(Received for publication, July 17, 1995; and in revised form, August 15, 1995)
From the
We have assayed the domains of the ERM protein radixin for
binding activities in vitro. Affinity columns bearing the
amino-terminal domain of radixin selectively bound a small subset of
the proteins of the chicken erythrocyte cytoskeleton. Two of those
proteins were identified as radixin itself and band 4.1. In contrast,
the carboxyl-terminal domain of the molecule bound neither protein, and
full-length radixin did not bind band 4.1 (binding of full-length
radixin to itself was not evaluated). Columns bearing a mixture of the
amino- and carboxyl-terminal domains of radixin also failed to bind
radixin and band 4.1. These results suggested that the amino- and
carboxyl-terminal sequences can interact with one another either in
cis or in trans, and so interfere with radixin's
interactions with other ligands. Using affinity co-electrophoresis, we
confirmed a direct interaction in solution between the two radixin
domains; the data are consistent with the formation of a 1:1 complex
with a dissociation constant of 5
10
M. Competition between intramolecular and intermolecular
interactions may help to explain the provocative and dynamic
localization of ERM proteins within cells.
The ERM proteins, ezrin, radixin, and moesin, are components of cortical cytoskeleton that are thought to play a role in linking cytoskeletal and membrane elements. They are found in ruffling edges, growth cones, and membrane extensions such as microvilli, filopodia and lamellipodia, regions rich in F-actin, but their precise positions do not coincide with those of F-actin or other major cytoskeletal elements (1, 2, 3, 4, 5) . For example, in neuronal growth cones, ERM and F-actin staining patterns are overlapping but distinct(3) . Moreover, drugs that depolymerize microtubules delocalize ERM proteins from growth cones, but not F-actin(3) .
It is not known what molecular interactions are responsible for correct localization of ERM proteins within cells. In vitro, ERM proteins do not behave like conventional actin-binding proteins(6) . Binding studies, and the isolation of complexes from cell extracts have suggested a number of potential ERM ligands. These include the transmembrane protein CD44 (7) and F-actin (which is reported to interact with the carboxyl-terminal domain of ezrin and moesin(8, 9) , although the details of that interaction are controversial(10) ). Apparently, ERM proteins may also self-associate into homo- and hetero-oligomeric complexes(11, 12) .
Assays in cells demonstrate that separable domains of the ERM proteins contribute information that specifies appropriate localization in the cell(13, 14) . For example, at low levels of expression, the carboxyl-terminal domain of radixin localizes to all of the structures in which ERM proteins are normally found, save the cleavage furrow, and associates quite clearly with one cellular element, stress fibers, where ERM proteins are not typically found. The information necessary to target radixin to the cleavage furrow is in the amino-terminal domain of the protein(14) .
These cellular assays also reveal evidence for regulatory interactions between the domains of ERM proteins. Expressed at high levels, the carboxyl terminus causes dramatic disruption of normal cell morphology and interferes with cell division, while the amino terminus has neither phenotype(14, 15, 16) . However, both consequences of high level expression of the carboxyl terminus are suppressed by the presence of the amino terminus, either in cis or in trans(14, 16) . Perhaps, then, the deleterious effects of one domain are prevented by an interaction with the other domain. We have tested this model using in vitro methods. Here, we present evidence that the amino- and carboxyl-terminal domains of radixin can bind each other in vitro. We also show that this binding event blocks the binding of other ligands.
We cloned chicken radixin cDNAs by polymerase chain reaction
(First Strand cDNA Synthesis Kit; Pharmacia Biotech Inc.) from chicken
embryo fibroblast mRNA. The clones representing the full-length,
amino-terminal (codons 1-318) and carboxyl-terminal (codons
319-585) sequences were ligated into PQE-70 (Qiagen) using the SphI and BglII sites, so that the last amino acid of
each sequence is followed by arginine, serine, and then 6 histidines
and a stop codon. Escherichia coli DH5F`IQ transformants
with the correct radixin insert were identified by restriction digests
and by DNA sequencing (Sequenase version 2.0 DNA Sequencing Kit; U. S.
Biochemical Corp.) at the junction of radixin insert and vector.
