From the Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853
Received for publication, July 27, 2000, and in revised form, November 15, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The neurofibromatosis 2 tumor suppressor
gene product merlin has strong sequence identity to the
ezrin-radixin-moesin (ERM) family over its ~300-residue N-terminal
domain. ERM proteins are membrane cytoskeletal linkers that are
negatively regulated by an intramolecular association between domains
known as NH2- and COOH-ERM association domains (N-
and C-ERMADs) that mask sites for binding membrane-associated proteins,
such as EBP50 and E3KARP, and F-actin. Here we show that merlin has
self-association regions analogous to the N- and C-ERMADs. Moreover,
the N-/C-ERMAD interaction in merlin is relatively weak and dynamic,
and this property is reflected by the ability of full-length
recombinant merlin to form homo-oligomers. Remarkably, the merlin
C-ERMAD has a higher affinity for the N-ERMAD of ezrin than the N-ERMAD
of merlin. Both the ezrin and merlin N-ERMAD bind EBP50. This
interaction with the ezrin N-ERMAD can be inhibited by the presence of
the ezrin C-ERMAD, whereas interaction with the merlin N-ERMAD is not
inhibited by either C-ERMAD. E3KARP binds tightly to the ezrin N-ERMAD
but has little affinity for the merlin N-ERMAD. The implications of these associations and the hierarchies of binding for the function and regulation of merlin and ERM proteins are discussed.
The disease neurofibromatosis 2 (NF2)1 is a
genetic disorder in which loss of heterozygosity of the NFZ gene
leads to bilateral schwannomas of the auditory nerve and other tumors
of the central nervous system (1). Genetic analysis and positional
cloning mapped the human NF2 gene to chromosome 22 and led
to the identification of the affected gene. The NF2 gene
product, a protein referred to as either merlin or schwannomin (2, 3),
shows sequence similarity to the ezrin-radixin-moesin (ERM) family of
cytoskeleton-plasma membrane linking proteins, sharing ~65% sequence
identity with the ERM family over the first 300 residues with ~45%
identity overall. Two major isoforms of human merlin have been
documented. Isoform I has 595 residues, resulting from translation of
exons 1-15 and 17, and isoform II has 590 residues and is a
translation of exons 1-16, replacing the last 16 residues from exon 17 of isoform I with 11 residues from exon 16 (4, 5).
Insight into the possible roles of merlin have come in part from
studies of mice and flies lacking merlin, as well as from the
identification of merlin-binding proteins. NF2 knockout mice are embryonic lethal as they do not develop proper extraembryonic tissues and fail to implant (7) and NF2+/-
heterozygous mice develop a wide range of metastatic tumors (8). Loss
of merlin in Drosophila melanogaster results in
overproliferation of cells (9). Interestingly, merlin shares many
biochemical and cell biological similarities with the ERM family. In
addition to their partly overlapping subcellular distributions
(10-13), they bind to several common proteins, such as CD44,
EBP50/human Na+/H+ exchanger regulatory
factor, and RhoGDI (13-19). Moreover, merlin can associate with
ERM family members (15, 20, 21). However, the nature of these
interactions have so far not been examined in detail, especially in
comparison with the better studied ERM proteins.
The three ERM family members are ~580 residues long and share ~75%
sequence identity. They provide a regulated linkage between membrane-associated proteins and the cortical actin cytoskeleton, and
they also participate in and are regulated by signal transduction pathways (for recent reviews, see Refs. 22-25). Biochemical and structural studies have revealed the nature of the conformational regulation of the ERM proteins. The NH2-terminal
~300-residue domain of any ERM member can form a tight association
with the COOH-terminal ~100 residues of any member, leading these
regions to be called NH2- and COOH-ERM association domains
(N- and C-ERMADs) (26, 27). In their dormant monomeric state, the
intramolecular interaction between the N- and C-ERMADs masks the
C-terminal F-actin binding site (28, 29) and N-terminal binding sites
for some membrane-associated proteins and Rho-GDI (19, 26, 30). Among the proteins known to bind the N-terminal domain of ezrin in a regulated fashion is the PSD-95/DlgA/Z0-1-like domain-containing scaffolding protein EBP50 (an ERM-binding phosphoprotein of 50 kDa)
(17, 30). Interestingly, merlin also binds EBP50 (also called human
Na+/H+ exchanger regulatory factor (16)).
Activation of ERM proteins to unmask binding sites involves
dissociation of the intramolecular N-/C-ERMAD, which can be achieved by
phosphorylation of a threonine 20 residues from the C terminus in
combination with specific inositol phospholipids (31-37).
The possibility that merlin might be subject to a similar regulatory
mechanism has been investigated in a number of studies. In support of
this possibility is the finding that the N-terminal half of merlin can
associate in vitro with the C-terminal half of isoform I but
not isoform II (6, 15, 20, 21), and ezrin and merlin have been shown to
exist as heterodimers in vivo (15). Moreover, EBP50/human
Na+/H+ exchanger regulatory factor binds better
to merlin isoform II than to isoform I, suggesting that the C terminus
is important in this intramolecular association (20). However, the idea
that merlin might be regulated by a mechanism equivalent to the
N-/C-ERMAD associations was initially considered unlikely, as isoform I
shares only 24% sequence identity with the ERMs over the region
corresponding to the C-ERMAD.
Therefore, we set out to determine whether the regions in the N- and
C-terminal halves of merlin described above might be functionally
analogous to the N- and C-ERMADs of the ERM proteins. Concurrently, the
availability of the atomic structure of the moesin N-/C-ERMAD complex
allowed us to use sequence alignments with merlin to examine whether
isoform I was likely to have N- and C-ERMADs. Remarkably, the alignment
showed that residues potentially on the N- and C-ERMAD interface were
strongly conserved between merlin and the ERM proteins, thereby
predicting a similar interaction for merlin (38). During the completion
of the present work, affinity coelectrophoresis studies reported a high
affinity interaction between the N- and C-terminal halves of merlin and
between the N-terminal half of merlin and the C-terminal half of moesin
(20). Analyses of homo- and heterotypic associations, as well as
interactions involving the scaffolding proteins EBP50 and E3KARP,
identify hierarchies of binding reactions between domains of these
proteins. These studies allow us to demonstrate regulated interactions
between merlin, ezrin, and their ligands that need to be integrated
into any model for tumor suppression by merlin.
