From the Department of Pathology, Yale University, New Haven, Connecticut 06510
Received for publication, October 11, 2000
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ABSTRACT |
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The anchorage of spectrin to biological
membranes is mediated by protein and phosphoinositol
phospholipid interactions. In epithelial cells, a nascent spectrin
skeleton assembles in regions of cadherin-mediated cell-cell contact,
and conversely, cytoskeletal assembly is required to complete the
cell-adhesion process. The molecular interactions guiding these
processes remain incompletely understood. We have examined the
interaction of spectrin with The spectrin-actin cytoskeleton contributes to membrane structure
and provides molecular linkages between organized membrane domains and
the filamentous cytoskeleton (reviewed in Refs. 1-4). Its assembly on
any given membrane is guided by interactions with membrane proteins and
phosphoinositol phospholipids (5-8). Best understood are linkages
involving the adapter protein ankyrin. Ankyrin binds spectrin with high
affinity ( In epithelial cells, assembly of the nascent cortical spectrin skeleton
occurs at zones of cell-cell contact, regions where there is productive
Ca2+-mediated homophilic adhesion between surface
E-cadherin molecules (8, 25, 26). Associated with E-cadherin is a group
of cytoplasmic proteins that include Construction of Expression Plasmids--
Molecular biological
procedures followed standard protocols (33). Recombinant peptides were
prepared in Escherichia coli as before using one of the pGEX
vector systems (Amersham Pharmacia Biotech, also see Refs. 29 and 34).
Inserts of cDNA encoding selected regions of Protein Purification--
Recombinant proteins were expressed in
the E. coli strains HB101 and DH5 Cell Culture--
A subcloned line of high resistance type II
strain of Madin-Darby canine kidney (MDCK) cells were cultured as
before (38). The human colon carcinoma cell line Clone A (39) was
provided as a gift by Dr. A. Mercurio, Harvard Medical School. Clone A cells were maintained in RPMI-H 1640 supplemented with 10% fetal calf
serum and 0.3% L-glutamine. The human colon carcinoma cell line HT-29 was provided by Dr. X-Y. Fu (Yale University). HT-29 cells
were maintained in McCoy's modified medium with 10% fetal calf serum
and 0.3% L-glutamine. Cell cultures were incubated at
37 °C in a humidified atmosphere of 5% CO2.
Immunoprecipitation--
Confluent monolayers of MDCK cells
(35-mm plates) were washed once in phosphate-buffered saline and lysed
at 4 °C in 600 µl of RIPA buffer (150 mM NaCl, 1%
Nonidet P-40, 0.5% deoxycholate, 50 mM Tris, pH 7.5) by
gentle rocking for 30 min. The lysate was spun for 10 min at
10,000 × g, and the supernatant was cleared by
treatment with normal rabbit serum (50 µl/ml lysate) and protein A-Sepharose for 30 min on ice. The lysate (100 µl) was then incubated with 1 µl of anti-spectrin antibody (RAF-A) for 1 h at 4 °C
and precipitated with 50 µl of a 50% suspension of protein
A-Sepharose. Immunoprecipitates were washed briefly 2× with RIPA
buffer, followed by 2 washes with 400 mM NaCl, 1% Nonidet
P-40, 0.5% deoxycholate, 50 mM Tris, pH 7.5. Pellets were
resuspended in 50 µl of SDS-PAGE sample buffer and evaluated by
SDS-PAGE.
Detergent Extraction--
Confluent cells were extracted
in situ with Triton X-100 as before (40). Briefly, cells
were washed with 0 °C phosphate-buffered saline, pH 7.5, and
extracted with buffer 1 (10 mM Pipes, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM
MgCl2, 0.5% Triton X-100, 1.2 mM
phenylmethylsulfonyl fluoride, 0.5 mM Perabloc SD) for 10 min, yielding the detergent-soluble cytoplasmic fraction. Extraction with buffer 2 (buffer 1 with 250 mM
NH4SO4 in lieu of NaCl) for 10 min yielded the
detergent-insoluble cytoskeletal fraction.
Precipitation Assays--
Affinity purified anti-GST antibodies
were used to precipitate quantitatively 125I-labeled
spectrin bound to GST- Overlay Binding--
GST was cleaved from recombinant
Surface Plasmon Resonance--
Binding detection by surface
plasmon resonance was implemented using a BiacoreTM 1000 or
2000 instrument (Biacore AB). This technique of detecting protein-protein interactions is fully described in several publications (e.g. see Refs. 42-44). Purified Transfection Procedure--
Clone A cells ( Immunofluorescence--
Cells were plated onto glass chamber
slides and grown to subconfluence. Cells were washed five times in TBS,
pH 8.0, and fixed in methanol for 20 min at 4 °C. After fixation,
cells were washed again and blocked with 0.3% BSA/TBS for 1 h at
RT. After blocking, cells were washed and incubated with various
primary antibodies (diluted in 0.3% BSA/TBS) for 1 h at RT.
