of erythrocyte
band 4.1, which connects the actin/spectrin network to the
membrane protein glycophorin C (Anderson and Lovrien,
1984; Leto and Marchesi, 1984; Marfatia et al., 1994, 1995).
Based on this homology and the cytoskeletal localization of
ezrin, Gould et al. (1989) suggested that the N-terminal
domain of ezrin associates with the membrane, whereas the
C-terminal half links it to the cytoskeleton. This hypothesis
was substantiated by transfection experiments which demonstrated that the N-terminal half of ezrin associates with the
plasma membrane, whereas the C-terminal half associates
with the actin cytoskeleton (Algrain et al., 1993). Because of
the high degree of homology among ERM members, many of
the properties that pertain to ezrin also apply to radixin and
moesin. However, their tissue distributions and primary structures indicate that these are not simply redundant proteins.
Although all three proteins are expressed in most cultured
cells (Amieva and Furthmayr, 1995; Franck et al., 1993; Sato
et al., 1992), cells in the body exhibit very distinct and
restricted patterns of expression (Amieva and Furthmayr,
The plasma membrane is the interface a cell has with its environment and its neighbors. It is the site of vectorial transport
of ions and nutrients, reception of signalling molecules, and
attachments to adjacent cells and the extracellular matrix. To
perform these functions, the membrane is supported and
organized into domains by the underlying actin cytoskeleton,
which together constitute the cell cortex. Thus, the cortical
cytoskeleton not only contributes to structural support, but
must also be regulated to coordinate the dynamic functions of
membranes, such as endocytosis, exocytosis, and transmembrane and other cortical signalling pathways. Of particular
interest are proteins that link the cortical cytoskeleton to
the membrane. The ERM (ezrin-radixin-moesin) family
of proteins appear to play structural and regulatory roles by
stabilizing specialized plasma membrane domains during
development and in adult tissues.
Here, we try to distil from an historical perspective what is
known about the ERM family, but with particular emphasis on
ezrin. We also include areas of uncertainty that will be clarified
through the study of diverse systems. The emphasis and flavor
of this discussion is intended to extend and complement other
reviews on ERM proteins (Arpin et al., 1994; Bretscher, 1991,
1993; Bretscher and Berryman, 1997; Tsukita et al., 1997a,b).
Key words: Ezrin, ERM protein, Band 4.1, Actin, Microvilli, Plasma
membrane
3012 A. Bretscher, D. Reczek and M. Berryman
1994; Berryman et al., 1993; Schwartz-Albiez et al., 1995; A.
Shcherbina et al., unpublished). In addition, a polyproline
stretch of unknown function that is present in ezrin and
radixin is absent from moesin. Although ezrin and radixin are
substrates for certain tyrosine kinases (Bretscher, 1989;
Crepaldi et al., 1997; Egerton et al., 1992; Fazioli et al., 1993;
Gould et al., 1986; Jiang et al., 1995; Louvet et al., 1996;
Thuillier et al., 1994), there is evidence to indicate that the
patterns of phosphorylation are distinct among family
members (Fazioli et al., 1993). In one example, treatment of
human A431 carcinoma cells with EGF results in the tyrosine
phosphorylation of ezrin but not moesin (Franck et al., 1993),
yet moesin contains one of the homologous tyrosines phosphorylated in ezrin (Krieg and Hunter, 1992). To date no clear
functional differences have been documented between
members of the family, so this discussion will be largely
devoted to ezrin’s biochemical properties and biological
functions.
DISCOVERIES LEADING TO EZRIN
The number of diverse systems in which ezrin was independently identified as a potentially interesting molecule is
remarkable. The first was as an 81 kDa polypeptide that
became phosphorylated very rapidly on tyrosine in response
to EGF stimulation of A431 human carcinoma cells (Hunter
and Cooper, 1981). It was subsequently identified as a
component of isolated chicken intestinal microvilli, from
where it was purified and characterized as a cytoskeletal
protein. Immunofluorescence microscopy showed that ezrin
is concentrated in actin-rich cell surface structures, such as
microvilli, filopodia and membrane ruffles (Bretscher, 1983).
It was then shown that the phosphoprotein from A431 cells
was identical to the microvillar cytoskeletal protein (Gould et
al., 1986).