Expression of the three His
-tagged radixin polypeptides and
His
-tagged dihydrofolate reductase was induced by growth in
1 mM isopropyl-1-thio-
-D-galactopyranoside for 3
h. Cell pellets were lysed (50 mM
NaH
PO
/NaHPO
, pH 8.0, 300 mM NaCl, 20 mM imidazole, 1 mM Pefabloc, 1 mg/ml
leupeptin, 1 mg/ml pepstatin, 0.009 TIU/ml aprotinin, and 1 mg/ml
lysozyme), sonicated, and clarified by centrifugation, then stored as
aliquots at -80 °C. High-speed supernatants containing the
His
-tagged polypeptides were incubated with 0.5 ml of
nickel-NTA (
)Sepharose CL-6B resin (Qiagen) in lysis buffer
supplemented with 10 mM
-mercaptoethanol. The resin was
washed batchwise twice with 15 ml of wash buffer (50 mM
NaH
PO
/NaHPO
, pH 8.0, 300 mM NaCl, 40 mM imidazole) plus 10% glycerol and once in wash
buffer alone.
For affinity adsorption experiments,
His-tagged proteins were left bound to nickel-NTA matrices
and incubated with a chicken erythrocyte cytoskeletal fraction prepared
as follows. Freshly washed erythrocytes were extracted with 0.1%
Nonidet P-40 in PM2G (100 mM Pipes, 1 mM
MgSO
, 2 mM EGTA, pH 6.9) containing 0.009 TIU/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mg/ml
leupeptin. After a wash in the same buffer, the pellet was extracted in
8 M urea in phosphate-buffered saline with protease
inhibitors. The urea extract was spun at 11,000
g and
the supernatant collected, dialyzed in phosphate-buffered saline, and
frozen as aliquots. The erythrocyte proteins were incubated with the
affinity matrices batchwise for 20 min 4 °C, then repeatedly washed
and centrifuged to remove unbound proteins. The matrices were loaded
into columns and eluted with two 0.5-ml aliquots of elution buffer (50
mM NaH
PO
/NaHPO
, pH 8.0,
300 mM NaCl, and 125 mM imidazole). Fractions were
boiled in sample buffer and analyzed by 7.5% SDS-PAGE. Proteins were
detected by silver stain or by immunoblotting as
described(14) , using antibodies that detect epitopes in the
amino-terminal domain of ERM proteins (#220), the carboxylterminal
domain of radixin (#457), and previously characterized antibodies
against chicken erythrocyte band 4.1(17, 18) .
For
affinity co-electrophoresis (ACE), high-speed supernatants of
His-tagged radixin amino- and carboxyl-terminal domains
were purified on nickel-NTA resin columns. Glutathione S-transferase was prepared by expression of pGEX-3X
(Pharmacia) in E. coli strain HB101. Protein concentrations
were determined by the Bradford assay (Bio-Rad). ACE gels were cast as
described (19, 20) using 1% low gelling temperature
agarose in 125 mM potassium acetate, 50 mM Hepes
adjusted to pH 7.5 with NaOH. Gels were run at 60 volts for 4 h and the
proteins then transferred to nitrocellulose by capillary action and
analyzed by immunoblotting. Retardation coefficients were calculated as
described previously, including the application of a correction for
``overrunning'' (electrophoresis of the detected species
beyond the end of the zones containing the retarding species (20) ). Dissociation constants were calculated from nonlinear
least squares fitting of plots of corrected retardation coefficient versus concentration of retarding protein(21) . Data
were fit to a general form of the binding equation that is appropriate
even when the concentration of the detected species is not K
(20) .
As a source of potential binding partners for radixin, we
used the proteins of the cytoskeletal fraction of chicken erythrocytes.
All of the cytoskeletal radixin in these cells is in a single
structure, the marginal band, from which it can only be extracted by
strong chaotropic agents(2) . The cells also are available in
large quantities. We prepared detergent-extracted cytoskeletons from
suspensions of chicken erythrocytes, solubilized these in 8 M urea, removed the urea by dialysis, and applied the extracts to
the three radixin affinity columns described above. After extensive
washing, columns were eluted with 125 mM imidazole and the
eluted proteins analyzed by SDS-PAGE. Several erythrocyte proteins that
bind to each of the columns were detected by silver stain, but we have
identified by immunoblotting two known proteins that bind either
exclusively or preferentially to the NH-terminal domain.