Antibodies were used at the following dilutions: monoclonal antibodies
Other Methods--
All reagents were from Sigma unless otherwise
stated. Restriction enzymes were obtained from New England Biolabs.
Ampicillin and HEPES were from U. S. Biochemical Corp. Synthetic
oligonucleotides were prepared by the Yale Critical Technologies
Laboratory. Other reagents and their suppliers were Pfu
polymerase from Stratagene; Taq polymerase from Cetus Corp.;
Sephacryl S-500 HR and DEAE-Sepharose chromatography media from
Amersham Pharmacia Biotech, Na125I from Amersham Pharmacia
Biotech; Enzymobeads from Bio-Rad; sucrose from ICN; and protein-A
Trisacryl-2000 beads from Pierce. Calf brain was obtained immediately
after death from a local abattoir, transported at 0 °C, washed in
0.32 M sucrose, frozen in liquid N2, and stored
at Spectrin Binds Directly to the NH2--
terminal 228 Residues of
To identify the site in
Finally, it was of interest to determine whether These findings establish that The demonstration of a direct interaction between -catenin, a component of the adhesion
complex. Spectrin (
II
II) and
-catenin coprecipitate from
extracts of confluent Madin-Darby canine kidney, HT29, and Clone A
cells and from solutions of purified spectrin and
-catenin in
vitro. By surface plasmon resonance and in vitro
binding assays, we find that
-catenin binds
II
II spectrin with
an apparent Kd of
20-100 nM. By
gel-overlay assay,
-catenin binds recombinant
II-spectrin
peptides that include the first 313 residues of spectrin but not to
peptides that lack this region. Similarly, the binding activity of
-catenin is fully accounted for in recombinant peptides encompassing
the NH2-terminal 228 amino acid region of
-catenin. An
in vivo role for the interaction of spectrin with
-catenin is suggested by the impaired membrane assembly of spectrin
and its enhanced detergent solubility in Clone A cells that harbor a
defective
-catenin. Transfection of these cells with wild-type
-catenin reestablishes
-catenin at the plasma membrane and
coincidentally recruits spectrin to the membrane. We propose that
ankyrin-independent interactions of modest affinity between
-catenin
and the amino-terminal domain of
-spectrin augment the interaction
between
-catenin and actin, and together they provide a polyvalent
linkage directing the topographic assembly of a nascent spectrin-actin
skeleton to membrane regions enriched in E-cadherin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
10-100 nM), linking spectrin to a variety of
transmembrane proteins including ion channels or pumps, such as the
anion exchanger AE1, the voltage-gated Na+ channel,
Na+/K+-ATPase,
H+/K+-ATPase (9-13), and cell adhesion
molecules of the Ig superfamily such as neuroglian, neurofascin, or
NrCAM (7, 14-16). Other recognized adapter proteins linking spectrin
to the membrane include adducin and members of the protein 4.1 superfamily, 4.1R, 4.1B, and 4.1G (reviewed in Refs. 4 and 17). Direct
interactions of spectrin with the membrane are mediated by at least
three distinct regions of
-spectrin, termed membrane association
domains 1, 2, and 3 (MAD1,1
MAD2, and MAD3). MAD1 is located in repeat unit 1 of both
I and
II spectrin (5) and contains a constitutive targeting signal that
together with sequences in region 1 of spectrin can direct spectrin to
either the Golgi or plasma membrane of
MDCK cells (18).2,3 MAD2,
found in the COOH-terminal pleckstrin homology domain of all spectrins
except
I
1 (4), binds both a protein ligand (5, 6) as well as
phosphatidylinositol-4,5-P2 phospholipid (20, 21). MAD2 may
participate in bestowing G-protein control on the assembly process
(21-23). MAD3 involves a region within
-spectrin repeats 3-9
(6)3; its function remains uncharacterized, although it has
been proposed to interact with a membrane calmodulin-binding protein
(24). Other modes of attachment and other adapter proteins almost
certainly exist; their identification remains central to a complete
understanding of spectrin assembly and function.
- and
-catenin (or
-catenin) and p120 (for review see Refs. 25 and 27).