Meanwhile, ~81 kDa polypeptides, later identified as ezrin,
were under study in several other systems. Pakkanen et al.
(1987) found that an antibody, generated to a peptide based on
a human endogenous retroviral DNA sequence (Suni et al.,
1984), recognized a ~75 kDa protein found in cell surface
structures and subsequently named it cytovillin (Pakkanen,
1988; Pakkanen et al., 1988; Pakkanen and Vaheri, 1989).
Sequence analysis revealed that ezrin and cytovillin are
identical (Gould et al., 1989; Turunen et al., 1989). Urushidani
et al. (1989) and Hanzel et al. (1989) identified an 80 kDa
polypeptide potentially involved in acid secretion in parietal
cells of the gastric glands. Stimulation of parietal cells induces
cytoplasmic tubulovesicles containing the proton pump to fuse
with the apical membrane to form microvilli (for review, see
Forte et al., 1989). During this remarkable transformation,
which involves activation of protein kinase A (PKA), the 80
kDa polypeptide becomes phosphorylated on serine and
threonine residues, and can be isolated from the apical
microvilli in association with actin and the pump. Hanzel et al.
(1991) established that the 80 kDa polypeptide is ezrin. Ullrich
et al. (1986) identified and purified an 82 kDa tumor antigen
from a methylcholanthrene-induced sarcoma; this protein was
subsequently shown to be ezrin (Fazioli et al., 1993). Ezrin was
also identified as a tyrosine kinase substrate in T-cells (Egerton
et al., 1992).
SUBCELLULAR LOCALIZATION OF EZRIN
A general theme that applies not only to ezrin, but to all ERM
proteins, is their presence in the apical domain of polarized
cells, a region usually characterized by the presence of
microvilli. Although ERM proteins can have overlapping distributions in certain epithelial cell types, such as the presence
of all three in the brush border of the kidney proximal tubule
epithelium, other epithelia preferentially express ezrin and
endothelia express moesin (Berryman et al., 1993; SchwartzAlbiez et al., 1995). In a careful study of liver tissue, Amieva
et al. (1994) have shown that hepatocytes only express radixin,
that the epithelial cells lining the bile ducts express both ezrin
and radixin, and that the endothelial cells only express moesin.
Significantly, they also reported that moesin is concentrated on
the apical (luminal) domain of endothelial cells. Thus, all three
ERM proteins are associated with microvilli. Although ERM
proteins are prominent in epithelial tissues, other cell types
within the body can also express one or more family members
(Berryman et al., 1993; A. Shcherbina et al., unpublished;
Schwartz-Albiez et al., 1995).
In contrast to adult tissues, which show distinct patterns of
ERM expression, cultured cell lines usually express all three
ERM proteins (Sato et al., 1992; Amieva and Furthmayr,
1995). Immunofluorescence studies with specific antibodies
has shown that all three are concentrated in actin-rich surface
structures such as microvilli, membrane ruffles, and filopodia
(Amieva and Furthmayr, 1995; Franck et al., 1993). Immunoelectron microscopy of ezrin in human placental syncytiotrophoblast and mouse mesothelia has shown that ezrin is highly
concentrated in the microvilli where it is associated with the
cytoplasmic aspect of the plasma membrane (Berryman et al.,
1993). The relatively low levels of ezrin on adjacent regions of
the apical membrane between microvilli supports the notion
that ezrin plays a special role in the attachment of microfilaments to the membrane specifically within microvilli.
Although ezrin has been reported to be concentrated in
adherens junctions (Takeuchi et al., 1994), we and others
cannot detect a significant enrichment of either ezrin or moesin
in these structures in cultured cells or in tissues, such as
intestine (Amieva and Furthmayr, 1995; Berryman et al.,
1993). One explanation for this discrepancy is that at the light
microscope level, ERM proteins can appear to be enriched in
adherens junctions or cleavage furrows of cultured cells simply
because microvilli can be particularly abundant at these sites
(Yonemura et al., 1993; our unpublished observations).