Antibodies and Reagents--
Rabbit antisera to ezrin have been
described (39). Antisera to N-terminal domain (residues 1-313) of
merlin were raised against purified recombinant protein in rabbits at
Cornell University (Ithaca, NY). cDNA for full-length merlin was a
kind gift from Dr. J. F. Gusella. MBP-E3KARP was a kind gift from
Dr. C. H. Yun. Restriction enzymes were purchased from Life
Technologies, Inc., and fast protein liquid chromatography
instrumentation was from Amersham Pharmacia Biotech.
Cloning of Ezrin and Merlin Constructs, EBP50, and
E3KARP--
The cloning of full-length ezrin (residues 1-586) and
Ez-1-297 has been described (30). The cDNA for merlin N-terminal
domain was purchased from Genome Systems Inc. (St. Louis, MO). This
cDNA was subcloned into expression vector pQE50 (Qiagen Inc.,
Chatsworth, CA) by polymerase chain reaction amplification and
restriction digest. The full cDNA for human merlin isoform I
(JJR-1) in BlueScript II/SK- plasmid from Dr. J. F. Gusella was
used to generate the full-length subclone of full-length merlin for
inducible overexpression in E. coli. Briefly, the
1.2-kilobase pair fragment from the partial digest of clone
JJR-1 with BamHI and HindIII, which contains the C-terminal half of merlin, was spliced to the N-terminal half of merlin
in pQE50 at the 865 HindIII site of the coding sequence of
merlin, and the 3'-end backbone of BSII/SK- was spliced to pQE50 at
the BamHI site. The subclone, named JJRR-4 (JJR-1 recloned, colony 4), was checked by restriction digest and found to be free of
errors by DNA sequencing. All pQE50-derived plasmids were propagated in
JM109 strain of E. coli (Qiagen). The cloning and expression of the COOH-terminal GST fusion constructs of ezrin, GST-Ez-475-586 and GST-Ez-475-584, have been described (26). The COOH-terminal GST
fusion constructs of merlin, GST-Mr-359-595ci (isoform I), GST-Mr-359-590cii (isoform II), GST-Mr-477-595, GST-Mr-477-593, GST-Mr-477-588, and GST-Mr-502-595 were made by polymerase chain reaction amplification of JJRR-4 plasmid at appropriate sites with
5'-BamHI and 3'-EcoRI overhangs and cloned into
vector pGEX-3X (Amersham Pharmacia Biotech). Polymerase chain reaction
products were inserted into pCRII Topo TA vector (Invitrogen Inc., San Diego, CA), and fidelity was confirmed by sequencing. These merlin C-terminal polymerase chain reaction products were inserted into pGEX-3X vector at the BamHI and EcoRI sites. All
pGEX-derived plasmids were propagated in DH5 Expression and Purification of Recombinant Proteins--
For
expression, untagged full-length and NH2-terminal
constructs of ezrin and merlin, Ez-1-586, Ez-1-297, Mr-1-595, and
Mr-1-313 were propagated in E. coli strain M15[pREP4]
(Qiagen). Bacteria were grown in LB medium containing 100 µg/ml
ampicillin and 25 µg/ml kanamycin. Freshly saturated overnight
cultures were diluted 1:20 or 1:30 and grown with vigorous shaking at
37 °C for 90-120 min until A595
reached 0.6-0.8, at which point the culture was induced with 1.5 mM isopropyl-
Full-length ezrin (Ez-1-586), Ez-1-297, Mr-1-595 and Mr-1-313 were
purified from total bacterial extracts by fast protein liquid
chromatography (Amersham Pharmacia Biotech) over hydroxyapatite (HA-Ultragel; Amersham Pharmacia Biotech) then S (Amersham Pharmacia Biotech) or Q (Amersham Pharmacia Biotech) Sepharose columns. Methods
for purifying full-length ezrin and Ez-1-297 have been described (30).
Because expression level is ~ Binding Assays--
Proteins purified off the ion exchange
columns were dialyzed into coupling buffer (0.1 M
NaHCO3, pH 8.3, 0.5 M NaCl) for covalent coupling to CNBr-activated Sepharose 4B beads (Sigma) according to the
manufacturer's protocol and as described (17). GST fusion proteins
were purified according to the manufacturer's protocol (Amersham
Pharmacia Biotech). Binding assays were performed in BA buffer (50 mM Tris-HCl, pH 7.4, 100 or 150 mM NaCl, 0.1%
TX-100). 10 µl of beads, with 1-2 mg of protein coupled per ml, were
used per reaction. The beads were equilibrated with 0.5 ml of BA buffer prior to the addition of normalized amounts of crude bacterial extract.
BA buffer was added to each reaction tube to normalize the final volume
to 0.5 ml. The beads were incubated in extract for 30-60 min, pelleted
by centrifugation at 16,000 × g, and washed 4-5 times
in BA buffer. Bound proteins were eluted by boiling in 30-50 µl of
2× Laemmli buffer for 2 min, and 10-20 µl were loaded for analysis
by gel electrophoresis.
For the mixing competition assays, bacterial extracts of the proteins
of interest, normalized to contain equal amount of the induced
proteins, were incubated together with protein coupled beads for 30-60
min. The beads were then pelleted and washed 4-5 times in BA buffer.
One-third of the bound fraction was analyzed by SDS-polyacrylamide gel
electrophoresis. For the challenge competition assays, beads were first
incubated with a saturating amount of the first protein for 30-60 min,
washed 4-5 times in BA buffer, and then challenged with the same
amount of a second protein.
Gel Electrophoresis and Western Blots--
All samples were
boiled in Laemmli buffer and analyzed on 10 or 12% gels following
standard protocols (41). For Western blots, proteins were transferred
to polyvinylidene fluoride membranes (Immobilon-P, Millipore, Bedford,
MA) with a semidry transfer system (Integrated Separation Systems, Hyde
Park, MA). Blots were blocked with 10% nonfat dry milk in rinse buffer
containing 150 mM NaCl, 0.1% Tween-20, 50 mM
Tris-HCl, pH 8.0, incubated with 1:5,000 rabbit antisera in 1% milk
followed by 1:10,000 peroxidase-conjugated goat anti-rabbit IgG (Sigma)
in 1% milk. Blots were developed using enhanced chemiluminescence
(ECL) (Amersham Pharmacia Biotech).
Gel Filtration--
Full-length merlin or ezrin was loaded onto
a Superose 6 HR 10/30 (Amersham Pharmacia Biotech) gel filtration
column equilibrated with 50 mM Tris-HCl, pH 7.4, 150 or 500 mM NaCl, 1 mM EDTA, 0.5 mM
dithiothreitol at a flow rate of 0.2 ml/min; 0.5-ml fractions were
collected. The column was calibrated with conventional gel filtration
standards (Sigma). Blue dextran was used to determine the void volume.