A 80-kDa erythrocyte protein is detected in the eluate from the
N-domain column by silver stain. That protein is identified as radixin
by immunoblotting with antibody 457, specific for the carboxyl terminus
of radixin, and antibody 220, which binds to an epitope present in the
amino termini of all ERM proteins (Fig. 1A, AMINO, both C and N lanes). This band is not
detectable in the eluates from the C-domain column, by either
immunoblotting (Fig. 1A, CARBOXY) or silver stain.
These data are consistent with the finding of Andreoli et
al.(12) , who demonstrated that full-length ezrin bound
more tightly to its amino terminus than to its carboxyl terminus. We do
not detect ezrin or moesin as proteins bound to the N-domain column,
perhaps because they are much less abundant in chicken erythrocytes
than radixin. (
)We cannot determine if radixin is bound to
the FL column, since the two proteins should co-migrate.
Figure 1:
A, Immunoblot
analyses of column eluates. High imidazole eluates from columns bearing
His-tagged polypeptides (AFFINITY COLUMN) plus bound chicken
erythrocyte proteins were collected. The His-tagged polypeptides
included: full-length radixin (FULL-LENGTH), the
NH- and COOH-terminal domains of radixin, and dihydrofolate
reductase. The eluates were separated on 7.5% SDS-PAGE, transferred to
nitrocellulose, and probed with antibodies recognizing the carboxyl
terminus (C) or amino terminus (N) of radixin, or
band 4.1 (4.1). The arrowhead indicates the position
of radixin, the arrow indicates the position of band 4.1, and
the numbers (kilodaltons) indicate mobilities of four
molecular weight markers. B, partitioning of vinculin,
radixin, and tubulin between flow-through and column-bound material,
detected by Western blots of fractions loaded to represent equal
starting material.
A
110-kDa polypeptide detected by silver stain binds preferentially
to the N-domain column. That protein is identified as band 4.1 in
immunoblots, using antibodies against chicken erythrocyte band 4.1 (Fig. 1A, amino, 4.1 lane). Granger et al.(22) demonstrated that in chicken erythrocytes, band 4.1
occurs in multiple isoforms with a wide range of molecular weights, but
that a species of
115 kDa (in their gel system) is the major one.
Our
110-kDa band co-migrates with the major band 4.1 element in
our hands. In some experiments, the antibodies identify a much less
intense band in the eluates from the C-domain column and the FL column.
Several properties of the observed protein binding suggest that it is specific. First, the binding is highly selective. Although some erythrocyte proteins appear to bind to all three columns, in fact they and the specific polypeptides named above account only for a small subset of the total complement of proteins in the extract. Second, we can detect both tubulin and vinculin by immunoblotting of the column flow-through, but we detect no signal above background among the proteins bound to the column (Fig. 1B). Third, neither band 4.1 nor radixin bind to column bearing an irrelevant His-tagged protein of comparable size, dihydrofolate reductase. In contrast, those erythrocyte proteins that bind to all three radixin columns also bind to the dihydrofolate reductase control (data not shown).
We do not know if band 4.1 and radixin are ligands of radixin in the cell. However, these experiments do suggest that, under the conditions of this assay, specific associations do occur between a small subset of chicken erythrocyte proteins and discrete domains of radixin.
Figure 2:
Affinity co-electrophoresis of the N- and
C-domains of radixin. A, column-purified
His-N-domain (10
M) was loaded
into a long gel slot perpendicular to the direction of electrophoresis.
Column-purified His
-C-domain was loaded, at the
concentrations given, into multiple lanes parallel to the direction of
electrophoresis. After electrophoresis, during which time the migrating
front of N-domain traversed the zones containing the C-domain, the
contents of the gels were transferred to nitrocellulose and visualized
by immunoblotting with antibody against the N-domain. B,
electrophoresis was carried out as in A, except that
glutathione S-transferase was loaded into the slot and was
detected using antibodies specific for that
protein.