Radixin was originally isolated as a component of adherens
junctions purified from liver (Tsukita et al., 1989). It has been
localized to these junctions in liver and intestine (Funayama et
al., 1991; Tsukita et al., 1989), and to focal contacts, cleavage
furrows (Sato et al., 1991), and the contractile ring of cultured
cells (Henry et al., 1995). We have also examined the subcellular distribution of radixin in cultured cells and found it concentrated in membrane ruffles and microvilli, but not in focal
contacts (our unpublished data). These results are consistent
with the results of Amieva et al. (1994), who showed that
radixin is associated with microvilli and not with adherens
junctions of hepatocytes in rat liver sections. However, since
different antibodies may recognize different activation states of
ERM proteins (see below), we cannot exclude the possibility
that additional locations exist, and thus the enrichment of ERM
Ezrin in assembly of cell surface structures 3013
proteins in adherens junctions in tissues, and in focal contacts
and the contractile ring of cultured cells, remains controversial. Nevertheless, the presence of ERM proteins in microvilli,
especially on the apical aspect of polarized cells, seems to be
a common feature that implicates their involvement in the
organization of this plasma membrane domain.
CONFORMATIONAL REGULATION OF EZRIN
Recently, several lines of investigation have indicated that ezrin
can exist in various ‘dormant’ or ‘active’ states (see Fig. 2).
Although the majority of the ezrin in soluble extracts of intestinal or placental tissues is monomeric (Bretscher, 1983, 1989),
a defined subset of ezrin can self-associate or associate with
moesin in cultured cells that express both proteins (Bretscher
et al., 1995; Gary and Bretscher, 1993). In baculovirus infected
insect cells that overexpress ezrin, a massive accumulation of
the protein occurs under the plasma membrane, also suggesting self-association under these circumstances (Andreoli et al.,
1994). As depicted in Fig. 1, the N-terminal 296 residues of
ezrin or moesin fold into a domain that associates with the Cterminal 107 residues of any ERM member; these domains are
known as N- and C-ERMADs (ERM association domains)
(Gary and Bretscher, 1995). In the native monomer the CERMAD is masked, thus precluding spontaneous oligomerization. Likewise, the F-actin binding site located at the C
terminus of ezrin (Pestonjamasp et al., 1995; Turunen et al.,
1994) is also masked (Gary and Bretscher, 1995). In accord
with the results for ezrin, Magendantz et al. (1995) have shown
that the native N-terminal domain of radixin binds to denatured
full-length radixin, conditions which expose the C-ERMAD.
These biochemical findings indicate that the native monomer
can exist in a ‘dormant’ state that needs to be ‘activated’ to
expose the C-ERMAD and allow for F-actin binding or association with an available N-ERMAD. Further examination of
ezrin forms in placenta has uncovered two relevant findings.
First, soluble ezrin from placenta can be isolated as a relatively
globular monomer or a highly extended dimer: both have
masked C-ERMADs (Bretscher et al., 1995). Second, in
isolated placental microvilli ezrin exists in oligomeric form,
with extended dimers, trimers etc. comprising the bulk of the
microvillar ezrin (Berryman et al., 1995). Moreover, during
EGF treatment of A431 cells there is a close temporal corre-
lation between the formation of microvilli and ezrin dimers,
suggesting that cell surface structures are enriched in
oligomeric forms of ezrin. We therefore proposed a working
model in which most of the ezrin in a resting cell exists in a
dormant state with masked C-ERMAD and F-actin binding
sites, and possibly also with a masked membrane association
site. Activation, perhaps involving multiple steps, can lead to
the exposure of self-assembly and/or F-actin and membrane
binding sites (Berryman et al., 1995; see Fig. 2).
In parallel with these studies, Martin et al. (1995) reported
that high level expression of the C-terminal half of ezrin in
insect cells induced the formation of long cellular protrusions,
whereas high level expression of the full length molecule did
not. They also found that the morphological changes induced
by the C-terminal construct could be suppressed by coexpression of the N-terminal domain. These results indicate that the
C-terminal half of the molecule has morphological effects that
can be masked by the N-terminal half in either the full length
molecule or supplied as a separate domain. More recently,
Martin et al. (1997) have shown that the situation is more
complex: expression of a construct which lacks the 21 Cterminal residues also induces long protrusions, and even
expression of the N-terminal domain alone (residues 1-310)
has similar effects. The coincidence of regions necessary for
morphological effects with those implicated in binding actin
(Roy et al., 1997) makes the physiological relevance of these
studies seem plausible. Likewise, Henry et al. (1995) found
that overexpression of the C-terminal half of radixin, but not
the entire protein, led to the formation of long surface structures on NIH 3T3 cells. These cell biological studies independently suggest that ERM proteins have biological activities that
are masked in the full length molecule.