Expression of Recombinant Ezrin and Merlin Constructs--
Ezrin
is regulated by an intramolecular association between the N- and
C-ERMADs, and these domains have been mapped to residues 1-297 and
475-586, respectively (Fig.
1A) (26). To determine whether
merlin has analogous domains, and whether domains of ezrin can interact
with domains of merlin, a number of ezrin and merlin constructs were
utilized (Fig. 1B).
We have described several ezrin constructs that were used to define the
N- and C-ERMADs as well as to demonstrate that the intramolecular
interaction masks the F-actin and EBP50 binding sites in the
full-length dormant molecule (26, 30). Among these (Fig. 1B)
were untagged ezrin N-ERMAD (Ez-1-297) and GST fusion proteins
containing the ezrin C-ERMAD (GST-Ez-475-586) and the mutated C-ERMAD
(GST-Ez-475-584) missing the last two residues.
Equivalent and additional constructs for merlin were generated based on
an alignment of the ERM and merlin protein sequences (38). Constructs
were made to express untagged full-length merlin isoform I (Mr-1-595)
and the N-terminal domain equivalent to the ezrin N-ERMAD, Mr-1-313.
In addition, we generated constructs to express GST fusion proteins
with the C-terminal regions of merlin isoforms I and II in which
similarity to the ERMs significantly declines (GST-Mr-359-595ci and
GST-Mr-359-590cii), as well as regions of merlin isoform I
corresponding to the ezrin C-ERMAD (GST-Mr-477-595), ones lacking the
last two residues (GST-Mr-477-593) or seven residues
(GST-Mr-477-588), and a shorter C-terminal construct (GST-Mr-502-595).
Expression of all of these constructs in bacteria resulted in readily
identifiable bands corresponding to the induced proteins (Fig.
1C, asterisks), except for expression of full-length merlin (Mr-1-595) and Mr-1-313. These proteins were detected in immunoblots using antibodies directed against the N-terminal domain of merlin (Fig.
1D). Ezrin, but not merlin, runs anomalously slowly on
SDS-polyacrylamide gel electrophoresis gels, and the region responsible
is contained within the ezrin C-ERMAD (26). Thus, although the
GST-Ez-475-586 construct is seven residues shorter than
GST-Mr-477-595, it runs with a much slower mobility in
SDS-polyacrylamide gel electrophoresis (compare Fig. 1C,
lanes 3 and 9, respectively).
Purificaton of Full-length Merlin and Its NH2-terminal
Domain--
The purification of full-length natural or recombinant
merlin has not been reported, and therefore the intact protein has not
yet been characterized biochemically. To aid our studies, we developed
methods for the purification of both untagged recombinant full-length
merlin and its free NH2-terminal domain (Fig.
2).
Like ezrin (42, 43), merlin and its NH2-terminal domain
bind to hydroxyapatite resins with high affinity. Immunoblot analysis revealed that full-length merlin in crude bacterial extracts bound quantitatively to this resin (data not shown) and could be eluted using
a potassium phosphate gradient. Subsequent chromatography on Q
Sepharose yielded the purified full-length protein (Fig. 2A). Likewise, the NH2-terminal domain
(Mr-1-313) bound tightly to hydroxyapatite and could be obtained in a
highly purified form after chromatography over S Sepharose (Fig.
2B), in a manner previously described for the ezrin N-ERMAD
(17) and shown in Fig. 2C. The yield for each merlin
construct was ~0.5 mg/liter of bacterial culture, whereas for the
ezrin N-ERMAD it was ~10 mg/liter bacterial culture.
Merlin Isoform I Has Functional Domains Corresponding to the N- and
C-ERMADs of Ezrin--
To determine whether the C-terminal domain of
merlin binds either the merlin or ezrin N-ERMAD, purified Ez-1-297 and
Mr-1-313 were coupled to beads and used in pull-down assays. Beads
were incubated with total soluble bacterial extracts expressing various constructs (shown in the summary diagram in Fig.
3D) and washed extensively,
and bound proteins were eluted and analyzed by gel electrophoresis. In
all of these experiments, the same molar concentration of the ezrin or
merlin N-terminal domain was immobilized on the beads and the bacterial
extracts contained a large excess of recombinant protein to allow for
the recovery of both tight and relatively weak interactions as a
starting point for our analysis (Fig. 3A).
Both sets of beads bound the ezrin C-ERMAD (Fig. 3A, lanes
1, GST-Ez-475-586), the C-terminal half of merlin isoform I
(lanes 3, GST-Mr-359-595ci), and the C-terminal region of
merlin corresponding to the ezrin C-ERMAD (GST-Mr-477-595). The
C-terminal half of merlin isoform II (lanes 4, GST-Mr-359-590cii) and the merlin constructs lacking the last two or
seven residues (GST-Mr-477-593 and GST-Mr-477-588) did not bind
either set of beads (lanes 6 and 7). Thus, the
C-terminal region of merlin isoform I, but not of isoform II, binds the
N-terminal domains of ezrin or merlin, and efficient binding is
dependent on the last two residues, as is the binding of the C-ERMAD of
ezrin to the N-ERMAD of ezrin (26). We conclude that merlin isoform I
has functional N- and C-ERMADs, and we use this nomenclature henceforth.
To compare the binding between homotypic and heterotypic N- and
C-ERMADs, the sensitivity of the interactions to increasing salt
concentrations was determined (Fig. 3B). Whereas the binding of either C-ERMAD to the ezrin N-ERMAD was relatively unaffected by
increasing NaCl washes up to 2 M, the binding of either
C-ERMAD to the merlin N-ERMAD was much more salt-sensitive, being
significantly reduced by 0.5 M NaCl.
During studies of the various deletion constructs, we were surprised to
find that the ezrin C-ERMAD construct lacking the last two residues
(GST-Ez-475-584) showed significant binding to the ezrin N-ERMAD (Fig.
3C, top panel) under physiological salt conditions.
Previously, we had reported that the presence of these two residues
were critical for the homotypic ezrin interaction, based on a blot
overlay assay (26). Thus, although a solid phase assay reveals this
requirement, the solution assay indicates that it is not absolute. We
also found that removal of the first 25 residues from the merlin
C-ERMAD construct, to generate GST-Mr-502-595, resulted in a fusion
protein that still bound both the ezrin and merlin N-ERMADs (Fig.