-Catenin (or
in some cells
-catenin) directly binds the cytoplasmic domain of
E-cadherin;
-catenin joins the membrane complex via a direct
association with
-catenin or
-catenin (28).
-Catenin binds and
bundles F-actin, an activity that presumably facilitates the attachment of actin filaments to the adhesion complex (29).
-Catenin also binds
-actinin, a distant member of the spectrin gene superfamily; this
interaction may facilitate the docking of actin at the adhesion complex
(30). The catenins, and their assembly with the cortical cytoskeleton,
are closely linked to the regulation of cadherin function (25, 31, 32).
Spectrin assembles with the adhesion complex soon after productive
cell-cell contact is established (26). The interactions guiding this
process remain undefined. We now report a direct interaction of
spectrin with
-catenin, and we demonstrate that
-catenin is
required for spectrin assembly at the plasma membrane in Clone A cells,
a human intestinal cell line. These studies thereby identify
-catenin as a novel adapter protein mediating spectrin-membrane
association, suggest that this association is necessary for maturation
of at least some types of cadherin-mediated junctions, and provide
insight into the molecular mechanisms by which spectrin participates in
the establishment of specialized membrane domains in polarized cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(E)-catenin were
isolated from a human colon cDNA library (GenBankTM
accession number L23805, see Ref. 35). The desired region of the
cDNA was digested with appropriate restriction endonucleases; the
inserts were purified from agarose gels with the Qiaex gel extraction
kit (Qiagen) and subcloned into pGEX-2T. The alignment with the
-catenin constructs used in relation to the full sequence is shown
in Fig. 2 and as used previously (29).
. For peptides
particularly sensitive to proteolysis, strain CAG-456 was used. The
expressed peptides were purified from bacterial lysates on
glutathione-agarose columns (36). Purified
-catenin-GST fusion
proteins were utilized as antigens for the generation of polyclonal
antibodies that were subsequently affinity purified by absorption to
immobilized immunogen.
II
II spectrin was purified by low ionic
strength extraction of fresh demyelinated bovine brain membranes,
followed by gel filtration on Sephacryl-S-500 HR (37).
-catenin fusion proteins. Binding assays were
performed in BSA-coated polypropylene tubes (41). Recombinant
-catenin peptides N576 or C447 (1 µg) were incubated with
increasing concentrations of 125I-labeled
II
II
spectrin for 2 h at room temperature in binding buffer (50 mM NaCl, 10 mM HEPES, 0.5 mM EGTA,
0.5 mM dithiothreitol, 1 mM NaN3,
0.2 mM phenylmethylsulfonyl fluoride, pH 6.8) containing 3% BSA. Anti-GST antibody (10 µg) was added, and the incubation was
continued for 1 h. Protein A-Trisacryl beads (100 µl of a 10%
final suspension) were added for an additional hour. The bound and free
samples were separated by centrifugation through a 20% sucrose cushion
at 5,000 rpm. Bound spectrin was quantified by
-counting using a
1282 Compugamma CS (LKB instruments). Control experiments ensured that
the GST fusion proteins were completely precipitated. Binding to GST
alone or to protein-A beads in the absence of fusion protein was used
as controls for nonspecific binding and were subtracted from all
results to obtain specific binding data.
-catenin by thrombin digestion (28). GST-spectrin peptides (1, 0.5, and 0.1 µg) were analyzed by SDS-PAGE on 7.5% gels and transferred
to PVDF membranes. Membranes were blocked for 1 h in 5% BSA in
Tris-buffered saline (TBS) , pH 7.5, and overlaid for 1 h with 0.5 mg/ml recombinant
-catenin. After five brief rinses with TBS, a
1:1000 dilution of monoclonal antibody 3H4 was overlaid for 1 h at
RT. After five additional washes, goat anti-mouse antibody at a
1:10,000 tagged with horseradish peroxidase was used with enhanced
chemiluminescence (Amersham Pharmacia Biotech) to detect the bound
-catenin.