ACTIN BINDING PROPERTIES OF EZRIN
Ezrin was originally purified from the microfilament bundle of
isolated intestinal microvilli, yet the native protein possessed
no convincing F-actin binding activity (Bretscher, 1983). Using
GST-ezrin fusion constructs, Turunen et al. (1994) identified
an F-actin binding site in the C-terminal 35 residues, which
was confirmed independently for all ERM members using Factin overlays (Pestonjamasp et al., 1995). The F-actin overlays
also indicated that ERM proteins bind with high affinity to the
Polyproline
1
N
Y145
296
N-ERMAD
Y353
α-helical domain
468-474
585
C-ERMAD
C
Band 4.1 Homology Domain
Fig. 1. Domain organization of ezrin.
Binding sites demonstrated to be
masked in dormant ezrin are indicated
(*). The two tyrosines whose
phosphorylation has physiological
consequences are shown. No function
has yet been ascribed to the polyproline
stretch.
Binding sites for:
* C-ERMADs (in ezrin, radixin & moesin)
* CD44 (integral membrane protein)
* EBP50 (PDZ-containing phosphoprotein)
* Rho-GDI (Rho pathway regulator)
PIP2 (lipid signalling molecule)
RII-regulatory
subunit of PKA
* N-ERMADs (in ezrin,
radixin & moesin)
* Binding site
for F-actin
3014 A. Bretscher, D. Reczek and M. Berryman
sides of filaments, consistent with immunoelectron microscopy
studies which show a uniform distribution of ezrin along the
length of microvilli (Berryman et al., 1993).
Although several investigators have successfully used
muscle α-actin in their assays (Pestonjamasp et al., 1995;
Turunen et al., 1994; Roy et al., 1997), others have provided
evidence that ezrin exhibits isoform specificity. Shuster and
Herman (1995) found that some of the ezrin from pericytes was
retained on a column containing immobilized nonmuscle F-βactin, but not immobilized muscle F-α-actin. They also
reported that purified pericyte ezrin did not bind F-β-actin
directly, and suggested that the interaction might be mediated
by a β-actin specific binding protein; in view of our current
understanding, perhaps the bulk of the isolated pericyte ezrin
was dormant and had a masked F-actin binding site. Yao et al.
(1995, 1996) showed that the distribution of ezrin in parietal
cells coincides with that of β-actin, and that ezrin isolated from
parietal cells bound β/γ- but not F-α-actin in vitro. In addition
to the interesting possibility that the isolated parietal cell ezrin
was in an activated form, it is also possible that ezrin simply
binds with greater affinity to β-actin than to the α-isoform, as
discussed by Yao et al. (1996).
Two reports suggest that ERM proteins have other actin
binding activities. Radixin was isolated as a barbed-end
capping protein making use of immobilized monomeric actin
in the purification procedure (Tsukita et al., 1989); however,
no monomeric actin binding site was detected in native ezrin
using this approach (Bretscher, 1986). Recently, Roy et al.
(1997) employed a solid phase assay to detect actin binding
activities in full length and truncated ezrin. Although no simple
C-terminal F-actin binding site was detected, they found one
F-actin binding site in the N-terminal domain (1-310), and a
second site present in the full length molecule and requiring
residues 13-30. In addition, a G-actin binding site was mapped
to residues 288-310. Clearly the nature of the binding of ezrin
to actin has not yet been resolved satisfactorily and appears to
be quite complicated. A perplexing and perhaps revealing
finding is that much of the ezrin and actin in isolated placental
microvilli is highly resistant to extraction (Berryman et al.,
1995). Perhaps under certain circumstances, ezrin has the
ability to bind avidly to F-actin and stabilize it against depolymerization. The possibility also exists that ezrin binding to Factin is enhanced by an additional factor in a ternary complex,
as is the case for the band 4.1/spectrin/F-actin complex
(Ungewickell et al., 1979).