3A, lanes 8), albeit with a lower recovery. We therefore
compared the retention of the modified ezrin and merlin C-ERMADs on
beads containing either N-ERMAD (Fig. 3C). Whereas the ezrin
N-ERMAD beads retain both C-ERMAD constructs efficiently, the merlin
N-ERMAD beads do not retain GST-Ez-475-584 and retain GST-Mr-502-595
only slightly (Fig. 3C, top panels). Moreover, the
interaction of the two modified constructs with the ezrin N-ERMAD is
much more salt-sensitive than the full C-ERMAD constructs (compare Fig.
3B, left panel, with 3C, bottom panels). We
conclude that for a high affinity interaction, the full C-ERMADs of
both ezrin (residues 475-585) and merlin (residues 477-595) are required.
In all of these experiments, the same molar amount of the ezrin or
merlin N-ERMAD was coupled to beads, yet the ezrin beads consistently
appear to have a somewhat higher binding capacity than the merlin beads
for the same ligand. We assume that this is due to differences in the
orientation of the two proteins during coupling to the resin.
Native Full-length Merlin Exists in Multiple Forms--
Earlier
work has documented that ezrin can be isolated from tissues as a
relatively globular dormant monomer or an elongated dormant dimer, and
the two forms have distinct and characteristic migration positions on
gel permeation chromatography. Interconversion between these two
purified forms is exceptionally slow, so they can be isolated and
characterized as stable species (43, 44). Equivalent forms of ezrin can
be isolated from bacterially expressed ezrin.2 The dormant monomers
have an intramolecular N-/C-ERMAD association, and the dimers are
associated through one or two N-/C-ERMAD associations. Because the
interaction between the isolated merlin N- and C-ERMADs is
salt-sensitive, being greatly reduced by 0.5 M salt, we
examined the migration behavior of native full-length recombinant
merlin under physiological salt (0.15 M NaCl) and high salt
(0.5 M NaCl) conditions.
Because ezrin and merlin have very similar polypeptide molecular
masses, we first calibrated our Sephacryl 6HR gel permeation column
with recombinant ezrin. The N-/C-ERMAD interaction in ezrin is little
affected by the inclusion of 0.5 M NaCl (Fig.
4), so the monomers and dimers present in
recombinant ezrin provide useful markers for the possible different
conformations of merlin. Chromatography of merlin in 0.5 M
salt revealed a single species with a Stokes radius slightly larger
than the ezrin monomer, but much smaller than the ezrin dimer,
consistent with merlin existing as a monomer under these conditions. In
fact, merlin comigrates with a recombinant ezrin in which the
N-/C-ERMAD interaction has been genetically destroyed revealing a more
extended monomeric form.3 We
conclude that merlin exists as a monomer in 0.5 M NaCl,
perhaps in an extended conformation in which the N-/C-ERMAD interaction is compromised.
Under physiological conditions of 0.15 M NaCl, merlin did
not migrate as a uniform species but migrated as a broad band
representing Stokes radii corresponding to greater than ezrin dimers
down to ezrin monomers. We interpret this finding as the ability of
merlin to self-associate through N-/C-ERMAD interactions to form dimers and higher form oligomers.
The C-ERMAD of Merlin Has a Higher Affinity for the N-ERMAD of
Ezrin than That of Merlin--
The finding that the C-ERMAD of
merlin has a higher salt resistance when binding the N-ERMAD of ezrin
compared with its own prompted us to investigate the relative
affinities under physiological conditions. Bacterial extracts
containing a mixture of equal amounts of the ezrin and merlin C-ERMADs
were used in pull down assays with N-ERMAD beads to see which bound
with higher efficiency (Fig. 5A). The ezrin N-ERMAD beads
selectively pulled down the ezrin C-ERMAD, whereas the merlin N-ERMAD
beads pulled down both C-ERMADs, but with a clear preference for the
ezrin C-ERMAD. In an alternative version of this experiment, the
respective N-ERMAD beads were saturated with either C-ERMAD and then
challenged by incubation with bacterial extract containing an excess of
the alternate C-ERMAD (Fig. 5B). Similar outcomes were found
regardless of the order of binding: the ezrin N-ERMAD beads showed
complete preference for the ezrin C-ERMAD, whereas the merlin N-ERMAD
beads showed a strong preference for the ezrin C-ERMAD. We conclude
that the N-ERMADs of ezrin and merlin bind the ezrin C-ERMAD with a
higher affinity than the C-ERMAD of merlin.
Because both N-ERMADs showed a preference for the ezrin C-ERMAD, we
wished to determine which of the two N-ERMADs bound preferentially to
the ezrin C-ERMAD. Therefore, the ability of immobilized ezrin C-ERMAD
(GST-Ez-475-586) to retain the two N-ERMADs was assessed (Fig.
5C). Both N-ERMADs bound to the ezrin C-ERMAD, whether added alone or together (Fig. 5C, left panel). When one N-ERMAD
was prebound and then challenged with the other, the resin still bound both, but with a small preference for the ezrin N-ERMAD.
Because our results indicate that the merlin C-ERMAD has a higher
affinity for the ezrin over the merlin N-ERMAD, it should be possible
to bind full-length merlin to immobilized ezrin N-ERMAD. In fact, when
a bacterial extract containing a very low level of full-length merlin
(Fig. 1C, lane 5) is passed over a resin containing
immobilized ezrin N-ERMAD, full-length merlin is retained (Fig.
3A, left panel, lane 2). Moreover, full-length merlin can also be affinity purified on immobilized merlin N-ERMAD (Fig. 3A,
right panel, lane 2). A similar experiment done with full-length ezrin over immobilized ezrin N-ERMAD does not retain significant amounts of ezrin (17), revealing how much more dynamic the N-/C-ERMAD association is in merlin than in ezrin.
Relative Binding of EBP50 and E3KARP to N-terminal Domains of Ezrin
and Merlin--
Human EBP50 was discovered by its ability to be
retained from placental extracts bound on immobilized ezrin N-ERMAD
(17). It was also discovered in a two-hybrid screen with merlin and called human NHE-RF (16), as it is the human homologue of rabbit NHE-RF
(45, 46). EBP50 binds ezrin through its C-terminal 30 residues (30), as
does the related protein E3KARP (30, 40). We were therefore interested
in determining the relative affinities of these proteins for the ezrin
and merlin N-ERMADs.