II
II spectrin was
immobilized onto a carboxymethylated dextran gold surface of the
BiacoreTM chip in 100 mM acetate buffer, pH
4.5, 50 mM N-hydroxysuccinimide, and
0.2 M
N-ethyl-N'-(dimethylaminopropyl) carbodiimide
hydrochloride. Several chip surfaces were prepared, ranging from 350 to
1500 RUs of bound spectrin. Purified recombinant human
-catenin or expressed
-catenin subdomains were injected onto the spectrin surface, and the binding was measured as an increase in the resonance units (RU). The kinetic constants, ka and
kd, for the binding of
-catenin to spectrin were
determined as described (45) from a plot of dRU/dt
versus R (s
1)
versus concentration. The slope of these plots are equal to ka and the abscissa intercept equal to
kd. The equilibrium dissociation value is determined
from the equation KD = kd/ka. Alternatively, the
sensograms were fit using the nonlinear fitting algorithms for
multisite binding provided by Biacore AB. Residuals were evaluated for
systematic divergences from the fitting algorithms as a measure of the
appropriateness of the binding model. The spectrin binding surfaces
were regenerated between determinations with 10 mM HCl or
10 mM NaOH. Control studies established the stability of
the binding surfaces over the course of these experiments. Binding
buffer conditions were 10 mM HEPES, 50 mM NaCl,
1 mM dithiothreitol, 0.5 mM EGTA, 1 mM NaN3, pH 6.8.
5 × 105) were plated in 60-mm Petri dishes, incubated
overnight, and transfected using the LipofectAMINE reagent (Life
Technologies, Inc.) with 3-6 µg of the plasmid pcDNA3
(Invitrogen) carrying full-length human
1(E)-catenin
(pcDNA3-
-catenin) (35). The manufacturer's transfection
protocol was followed without modification. When stable lines were
desired, neomycin-resistant clones were isolated by selective growth in
medium containing 0.6 mg/ml of G418 (Life Technologies, Inc.).
Subclones were identified on the basis of their assumption of a more
sheet-like morphology, a phenotypic change in Clone A cells
characteristic of full-length wild-type
-catenin expression (46).
Alternatively, since stable lines often proved unstable, transiently
expressing cells were used for most experiments. The expression of
-catenin was verified by immunofluorescence and Western blotting.
-catenin (3H4) and 7A11 were used as undiluted culture supernatants;
polyclonal antibody (RAFA) to
II spectrin was used at 1:500
dilution. After incubation with primary antibodies, cells were washed
and incubated with CY3- or CY2-conjugated secondary antibodies (Jackson
ImmunoResearch) diluted 1:500 in 0.3% BSA/TBS for 1 h at RT.
Cells were finally washed, mounted, and viewed by phase and
epifluorescence using an Olympus AX-70 microscope.
80 °C until use.
II
II spectrin was labeled with
125I by immobilized lactoperoxidase and glucose oxidase
using Enzymobead reagent as per the manufacturer's instructions. Final
specific activity of the labeled proteins ranged between
0.01 and 1 mCi/mg. Protein concentrations were determined by the Coomassie Blue
Protein Assay reagent (Pierce) using BSA as a standard. When needed for purposes of precise quantitation, protein concentrations were verified
by triplicate amino acid analysis at the Keck Biotechnology Laboratory
(Yale University).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
II
II Spectrin and
-Catenin Associate in Vivo--
Several
studies have established the coincidence at the light microscopic level
of plasma membrane-associated spectrin and the cadherin-catenin
adhesion complex along zones of cell-cell contact. A comparison of the
pattern of spectrin staining in Clone A cells and HT29 cells (a closely
related colonic epithelial cell line) suggests that
-catenin may
mediate the linkage of spectrin to the adhesion complex. In poorly
confluent HT29 cells, spectrin is largely cytoplasmic, as is
-catenin (Fig. 1, A-C).
When these cells become more confluent and establish effective
intercellular cadherin-mediated junctions,
-catenin is recruited to
zones of cell-cell contact (Fig. 1D). Coincident with this
recruitment of
-catenin, there is recruitment of spectrin from
cytoplasmic to membrane pools, in a distribution indistinguishable from
-catenin (Fig. 1, D and E; Fig.
2). Conversely, in Clone A cells, an
internal deletion of exons 4 and 5 in the transcribed cDNA of
-catenin generates a shortened and mutated
-catenin transcript
(Fig. 3, also see Refs. 46 and 47). Clone
A
-catenin is unstable and fails to associate with the
cadherin-based adhesion complex (39, 46), even though its ability to
associate with
-catenin and actin appear qualitatively unimpaired
(46). As a result, intercellular adhesion in Clone A cells is impaired.
Also impaired is the assembly of spectrin to the plasma membrane. This
is evident both by its persistent cytoplasmic intracellular
distribution even in confluent monolayers of Clone A cells (Fig.
2A), as well as by its reduced resistance to extraction with
Triton X-100 (compared with HT29 cells, an intestinal line with normal
-catenin, Fig. 2B).