MEMBRANE ASSOCIATION OF EZRIN
Since ezrin shares homology with the membrane binding
domain of erythrocyte band 4.1, a major goal has been to
identify the membrane protein(s) to which it binds. Tsukita et
al. (1994) used a monoclonal antibody to co-immunoprecipitate moesin together with several additional proteins from
BHK cells, among which was the hyaluronate receptor CD44.
Immunoprecipitation of CD44 recovered all three ERM
members, suggesting that they all can bind to this transmembrane protein. Hirao et al. (1996) extended these findings to
show in vitro that full-length ERM members require PIP2 to
bind the cytoplasmic tail of CD44, whereas the isolated Nterminal domains bind with high affinity in the absence of PIP2.
Apparently, PIP2 can activate the dormant protein and expose
the membrane binding site. Niggli et al. (1995) have shown
that full length ezrin binds PIP2 in vitro, and have mapped the
binding site to the N-terminal domain. It is likely that other
membrane attachment proteins exist for ERM members,
because CD44 is not found in some cells that are highly
enriched in ezrin, such as the placental syncytiotrophoblast (St
Jacques et al., 1993). In addition, the subcellular distribution
of ERM proteins in polarized epithelial tissues does not always
correspond to that of CD44, which is usually found in the basolateral membrane. In another system, Helander et al. (1996)
have shown that transfection of human ezrin into mouse
thymoma BW5147 cells induces the formation of the actinbased uropod and redistribution of the intercellular adhesion
molecule ICAM-2 to uropods which renders the cells susceptible to attack by IL-2-activated killer cells. It is therefore
possible that ezrin also binds directly or indirectly to the cytoplasmic domain of ICAM-2.
Reczek et al. (1997) have isolated a family of 50-55 kDa
phosphoproteins from human placenta and bovine brain that
bind to the immobilized N-terminal domains of ezrin or moesin
with high affinity. The human protein, called EBP50 (ERM
binding phosphoprotein 50) is 357 residues long and contains
two PDZ domains in the N-terminal half of the molecule. Since
PDZ domains can associate with the cytoplasmic tails of transmembrane proteins (Saras and Heldin, 1996), it seems likely
that EBP50 links ERM members to one or more integral
membrane proteins. The probable rabbit homolog of EBP50,
referred to as NHE-RF (Na+/H+ exchanger regulatory factor),
was identified as a cofactor necessary for the PKA regulation
of the renal Na+/H+ exchanger (Weinman et al., 1993) and
shares 84% identity with EBP50 (Reczek et al., 1997;
Weinman et al., 1995). Using the yeast two-hybrid system to
search for proteins that bind to the cytoplasmic domain of the
NHE3 isoform of the exchanger, Yun et al. (1997) recovered a
clone encoding a protein (called E3KARP) having two Nterminal PDZ domains with >70% identity over this region to
both EBP50 and NHE-RF. The second PDZ domain of
E3KARP appears to be sufficient for interaction with NHE3.
Since the N-terminal domain of ezrin binds to the C-terminal
region of EBP50 (D. R. and A. Bretscher, unpublished), it is
possible that EBP50 links ezrin to NHE3, in tissues containing
this exchanger (Tse et al., 1992). Given the relatively high
levels of EBP50 expression in organs rich in epithelia (Reczek
et al., 1997), it seems reasonable to speculate that it may also
link ERM proteins to alternate transmembrane proteins in other
cells.
The finding of a protein containing PDZ domains that
interacts with the N-terminal domains of ERM members is
reminiscent of membrane associations in other cortical
systems. A protein known as p55, containing a single PDZ
domain, an SH3 domain and a guanylate kinase (GUK)
domain, is involved in the association of the N-terminal domain
of band 4.1 with glycophorin C in erythrocytes (Marfatia et al.,
1994, 1995). The Drosophila discs-large tumor suppressor
protein has three PDZ domains, one SH3 domain and a GUK
domain (Woods and Bryant, 1991). Its human homolog, hDlg,
has a similar domain organization, binds to the N-terminal
domain of band 4.1, and localizes to membranes and regions
of cell-cell contact (Lue et al., 1994). One binding site for band
4.1 resides in a basic region between the SH3 and GUK
Ezrin in assembly of cell surface structures 3015
domains, and the first two PDZ domains associate with the
Shaker K+ channel (Lue et al., 1996; Marfatia et al., 1996).