Bacterial extracts expressing GST-EBP50 or MBP-E3KARP were applied to
beads containing the immobilized ezrin or merlin N-ERMAD and subjected
to washes with increasing salt concentrations, and the retained protein
was eluted and analyzed (Figs. 6,
A and B). The binding of EBP50 to the ezrin
N-ERMAD is highly resistant to salt washes, whereas the interaction
with the merlin N-ERMAD is a bit more sensitive. Ezrin N-ERMAD beads
retained E3KARP efficiently up to 1 M salt, whereas binding
to the merlin N-ERMAD was inefficient and very salt-sensitive. To
determine which ligand bound the N-ERMADs preferentially, extracts
expressing GST-EBP50 and MBP-E3KARP were either mixed and applied to
the N-ERMAD beads (Fig. 6C, left panel) or applied
sequentially in excess (Fig. 6C, right panel). In either case, ezrin N-ERMAD beads bound about equivalent amounts of EBP50 and
E3KARP, whereas merlin N-ERMAD beads had a strong preference for
EBP50.
We next examined whether EBP50 exhibits a preference for the ezrin or
merlin N-ERMAD by incubating beads containing immobilized GST-EBP50
tail with the two N-ERMADs simultaneously or sequentially and then
analyzed the retained proteins (Fig.
7A). Although both N-ERMADs
were retained on the beads, there was some preference for retention of
the ezrin N-ERMAD.
We have shown that EBP50 bound to the ezrin N-ERMAD can be largely
displaced by subsequent addition of the ezrin C-ERMAD (30). We wished
to examine whether the same situation existed with EBP50 bound to the
merlin N-ERMAD as well as the ability of either C-ERMAD to displace
bound EBP50. These experiments were performed with the two ligands
added competitively or sequentially and similar results were obtained.
Interestingly, although the ezrin C-ERMAD can displace EBP50 from the
ezrin N-ERMAD, the merlin C-ERMAD cannot do this (Fig. 7B).
Moreover, EBP50 bound to the merlin N-ERMAD is not displaced by either
the ezrin or merlin C-ERMAD (Fig. 7C). Competitive binding
between E3KARP with the C-ERMADs on ezrin N-ERMAD beads shows results
similar to those for EBP50 (data not shown).
The studies reported here establish that merlin has functional
domains equivalent to the N- and C-ERMADs of ezrin, refining the
earlier interaction results (6, 15, 20, 21) and confirming the
prediction, based on conserved residues expected to lie on the
N-/C-ERMAD interface, that merlin is likely to have functional N- and
C-ERMADs (38). The fact that the last two residues of the merlin
C-ERMAD are required for this interaction and the lack of
interaction between the equivalent C-terminal region of merlin isoform
II are consistent with the N-/C-ERMAD model developed for ERM proteins.
The questions naturally arose as to how robust this interaction is in
merlin, whether ERMAD interactions might exist between merlin and ERM
proteins, and how these interactions might affect binding of EBP50 and
E3KARP to the N-ERMADs.
By analyzing isolated domains in solution, we have been able to
establish the following binding hierarchy for the association domains of merlin, ezrin, and EBP50/E3KARP, from low to high: Mr-N-ERMAD/E3KARP < Mr-N-ERMAD/Mr-CERMAD < Mr-N-ERMAD/Ez-C-ERMAD The merlin N-/C-ERMAD interaction is of lower affinity than is seen for
ezrin and is quite dynamic. This is revealed both by studying the
interaction between the two separated domains in vitro, as
well as the ability of recombinant full-length merlin to be retained on
immobilized merlin N-ERMAD. It is also consistent with the finding that
full-length merlin migrates as a monomer by gel filtration in 0.5 M NaCl under conditions that inhibit the N-/C-ERMAD
interaction, but as oligomeric species under physiological conditions,
in which the N- and C-ERMADs can interact. Although merlin oligomers
can be prepared in vitro, there is so far no evidence for
their existence in vivo. Moreover, because merlin is a
relatively minor protein, the interaction of merlin with the much more
abundant ERM proteins may be more physiologically relevant.
If the homotypic N-/C-ERMAD of merlin interaction had been of higher
affinity than the heterotypic interaction with the domains of ezrin,
this would have suggested that the self-association of merlin may
simply be for self-regulation rather than participating in another
pathway. Thus, we were very surprised and interested to find that the
N-ERMAD of merlin has a strong preference for binding the C-ERMAD of
ezrin over its own, and the C-ERMAD of merlin has a slight preference
for the N-ERMAD of ezrin over its own. This has interesting
implications for cells that express both ezrin and merlin. The bulk of
ezrin in the cytoplasm is present in a dormant state, i.e.
with its N- and C-ERMADs associated. Activation of ezrin by
inactivating its C-ERMAD, perhaps involving phosphorylation and
phosphatidylinositol 4,5-bisphosphate binding (32-34, 36, 37),
will unmask the N-ERMAD. Because the N-/C-ERMAD association in merlin
is dynamic and the C-ERMAD has a preference for the N-ERMAD of ezrin
over its own, a relatively stable heterodimer is expected to form in
which the C-ERMAD of merlin binds to the N-ERMAD ezrin.
A well documented ligand for the N-terminal domains of ezrin and merlin
is EBP50/human Na+/H+ exchanger regulatory
factor (16, 17). Interestingly, we find that the closely related
protein E3KARP, which binds with high affinity to ezrin (30, 40), has
little affinity for merlin and is therefore probably not
physiologically relevant. The region of EBP50 involved in binding to
the ezrin N-ERMAD resides in the C-terminal 30 residues, a region that
is especially well conserved (55% identity) between EBP50 and E3KARP.
It will be very interesting to identify the residues in this region
that contribute to the binding discrimination and examine the
complementary surface residues on the ezrin and merlin N-ERMADs to
which they bind. Structural studies, currently under way, that reveal
the ERM/EBP50 interface should provide insight into this question.
How might the presence of EBP50 affect the molecular forms of merlin
and ezrin present in a cell? EBP50 binds to the merlin N-ERMAD, and
this interaction cannot be readily displaced by the merlin C-ERMAD,
implying that the association of merlin with EBP50 is not as completely
blocked as in ezrin. Merlin isoform II is expected to have a
constitutively active N-ERMAD because it does not have an active
C-ERMAD, and this difference in EBP50 retention between isoform I and
isoform II has been reported (20). By contrast, ezrin can only bind
EBP50 when it is activated. Our binding hierarchy indicates that
activation of ezrin could lead to EBP50 switching its binding partner
from merlin to activated ezrin. However, the complexes that form
in vivo will be highly dependent on the local concentrations
and activation states of ezrin, merlin, and EBP50.