From measurements of
mobility retardation in Fig. 2A, we can estimate the
dissociation constant for the interaction of the amino- and
carboxyl-terminal polypeptides of radixin(20) . Fig. 3shows the analysis of one such experiment. As
described(21) , to avoid problems arising from saturation of
the film, we used a PhosphorImager to determine the true midpoint of
each of the bands. The data have been fit by an equation that assumes
1:1, noncooperative binding, and a concentration of N-domain of 5
10
M. The curve indicates an
apparent value for K
of 4.5
10
M. A second experiment, analyzed in the
same way, gave a value for K
of 4.2
10
M (not shown). Equations that assume
higher order or cooperative binding (21) fit the data
significantly less well (not shown).
Figure 3:
Analysis of the binding of the N- and
C-domains of radixin as revealed by affinity co-electrophoresis. R, the corrected value of the
retardation coefficient, quantifies the electrophoretic retardation of
the N-domain. C-Term gives the nominal concentration of the
C-domain in the lanes. Typically, the concentration of the detected
species can be ignored in ACE experiments, because it is K
. Here, that assumption does not apply
because detection of the N-domain required relatively high
concentrations. The nominal initial concentration of the N-domain was
10
M, establishing an upper limit. Because
the band broadens and diffuses during electrophoresis, the actual
concentration of N-domain was likely lower. Varying the assumed
concentration of the N-domain in the analysis from its highest possible
value (10
M) to 1.25
10
M yielded values for K
that varied about 4-fold range. The
data have been fit to an equation derived from the definition of K
, using the assumption that bound
fraction is equal to R
/R
, where R
, represents the limiting value of R
when the concentration of the
carboxyl-terminal domain is arbitrarily large. The curve was obtained
using nonlinear least squares methods, in which K
and R
were
taken as variables to be fit
simultaneously.
In summary, the data indicate
that radixin has separable domains capable of specific binding
interactions in vitro. Binding partners for radixin's
domains include band 4.1 and radixin itself. Furthermore, the N- and
C-domains of radixin can bind each other in solution with high
affinity. Like the intact full-length protein, the reconstituted
complex of the N- and C-domains fails to interact with band 4.1. Since
the estimated concentration of the N- and C-domain polypeptides in
bacterial extracts is 2.5
10
M, it is likely that, in the experiment in which both
polypeptides were mixed and applied to a single column (Fig. 1),
complexes had formed before adsorption to the column (Fig. 1).
Taken together with the results of transfection experiments, in which
high level expression of the carboxyl-terminal domain of radixin had
deleterious consequences that the full-length protein did not have, and
that could be suppressed by co-expression of the amino-terminal
domain(14, 16) , these data strongly suggest that
direct interactions between the amino- and carboxyl-terminal domains of
the radixin molecule in vivo inhibit the interaction of
radixin with other molecules. Presumably, such inhibition is overcome
under appropriate circumstances, either because ligands with higher
affinity or effective local concentration successfully compete with the
interaction between the amino- and carboxyl-terminal domains or because
regulatory modifications (e.g. phosphorylation) modulate the
affinity of that interaction.
It is reasonable to propose that the interaction between amino- and carboxyl-terminal domains of radixin is intramolecular. Such a situation would be strikingly similar to what has been observed in studies of vinculin, in which intramolecular interaction between head and tail domains has been shown to compete with the binding of presumptive ligands(23, 24) . Since ERM proteins can self-associate, however, we cannot rule out the possibility that interactions of the amino- and carboxyl-terminal domains of radixin may also be intermolecular. In this regard it is noteworthy that, in the present study, affinity columns of the amino-terminal domain of radixin bound full-length radixin, but columns of the carboxyl-terminal domain did not. Similarly, Andreoli et al.(12) reported that full-length ezrin binds its own amino terminus substantially more tightly than its own carboxyl terminus. Although negative results must be interpreted with caution (protein fragments, and proteins immobilized on solid supports, may have artifactually altered binding properties), it is possible that self-association of full-length ERM proteins involves activities other than those demonstrated in the present study. A detailed analysis of binding domains and their activities, both in vivo and in vitro, will resolve these issues.