Although ezrin has been reported to be a component of a
complex recovered by immunoprecipitation with hDlg antibodies, probably by direct interaction with hDlg (Lue et al.,
1996), no direct binding of ezrin to hDlg was detected in vitro
(Marfatia et al., 1996). Therefore, PDZ-containing proteins
constitute another mechanism by which ezrin associates with
transmembrane proteins.
ADDITIONAL EZRIN BINDING PARTNERS
Ezrin has been identified recently as an anchoring protein for
the regulatory RII subunit of PKA in parietal cells, suggesting
it might be involved in the functional localization of PKA
(Dransfield et al., 1997). Interestingly, Dransfield et al. (1997)
reported that the RII subunit of PKA appears to be capable of
binding to essentially all the ezrin in cell extracts, suggesting
that it is the first example of a protein that can bind to dormant
ezrin. Putting this study together with those of Reczek et al.
(1997) and Yun et al. (1997), an attractive model can be
envisaged whereby the PKA catalytic and regulatory complex
bound to ezrin is recruited by EBP50 (or E3KARP) to transporters such as NHE3, thus localizing the kinase for cAMPdependent regulation of their activities.
Another ERM binding partner has been discovered recently:
Hirao et al. (1996) reported that moesin immunoprecipitates
contain Rho-GDI (Rho-GDP-dissociation inhibitor) in addition
to CD44. Further evidence that the Rho signalling pathway
involves ERM proteins came from experiments which showed
that the association of exogenous ERM proteins with CD44 in
permeabilized BHK cells was reduced when Rho was inactivated and enhanced when activated Rho was present. Most
recently, Takahashi et al. (1997) have found that binding of
Rho-GDI to the N-terminal domain of ERM members releases
Rho to activate Rho-dependent processes. By contrast, Mackay
et al. (1997) have reported that in permeabilized quiescent 3T3
fibroblasts ERM proteins are an essential downstream
component of active Rho and Rac to induce the assembly of
stress fibers and lamellipodia. From these studies, it seems
possible that ERM proteins may play dual roles (Fig. 2) acting
in the upstream activation of the Rho pathway (e.g. binding
Rho-GDI), and as downstream targets of activated Rho and Rac
(e.g. stimulation of membrane-cytoskeletal linkages and the
assembly of actin-based cortical structures).
REGULATION OF CORTICAL STRUCTURE BY EZRIN
There is considerable evidence to indicate that ezrin regulates
the structure of the cortical cytoskeleton to control cell surface
topography. For example, there is a good correlation between
tyrosine or serine/threonine phosphorylation and the formation
Fig. 2. A working model for the involvement of ezrin in the assembly of actin-rich surface structures and in Rho signalling pathways. For both
of these pathways, dormant ezrin has to be activated in one or more steps, perhaps through interaction with the lipid PIP2 or as a substrate of
tyrosine and serine/threonine kinases (1); the number and types of activated forms is not yet known. Following activation to unmask binding
sites, ezrin can associate with itself and/or membrane and cytoskeletal components (2); some of these associations require active Rho (3).
Activation of a sufficient number of ezrin molecules (4) may lead to cytoskeletal membrane associations (adaptor and membrane proteins are
not shown), and together with an F-actin crosslinker such as α-actinin, induces the formation of cell surface structures (5). An activated ezrin
form appears to be involved indirectly in the activation of Rho through the sequestration of Rho-GDI (6) which results in the release of free Rho
to associate with the membrane and initiate downstream events, some of which might also require ERM proteins (7). Note that in this diagram,
each solid arrow may represent more than one step, and broken lines indicate less well defined pathways.
3016 A. Bretscher, D. Reczek and M. Berryman
of microvilli and membrane ruffles that contain abundant ezrin
(Bretscher, 1989; Hanzel et al., 1989; Thuillier et al., 1994).
Although mutation of the two major tyrosine phosphorylation
sites to phenylalanine renders transfected LLC-PK1 cells unresponsive to hepatocyte growth factor (HGF, also known as
scatter factor), which normally induces tyrosine phosphorylation of ezrin and dramatic morphological changes, this does
not preclude the localization of ezrin to microvilli (Crepaldi et
al., 1997).