Our results provide compelling biochemical evidence for an
interrelationship between the functions of ezrin and merlin. It is
interesting to note that whereas merlin is a tumor suppressor protein,
overexpression of ezrin is correlated with uncontrolled growth
(47-49). How the functions and regulation of these proteins are
intertwined remains to be elucidated, but any model must account for
the biochemical interactions documented here. Moreover, this represents
an important step in an analysis of binding hierarchies, as merlin and
ERM proteins have several additional common ligands, and more are sure
to follow. Finally, we have presented a method for expressing and
purifying full-length untagged recombinant merlin. As far as we are
aware, this is the first report on the purification of the native
protein and will provide a valuable reagent for future detailed
biochemical and biophysical studies of this tumor suppressor gene product.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
. The cloning
of GST-EBP50 (30) and MBP-E3KARP (40) has been described.
-D-thiogalactopyranoside
(Sigma) and allowed to grow for another 2-3 h. pGEX-derived plasmids
were transformed into E. coli strain BL21, grown in LB
medium containing 100 µg/ml ampicillin. Growth condition was the same
as in M15 strain, except protein expression was induced with 0.4 mM isopropyl-
-D-thiogalactopyranoside. Cells
were harvested by centrifugation at 3000 × g and
washed with 4 °C phosphate-buffered saline, and pellets were
quick-frozen on dry ice/ethanol for later use.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (49K):
[in a new window]
Fig. 1.
Summary of recombinant proteins used.
A, diagram of ezrin and merlin isoform I. B,
diagram of recombinant proteins used. 1, full-length ezrin
(residues 1-586); 2, Ez-1-297; 3, GST-Ez-475-586; 4, GST-Ez-475-584; 5, full-length merlin isoform I (1); 6, Mr-1-313;
7, GST-Mr-359-595ci isoform I; 8, GST-Mr-359-590cii isoform II; 9, GST-Mr-477-595;
10, GST-Mr-477-593; 11, GST-Mr-477-588;
12, GST-Mr-502-595; 13, GST; 14, GST-EBP50; 15, MBP-E3KARP. C, induced bands (*)
of appropriate sizes can be seen after Coomassie staining of total
induced bacterial extracts, except for full-length merlin (lane
5) and Mr-1-313 (lane 6). D, expression of
full-length merlin and Mr-1-313 can be detected by immunoblot with
antibodies raised against merlin 1-313. Extracts were prepared from
uninduced (U) and induced (I) bacteria. In
C and D, molecular masses in kDa are shown at the
left.
View larger version (40K):
[in a new window]
Fig. 2.
Purification of full-length merlin (1),
merlin 1-313, and ezrin 1-297. A, purification of
full-length recombinant merlin over hydroxyapatite and Q Sepharose
columns. B and C, final elution profiles over S
Sepharose of merlin 1-313 (B) and ezrin 1-297
(C). Merlin 1-313 migrates with an apparent
molecular mass of ~34 kDa and ezrin 1-297 with an apparent molecular
mass of 32 kDa.
View larger version (47K):
[in a new window]
Fig. 3.
Merlin has N- and C-ERMADs.
A, extracts of bacteria expressing GST-Ez-475-586
(1), 2. full-length merlin (2), GST-Mr-359-595ci
isoform I (3), GST-Mr-359-590cii isoform II (4),
GST-Mr-477-595 (5), GST-Mr-477-593 (6),
GST-Mr-477-588 (7), and GST-Mr-502-595 (8) were
incubated with either Ez-1-297 or Mr-1-313 coupled to Sepharose
beads, and the beads were washed extensively. The Coomassie gel shows
the bound fraction eluted by boiling in SDS sample buffer.
B, to determine the salt sensitivity of the homo- and
heterotypic N-/C-ERMAD interactions, extracts expressing
GST-Ez-475-586 and GST-Mr-477-595 were incubated with ezrin or merlin
N-ERMAD beads and subjected to washes of the following ionic strengths:
0.15, 0.3, 0.5, 1.0, or 2.0 M NaCl. C, top
panel, the ability of truncated C-ERMAD constructs of ezrin
(GST-Ez-475-584) and merlin (GST-Mr-502-595) to bind to ezrin and
merlin N-ERMAD beads. Bottom panel, salt sensitivity of the
interaction of the truncated C-ERMAD constructs to bind ezrin N-ERMAD
beads. After incubating the beads with bacterial extracts expressing
the constructs, the beads were washed with various concentrations
(0.15, 0.3, 0.5, 1.0, or 2.0 M) of NaCl, and the retained
proteins were eluted and analyzed by gel electrophoresis. D,
summary of the constructs used and binding results obtained.
View larger version (30K):
[in a new window]
Fig. 4.
Merlin exists as oligomers under conditions
of physiological salt. Full-length ezrin or full-length merlin
were chromatographed on a Superose 6HR gel filtration column in either
0.15 or 0.5 M NaCl. The migration of standards (bovine
serum albumin (BSA) at 66 kDa, and carbonic anhydrase
(CA) at 29 kDa) and of ezrin monomers (M) and
dimers (D), as described (43), are indicated.
View larger version (55K):
[in a new window]
Fig. 5.
Competitive binding between N- and C-ERMADs
of ezrin and merlin. A, competition between homo- and
heterotypic C-ERMADs. Ez-1-297 (EzN), Mr-1-313
(MrN), or bovine serum albumin (BSA) beads were
incubated for 30 min with a saturating amount of bacterial extract
containing an equal mixture of Ez-475-586 and Mr-477-595
(Load). After washing, bound protein was eluted and analyzed
by SDS-polyacrylamide gel electrophoresis and Coomassie staining.
B, sequential competition between the two C-ERMADs.
Ez-1-297 or Mr-1-313 beads were incubated first with saturating
amounts of Ez-475-586 or Mr-477-595, washed, and then challenged with
a second round of the alternative C-ERMAD ligand. C,
competition between N-ERMADs for the ezrin C-ERMAD. Left
panel, purified Ez-1-297 and Mr-1-313 were incubated with
GST-Ez-475-586 beads alone (lanes 1 and 2) or
together (lane 3), the beads were washed and eluted, and
released proteins were analyzed. Right panel, the two
N-ERMADs were incubated with GST-Ez-475-586 beads (GST-EzC)
and then challenged with buffer alone (lanes 4 and
5) or with the alternate N-ERMAD (lanes 6 and
7). L, load (mixture of the N-ERMADs used in
these experiments).
View larger version (55K):
[in a new window]
Fig. 6.