-Catenin can also be directly
demonstrated in immunoprecipitates of
II
II spectrin solubilized
from confluent MDCK, HT29, and Clone A cells (Fig. 2C); MDCK
cells are an epithelial line with well documented spectrin
association at cadherin-based junctions (e.g. see Ref. 8).
Collectively, these in vivo observations indicate a tight and possibly direct linkage between spectrin and
-catenin and also
suggest that these two proteins may associate both in the cytosol and
at the membrane.
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Fig. 1.
II
II
spectrin and
-catenin are coincident only
along zones of cell-cell contact. The distribution of
-catenin
(A and D) or
II
II spectrin (B
and E) were evaluated by indirect immunofluorescence in
sparse (A-C) or partially confluent
(D--F) cultures of HT29 cells. Note that whereas
sparse cultures display high cytoplasmic concentrations of both
-catenin and spectrin, these proteins assemble together along zones
of productive cell-cell contact, with reduction of the cytoplasmic
concentrations as cells grow to confluence. This is most evident in the
merged images (C and F). Bar = 10µm.
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Fig. 2.
II
II spectrin and
-catenin associate in vivo.
A,
II
II spectrin fails to assemble efficiently at the
plasma membrane into a Triton-insoluble cortical membrane skeleton in
fully confluent Clone A cells (right), as it does in other
confluent epithelia with competent intercellular adhesion such as HT29
colonic cancer cells (left) or MDCK cells (not shown).
B, anti-
II
II spectrin (spec) Western blot
of the Triton X-100 soluble and insoluble extracts from HT29 cells
versus Clone A cells. In cell monolayers at confluence, the
majority of spectrin is typically Triton X-100-insoluble. In the
experiment shown, 85% of the spectrin is insoluble in HT29 cells
versus 52% in Clone A cells. The lane marked
(Sp) is purified
II
II spectrin. s,
soluble; p, pellet. C, immunoprecipitates of confluent MDCK,
HT29, or Clone A cells with anti-spectrin antibodies, Western-blotted
with the anti-
-catenin monoclonal antibody 3H4. Note the presence of
-catenin in the whole cell lysates (lys) and in the
immunoprecipitate with
II
II spectrin antibodies but not in the
immunoprecipitate using antibodies to
I
I spectrin or nonreactive
antibodies (nrs). Also apparent is the lower molecular
weight of the Clone A
-catenin
(
-cat).
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Fig. 3.
II spectrin binds directly to
the NH2-terminal 228 amino acids (aa)
of
-catenin. A, schematic
representation of the structure of
-catenin, the mutation found in
Clone A cells (shaded box (46)), and the recombinant
-catenin peptides used in this study. On the right is
shown an SDS-PAGE analysis, Coomassie Blue-stained, of the purified
II
II spectrin (S) and each of the
-catenin peptides
(with GST removed). Their Mr
1000 is
shown. B, surface plasmon resonance analysis of the
interaction of GST-
-catenin with immobilized
II
II spectrin. A
representative analysis is shown. Binding was evaluated on sensor chips
containing three different concentrations of spectrin (see
"Experimental Procedures"). Each curve (from bottom to
top) represents the sensogram trace of the binding of 0.15, 0.30, 0.60, 1.2, and 2.4 µM recombinant GST-
-catenin,
respectively. Similar experiments carried out with different levels of
immobilized spectrin, and with recombinant
-catenin in which the GST
moiety had been removed by thrombin treatment, gave comparable results.
Interestingly, analysis of recombinant
-catenin representing the
form found in Clone A cells, with the internal deletion of sequences
encoded by exons 4 and 5, did not change its affinity for spectrin
(data summarized in Table I). C, comparison sensogram of the
binding of various GST-
-catenin peptides at 5.0 µM to
II
II spectrin. The
-catenin peptides are as indicated. Note
that only those peptides containing the NH2-terminal 228 residues demonstrate appreciable binding and that the level of binding
achieved in the sensogram at saturation (for active peptides) is
roughly proportional to the mass of each active peptide. D,
solution binding of
II
II spectrin to
-catenin peptides.
Increasing concentrations of 125I-labeled
II
II
spectrin were incubated with
-catenin peptides N576 (
) or C447
(
). The binding of spectrin to GST alone or to the protein A
beads in the absence of fusion protein was taken as a measure of
nonspecific binding.
-Catenin
To obtain a more precise analysis of the
interaction between
-catenin and spectrin, the ability of
recombinant
-catenin peptides to bind to
II
II spectrin was
investigated by surface plasmon resonance. A series of recombinant
-catenin peptides were prepared as fusions with GST (Fig.