Administration of antisense oligonucleotides to suppress
expression of all ERM proteins results in the loss of cell surface
microvilli and cell contacts (Takeuchi et al., 1994). As elaborated above, overexpression of C-terminal domains of ezrin or
radixin with unmasked activities leads to a dramatic reorganization of cortical microfilaments (Henry et al., 1995; Martin et
al., 1995, 1997). It follows that overexpression of just the NERMAD in an otherwise wild type cell might be expected to
rapidly bind and mask any C-terminal sites uncovered by signal
transduction pathways. Such a situation has just been reported:
Crepaldi et al. (1997) found that overexpression of the ezrin NERMAD in kidney-derived LLC-PK1 cells acts as a dominant
negative molecule to greatly reduce the number of cell surface
microvilli and render the cells unresponsive to HGF/scatter
factor.
Given these findings, it is not surprising that ezrin undergoes
reorganization during development in cells that assemble
microvilli. During mouse embryogenesis, ezrin is localized to
microvilli over the whole cell surface from the oocyte until
compaction, when the cells become polarized and ezrin
becomes restricted to the abundant apical microvilli (Louvet et
al., 1996). Likewise, following fertilization of sea urchin eggs,
a massive assembly of moesin-containing surface microvilli
occurs (Bachman and McClay, 1995). It will be very interesting to uncover the signals that activate the ERM proteins in
these systems.
Even less is known about the disassembly of dynamic
cortical structures. However, Chen et al. (1995) have shown
that dephosphorylation of ezrin on serine/threonine residues
correlates with the disassembly of microvilli in kidney during
anoxia. It is also noteworthy that ezrin is also very susceptible
to calpain cleavage, so this protease may play an active role in
the disassembly of cortical structures (Shuster and Herman,
1995; Yao et al., 1993). Interestingly, moesin is much more
resistant to calpain, and in fact survives during platelet activation (Nakamura et al., 1996; A. Shcherbina et al., unpublished).
Perhaps one of the functional differences between ezrin and
moesin is their different susceptibilities to regulatory proteases,
possibly explaining the absence of ezrin (and radixin) from
platelets (A. Shcherbina et al., unpublished).
PERSPECTIVES
Nearly 15 years ago, ezrin was simply known as a minor
component of cell surface structures. Now it is recognized as
the prototypic member of the ERM family - a family of highly
regulated molecules playing a central role in determining the
structure and function of the plasma membrane (Fig. 2). The
models that apply to ezrin, radixin and moesin will undoubtedly be tested for merlin/schwannomin, the neurofibromatosis
type 2 (NF2) tumor suppressor gene product (Rouleau et al.,
1993; Troffater et al., 1993). This protein shares 61% sequence
identity with ezrin over the N-terminal domain, but is more
divergent in its C-terminal half. Currently, little is known about
the biochemistry and cell biology of merlin/schwannomin,
although it does appear to be enriched in ruffling membranes
(Gonzalez-Agosti et al., 1996; Sainio et al., 1997).
There are many fascinating questions that remain to be
answered, some of which are indicated in Fig. 2. For example,
how is ezrin activated, and how many different activated forms
are there? Is activation specifically elicited at the membrane (by
phosphorylation, PIP2 or perhaps downstream of activated
membrane-bound Rho), or can it occur as a result of a cytoplasmic signalling pathway? Does the mode of activation
determine the specificity of ligand binding? Does the mode of
activation vary during development, and does it differ among
cell types or ERM family members? Ezrin associates with
CD44 and EBP50 for membrane association, but what is the
full repertoire of membrane-associated ligands? Do ERM
proteins help regulate the activities of their membrane binding
partners, perhaps through association with kinases, such as
PKA? How is ezrin restricted to the apical aspect of polarized
cells? In other parts of the molecule, how is F-actin binding
regulated, and how is ezrin restricted to surface structures that
contain an actin cytoskeleton? Why are there three members of
this family with very distinct tissue distributions? The full story
of how conformational activation of ERM proteins regulates
plasma membrane topography is therefore far from complete.
The work in the authors’ laboratory was supported by National
Institutes of Health grant GM36652.
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(Acceoted 27 October 1997)