Comparative binding of EBP50 and E3KARP to
ezrin and merlin N-ERMADs. A and B,
bacterial extracts containing GST-EBP50 (EBP) (A)
or MBP-E3KARP (E3K) (B) were incubated with
Ez-1-297 or Mr-1-313 beads and subjected to salt washes at
concentrations 0.15, 0.3, 0.5, 1.0, or 2.0 M NaCl. Retained
proteins were eluted and analyzed. C, bacterial extracts
containing equal molar amounts of GST-EBP50 (EBP) or/and
MBP-E3KARP (E3K) were incubated with Ez-1-297-coupled
(EzN) or Mr-1-313-coupled (MrN) beads.
Left panel, proteins were incubated either separately or
together as indicated. Right panel, beads were saturated
with one ligand and the either challenged with a buffer control, or an
extract containing saturating amount of the alternate ligand. The load
(L) shows the bacterial extract expressing the two fusion
proteins used in these experiments.
View larger version (66K):
[in a new window]
Fig. 7.
Binding of EBP50 to the ezrin and merlin
N-ERMADs, and the effect of competition by the C-ERMADs.
A, left panel, equal amounts of Ez-1-297
(EzN) and Mr-1-313 (MrN) were incubated either
alone (lanes 1 and 2) or together (lane
3), with the GST fusion construct of the ERM binding domain of
EBP50 (GST-EBP50-C) immobilized on glutathione beads. The
beads were washed and eluted, and recovered proteins were analyzed.
Because the GST-EBP50-C was not covalently bound to the beads, it is
also recovered in the SDS elution. Right panel, GST-EBP50-C
beads were incubated with one N-ERMAD and then challenged with either
buffer or an extract expressing an excess of the alternate N-ERMAD. A
mixture of the two N-ERMADs used is shown in lane L. B, competitive binding of GST-EBP50 (EBP) and
ezrin C-ERMAD (EzC) or merlin C-ERMAD (MrC) to
ezrin N-ERMAD beads. Binding competition between ezrin C-ERMAD and
GST-EBP50 to Ez-1-297 (left panel) or Mr-1-313
(right panel) was done both competitively and sequentially.
C, competitive binding of GST-EBP50 (EBP) and
ezrin C-ERMAD (EzC) or merlin C-ERMAD (MrC) to
merlin N-ERMAD beads. The experiment was similar to that shown in
B except that merlin N-ERMAD beads were used. Bacterial
extracts showing the mixture of recombinant proteins used are shown in
lane L.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Ez-N-ERMAD/Mr-C-ERMAD < Mr-N-ERMAD/EBP50
Ez-N-ERMAD/E3KARP
Ez-N-ERMAD/EBP50 < Ez-N-ERMAD/Ez-C-ERMAD. The competitive binding studies have provided a
demonstration of the potential dynamic exchange between binding
partners, even though associations may appear to be relatively stable
in the absence of a competing species. In a broader sense, this
establishes how proteins can select between multiple binding partners
on the simple basis of thermodynamic favorability.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM36652.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Biology and Genetics, Biotechnology Building, Cornell University, NY 14853. Tel.: 607-255-5713; E-mail: apb5@cornell.edu.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M006708200
2 D. Chambers and A. Bretscher, unpublished data.
3 D. Chambers and A. Bretscher, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NF2, neurofibromatosis 2; ERM, ezrin-radixin-moesin; ERMAD, ERM association domain; GST, glutathione S-transferase; MBP, maltose-binding protein; Ez, ezrin; Mr, merlin; MES, 2-[N-Morpholino]ethanesulfonic acid; HA, hydroxyapatite.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Martuza, R. L., and Eldridge, R. (1988) N. Engl. J. Med. 318, 684-688[Medline] [Order article via Infotrieve] |
2. | Rouleau, G. A., Merel, P., Lutchman, M., Sanson, M., Zucman, J., Marineau, C., Hoang-Xuan, K., Demczuk, S., Desmaze, C., Plougastel, B., Pulst, S. M., Lenoir, G., Bijlsma, E., Fashold, R., Dumanski, J., de Jong, P., Parry, D., Eldrige, R., Aurias, A., Delattre, O., and Thomas, G. (1993) Nature 363, 515-521[CrossRef][Medline] [Order article via Infotrieve] |
3. | Trofatter, J. A., MacCollin, M. M., Rutter, J. L., Murrell, J. R., Duyao, M. P., Parry, D. M., Pulaski, K., Haase, V. H., Ambrose, C. M., Munroe, D., Bove, C., Haines, J. L., Martuza, R. L., MacDonald, M. E., Seizinger, B. R., Short, M. P., Buckler, A. J., and Gusella, J. F. (1993) Cell 72, 791-800[Medline] [Order article via Infotrieve] |
4. | Bianchi, A. B., Mitsunaga, S.-I., Cheng, J. Q., Klein, W. M., Jhanwar, S. C., Seizinger, B., Kley, N., Klein-Szanto, A. J., and Testa, J. R. (1994) Nat. Genet. 6, 185-192[Medline] [Order article via Infotrieve] |
5. | Pykett, M. J., Murphy, M., Harnish, P. R., and George, D. L. (1994) Hum. Mol. Genet. 3, 559-564[Abstract] |
6. | Sherman, L., Xu, H. M., Geist, R. T., Saporito-Irwin, S., Howells, N., Ponta, H., Herrlich, P., and Gutmann, D. H. (1997) Oncogene 15, 2505-2509[CrossRef][Medline] [Order article via Infotrieve] |
7. | McClatchey, A. I., Saotome, I., Ramesh, V., Gusella, J. F., and Jacks, T. (1997) Genes Dev. 11, 1253-1265[Abstract] |
8. |
McClatchey, A. I.,
Saotome, I.,
Mercer, K.,
Crowley, D.,
Gusella, J. F.,
Bronson, R. T.,
and Jacks, T.
(1998)
Genes Dev.
12,
1121-1133 |
9. |
LaJeunesse, D. R.,
McCartney, B. M.,
and Fehon, R. G.
(1998)
J. Cell Biol.
141,
1589-1599 |
10. |
Berryman, M.,
Franck, Z.,
and Bretscher, A.
(1993)
J. Cell Sci.
105,
1025-1043 |
11. | Huynh, D. P., Tran, T. M., Nechiporuk, T., and Pulst, S. M. (1996) Cell Growth Differ. 7, 1551-1561[Abstract] |
12. |
den Bakker, M. A.,
Vissers, K. J.,
Molijn, A. C.,
Kros, J. M.,
Zwarthoff, E. C.,
and van der Kwast, T. H.
(1999)
J. Histochem. Cytochem.
47,
1471-1480 |
13. |
Sainio, M.,
Zhao, F.,
Heiska, L.,
Turunen, O.,
den Bakker, M.,
Zwarthoff, E.,
Lutchman, M.,
Rouleau, G. A.,
Jaaskelainen, J.,
Vaheri, A.,
and Carpen, O.