3A). Also prepared was an
-catenin of the type found in
the Clone A cells, incorporating an internal deletion. These peptides
were used in binding assays either as a GST fusion peptide or as the recombinant proteins alone after removal of the GST by thrombin. Bovine
II
II spectrin was immobilized on the BiacoreTM sensor
chip surface, and the changes in resonance units were monitored for
different concentrations of
-catenin (Fig. 3B) or for
different
-catenin subdomains (Fig. 3C). Sensograms (not shown) were also obtained for different surface loadings of spectrin, to evaluate artifacts arising from limitations of mass transport to the
sensor surface. In each analysis, the earliest portions of the
association and dissociation phases were analyzed. These are the
regions where mass transport (during association) and rebinding (during
dissociation) do not dominate the sensogram. Each sensogram was fit to
several different binding models, as provided in the Biacore software
package, including simple one-to-one Langmuir binding isotherms and
two-exponential models. All models generated reasonable fits, with
apparent values of ka, kd, and
KD within a factor of
3 of each other for a given
peptide. However, no model generated fits with fully random residuals,
indicating that the binding of
-catenin to immobilized spectrin,
although real and of high affinity, is complex and does not conform to
any simple binding model. A summary of the apparent kinetic values and
derived apparent KD values for
-catenin binding
to
II
II spectrin is presented in Table I. In this analysis, wild-type
-catenin bound spectrin with an apparent KD of
19-80 nM, GST-
-catenin bound with an apparent
KD of 19-24 nM, and the mutant Clone A
-catenin, devoid of GST, bound with an apparent
KD of 15-25 nM. The differences in
apparent binding affinity between the GST-
-catenin versus
peptides without GST presumably reflects the propensity of GST to
induce homodimerization. Oligomers of
-catenin generated by this
mechanism would bind with enhanced affinity, a phenomenon that has now
been well documented (19). A surprise is the apparently greater
affinity of GST-free Clone A
-catenin for spectrin versus wild-type
-catenin. The genesis of this effect is unknown and was
not further studied, although such a finding does suggest the
possibility that the Clone A mutation might affect the oligomerization pathway of native
-catenin (28).
Kinetics and derived dissociation constants as measured by surface
plasmon resonance
-catenin to
spectrin.
-catenin that interacts with spectrin,
additional recombinant peptides derived from the NH2 and
COOH termini of
-catenin (Fig. 3A) were prepared and
assayed qualitatively for their ability to bind directly to
II
II
spectrin using surface plasmon resonance (Fig. 3C). As
before, full-length
-catenin (peptide a907) bound avidly. Peptide
N576, representing the NH2-terminal half of
-catenin,
bound in a similar way, achieving approximately half of the RUs of
peptide a907. A peptide representing the NH2-terminal 228 residues of
-catenin also bound. Since the BiacoreTM
instrument measures a mass change at the sensor surface, the decrease
in RU values seen with the various NH2-terminal peptides are in proportion to their relative molecular weights and indicate that
all of these peptides, loaded at equal concentrations, are saturating
the spectrin surface to the same extent. Conversely, the C447 peptide,
encompassing the COOH-terminal half of
-catenin, did not bind to
spectrin any better than did GST or BSA alone. Thus, it appears that a
site near the NH2 terminus of
-catenin fully accounts
for its interaction with spectrin.
-catenin would also
bind
II
II spectrin in vitro in solution
(versus immobilized spectrin on the Biacore sensor surface).
Increasing concentrations of purified 125I-labeled
II
II spectrin were mixed with either of the recombinant GST
fusion proteins N576 or C447. Antibodies to GST were used to
co-precipitate GST-catenin along with bound 125I-labeled
spectrin, which was quantified by
-counting (Fig. 3D). Nonlinear regression analysis of the binding isotherm (fitted line)
revealed KD values of 164 ± 86 (2 S.D.)
nM for spectrin binding to GST-N576, with an estimated
Kmax of 0.50 ± 0.12 (2 S.D.) mol of
spectrin dimer bound per mol of GST-N576. In these assays, there was no
binding of spectrin to GST-C447 or GST alone. The KD
determined from this assay agreed reasonably well with those from the
Biacore studies, especially considering the differences in technique.
Collectively, they demonstrate a strong and direct interaction between
II
II spectrin and the NH2-terminal 228 residues of
-catenin. Consistent with this binding locus, no differences in
spectrin binding by Clone A
-catenin were detected. Clone A
-catenin deletes residues 197-354 of the native protein, suggesting
that the actual interaction site in
-catenin for spectrin is
proximal to residue 197.