(1997)
J. Cell Sci.
110,
2249-2260 |
14. | Maeda, M., Matsui, T., Imamura, M., Tsukita, S., and Tsukita, S. (1999) Oncogene 18, 4788-4797[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Gronholm, M.,
Sainio, M.,
Zhao, F.,
Heiska, L.,
Vaheri, A.,
and Carpen, O.
(1999)
J. Cell Sci.
112,
895-904 |
16. |
Murthy, A.,
Gonzalez-Agosti, C.,
Cordero, E.,
Pinney, D.,
Candia, C.,
Solomon, F.,
Gusella, J.,
and Ramesh, V.
(1998)
J. Biol. Chem.
273,
1273-1276 |
17. |
Reczek, D.,
Berryman, M.,
and Bretscher, A.
(1997)
J. Cell Biol.
139,
169-179 |
18. | Tsukita, S., Oishi, K., Sato, N., Sagara, J., Kawai, A., and Tsukita, S. (1994) J. Cell Biol. 126, 391-401[Abstract] |
19. |
Takahashi, K.,
Sasaki, T.,
Mammoto, A.,
Takaishi, K.,
Kameyama, T.,
Tsukita, S.,
Tsukita, S.,
and Takai, Y.
(1997)
J. Biol. Chem.
272,
23371-23375 |
20. |
Gonzalez-Agosti, C.,
Wiederhold, T.,
Herndon, M. E.,
Gusella, J.,
and Ramesh, V.
(1999)
J. Biol. Chem.
274,
34438-34442 |
21. | Huang, L., Ichimaru, E., Pestonjamasp, K., Cui, X., Nakamura, H., Lo, G. Y., Lin, F. I., Luna, E. J., and Furthmayr, H. (1998) Biochem. Biophys. Res. Commun. 248, 548-553[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Tsukita, S.,
and Yonemura, S.
(1999)
J. Biol. Chem.
274,
34507-34510 |
23. | Mangeat, P., Roy, C., and Martin, M. (1999) Trends Cell Biol. 9, 187-192[CrossRef][Medline] [Order article via Infotrieve] |
24. | Bretscher, A. (1999) Curr. Opin. Cell Biol. 11, 109-116[CrossRef][Medline] [Order article via Infotrieve] |
25. | Bretscher, A., Chamber, C., Nguyen, R., and Reczek, D. (2000) Annu. Rev. Cell Dev. Biol. 16, 113-143[CrossRef][Medline] [Order article via Infotrieve] |
26. | Gary, R., and Bretscher, A. (1995) Mol. Biol. Cell 6, 1061-1075[Abstract] |
27. |
Magendantz, M.,
Henry, M. D.,
Lander, A.,
and Solomon, F.
(1995)
J. Biol. Chem.
270,
25324-25327 |
28. | Turunen, O., Wahlstrom, T., and Vaheri, A. (1994) J. Cell Biol. 126, 1445-1453[Abstract] |
29. | Pestonjamasp, K., Amieva, M. R., Strassel, C. P., Nauseef, W. M., Furthmayr, H., and Luna, E. J. (1995) Mol. Biol. Cell 6, 247-259[Abstract] |
30. |
Reczek, D.,
and Bretscher, A.
(1998)
J. Biol. Chem.
273,
18452-18458 |
31. |
Nakamura, F.,
Amieva, M. R.,
and Furthmayr, H.
(1995)
J. Biol. Chem.
270,
31377-31385 |
32. |
Nakamura, F.,
Huang, L.,
Pestonjamasp, K.,
Luna, E. J.,
and Furthmayr, H.
(1999)
Mol. Biol. Cell
10,
2669-2685 |
33. |
Matsui, T.,
Maeda, M.,
Doi, Y.,
Yonemura, S.,
Amano, M.,
Kaibuchi, K.,
Tsukita, S.,
and Tsukita, S.
(1998)
J. Cell Biol.
140,
647-657 |
34. | Matsui, T., Yonemura, S., Tsukita, S., and Tsukita, S. (1999) Curr. Biol. 9, 1259-1262[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Pietromonaco, S. F.,
Simons, P. C.,
Altman, A.,
and Elias, L.
(1998)
J. Biol. Chem.
273,
7594-7603 |
36. | Simons, P. C., Pietromonaco, S. F., Reczek, D., Bretscher, A., and Elias, L. (1998) Biochem. Biophys. Res. Commun. 253, 561-565[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Huang, L.,
Wong, T. Y.,
Lin, R. C.,
and Furthmayr, H.
(1999)
J. Biol. Chem.
274,
12803-12810 |
38. | Pearson, M., Reczek, D., Bretscher, A., and Karplus, P. (2000) Cell 101, 259-270[Medline] [Order article via Infotrieve] |
39. | Bretscher, A. (1989) J. Cell Biol. 108, 921-930[Abstract] |
40. |
Yun, C. H.,
Lamprecht, G.,
Forster, D. V.,
and Sidor, A.
(1998)
J. Biol. Chem.
273,
25856-25863 |
41. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
42. | Bretscher, A. (1983) J. Cell Biol. 97, 425-432[Abstract] |
43. | Bretscher, A., Gary, R., and Berryman, M. (1995) Biochemistry 34, 16830-16837[Medline] [Order article via Infotrieve] |
44. | Berryman, M., Gary, R., and Bretscher, A. (1995) J. Cell Biol. 131, 1231-1242[Abstract] |
45. | Weinman, E. J., and Shenolikar, S. (1997) Exp. Nephrol. 5, 449-452[Medline] [Order article via Infotrieve] |
46. | Weinman, E. J., Steplock, D., Wang, Y., and Shenolikar, S. (1995) J. Clin. Invest. 95, 2143-2149[Medline] [Order article via Infotrieve] |
47. | Lamb, R. F., Ozanne, B. W., Roy, C., McGarry, L., Stipp, C., Mangeat, P., and Jay, D. G. (1997) Curr. Biol. 7, 682-688[Medline] [Order article via Infotrieve] |
48. | Akisawa, N., Nishimori, I., Iwamura, T., Onishi, S., and Hollingsworth, M. A. (1999) Biochem. Biophys. Res. Commun. 258, 395-400[CrossRef][Medline] [Order article via Infotrieve] |
49. | Kaul, S. C., Kawai, R., Nomura, H., Mitsui, Y., Reddel, R. R., and Wadhwa, R. (1999) Exp. Cell Res. 250, 51-61[CrossRef][Medline] [Order article via Infotrieve] |