-Catenin Binds to the First 313 Residues of
II
Spectrin--
The site to which
-catenin binds in
II spectrin
was identified by gel-overlay assay (Fig.
4). Recombinant GST fusion peptides representing all regions of human
II spectrin were transferred to
PVDF membranes and overlaid with
-catenin (from which GST had been
removed) (Fig. 4). Of the peptides examined, only those (
IIN-1,
IIN-4, and
IIN-6)
that included the NH2-terminal region of
II spectrin
bound
-catenin (Fig. 4B, center). To assess further the
relative affinities of
-catenin for this region of
II spectrin, overlay experiments were designed using a range of peptide
concentrations (Fig. 4B, right). Regardless of
concentration,
-catenin did not bind to GST alone or to
II9-C. Conversely, strong binding was detected at every
concentration of the
IIN-1 peptide. This active peptide,
the smallest one examined in these experiments, spans residues 1-313
of
II spectrin and places the
-catenin-binding site within this
region.
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Fig. 4.
The first 313 residues of
II spectrin effect
-catenin binding. A, summary of the
recombinant
II spectrin peptides used in relation to the overall
functional domain structure of
II spectrin. The major known
ligand-binding regions in
II spectrin are depicted, as are the
codons at the beginning and end of each of the recombinant peptides.
ABD, actin-binding domain; MADn, membrane
association domain n; ANK, ankyrin binding domain;
PH, pleckstrin homology domain. B, solid phase
blot assay of recombinant
-catenin binding to
II spectrin
peptides. Left, Coomassie Blue-stained SDS-PAGE analysis of
the GST-spectrin peptides, with their molecular weights.
Center, PVDF transfer of spectrin recombinant peptides,
overlaid with
-catenin and developed with the 3H4 antibody to
-catenin. Note the strong binding of
-catenin to
IIN-6,
IIN-4, and
IIN-1
and the absence of binding to the other spectrin peptides or to GST
alone. Data from peptides prepared as a fusion with GST or as the
peptide alone after removal of the GST are shown. Right,
PVDF transfer of
II9-C,
IIN-1, or GST
alone at three loadings (1.0, 0.5, or 0.1 µg) overlaid with
-catenin (GST-free). Note the strong binding to
IIN-1
with no detectable binding to
II9-C or GST at any
concentration.
-Catenin Facilitates Spectrin Membrane Assembly in
Vivo--
Clone A cells are defective in cell-cell adhesion and harbor
an internal deletion in the expressed
-catenin (39, 46). This
mutation leads to the loss of
-catenin associated with the plasma
membrane, reduced cell-cell adhesion, and coincidentally, reduced
assembly of
II
II spectrin at the membrane (Figs. 2 and 5). These
cells do, however, form epithelial-like sheets at confluence (albeit
with altered morphology and highly refractile membranes) and display
surface E-cadherin and
-catenin staining (46). To test whether the
failure of spectrin assembly in these cells was due to the defect in
-catenin, Clone A cells were transiently transfected with wild-type
-catenin, and the assembly of
-catenin and
II
II spectrin at
the membrane was monitored (Fig. 5). The transfected wild-type
-catenin was fully competent for assembly with
the cadherin adhesion complex at the plasma membrane (Fig. 5,
A and C), and coincident with its appearance,
II
II spectrin was restored to its plasma membrane location (Fig.
5, B and D). Thus, wild-type
-catenin is fully
competent to rescue the impaired membrane assembly of spectrin in Clone
A cells.
View larger version (113K):
[in a new window]
Fig. 5.
Wild-type -catenin
restores spectrin assembly at the membrane in Clone A cells. Clone
A cells were transiently transfected with wild-type
-catenin and
stained with the monoclonal antibody 7A11 which only recognizes
wild-type
-catenin (46) (A and C) or with
RAF-A, an antibody to
II
II spectrin (B and
D). Note the cluster of transfected cells that express
-catenin at the membrane and assume a more epithelial morphology. As
shown in higher power in the bottom panels (C and
D), the spectrin in these cells shifts from a largely
cytoplasmic distribution in the untransfected Clone A cells
(arrows, D) to a predominantly plasma membrane association
(arrowheads, D).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin can bind directly to
spectrin, that these proteins are associated in cultured epithelial cells in vivo, and that
-catenin facilitates the plasma
membrane assembly of spectrin in regions of direct cell-cell contact.
These conclusions are supported by several lines of evidence as
follows. (i) In vitro coprecipitation, surface plasmon
resonance, and gel overlay assays detect a direct interaction of
moderate to high affinity between the NH2-terminal domain
of
-catenin and the first 313 residues of
II spectrin. (ii)
Spectrin and
-catenin co-localize and co-precipitate in confluent
monolayers of Clone A, HT29, and MDCK cells. (iii) Spectrin and
-catenin do not assemble into a detergent-insoluble matrix at the
plasma membrane in clone A cells, a defect restored by transfection of
wild-type
-catenin. Collectively, these findings suggest that in
addition to its other roles,
-catenin acts as a novel adapter
protein directing the assembly of a nascent cortical spectrin membrane
skeleton to zones of productive cell-cell adhesion. It is also likely
that the binding of spectrin to the adhesion complex stabilizes the
complex itself and facilitates adhesion by linking adjacent adhesion
complexes into macromolecular membrane mosaics centered at
cadherin-based junctions. In this respect, the interaction of spectrin
with the adhesion complex is but a specific example of the more general role of spectrin as an organizer of linked membrane mosaics (2, 4).
-catenin and
spectrin is reminiscent of the binding of
-catenin to actinin (30).
A member of the spectrin gene superfamily,
-actinin shares the
repeat structure of spectrin and binds F-actin. However, unlike for
spectrin, a sequence in the two central
-actinin repeat units appears to interact with a region in
-catenin that is downstream of
the spectrin-binding site identified here. This result is a bit
surprising given the similarity of
-actinin to spectrin and suggests
that spectrin and
-actinin, despite their similarities, play
distinct roles in the physiology of the adhesion complex. The presence
of distinct binding sites for both
-actinin and spectrin (and actin,
Ref. 29) in
-catenin, as well as independent binding sites for actin
in both spectrin and
-actinin, suggests that these molecules can
bind simultaneously and independently (although this premise has not
been formally examined). Thus, it is likely that a cooperative and
redundant interaction of
-catenin with spectrin, F-actin, and
-actinin guides the assembly of a spectrin-actin skeleton to regions
of cell-cell contact. The findings in Clone A cells lend support to
this notion. Although
-catenin from Clone A cells binds spectrin
normally (as it does F-actin and
-catenin (46)), the deletion in
this catenin (residues 197-354) overlaps a region previously
demonstrated to bind
-actinin (residues 325-394 (30)). Perhaps a
loss of
-actinin binding leads to an impairment of actin assembly at
the membrane, with consequential impairment of spectrin-actin assembly
in zones of cell-cell contact.
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FOOTNOTES |
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* This work was supported in part by grants from the National Institutes of Health (to J. S. M. and D. L. R.), a National Institutes of Health postdoctoral fellowship (to C. R. L.), and an independent investigator award (to D. L. R.) from the Patrick and Catherine Weldon Donaghue Medical Research Foundation.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.
These authors contributed equally to this work.
§ Current address: The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92137.
¶ Current address: Genetics Institute, CMS/DSD, One Burrt Rd., Andover, MA 01810.
To whom correspondence and requests for reprints should be
addressed: Dept. of Pathology, Yale University, 310 Cedar St., New
Haven, CT 06510. Tel.: 203-785-3624; Fax: 203-785-7037; E-mail: jon.morrow@yale.edu.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M009259200
2 C. Cianci, P. Stabach, S. Kennedy, P. Devarajan, and J. S. Morrow, unpublished observations.
3 Y. Ch'ng, M. C. Stankewich, and J. S. Morrow, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: MAD, membrane association domain; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; MDCK, Madin-Darby canine kidney; GST, glutathione S-transferase; Pipes, 1,4-piperazinediethanesulfonic acid; RU, resonance units; TBS, Tris-buffered saline; RT, room temperature; PVDF, polyvinylidene difluoride.
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REFERENCES |
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---|
1. | Luna, E. J., and Hitt, A. L. (1992) Science 258, 955-964[Medline] [Order article via Infotrieve] |
2. | Morrow, J. S., Rimm, D. L., Kennedy, S. P., Cianci, C. D., Sinard, J. H., and Weed, S. A. (1997) in Handbook of Physiology (Hoffman, J. , and Jamieson, J., eds) , pp. 485-540, Oxford University Press, Oxford |
3. | Dubreuil, R. R., and Grushko, T. (1998) BioEssays 20, 875-878[CrossRef][Medline] [Order article via Infotrieve] |
4. |
De Matteis, M. A.,
and Morrow, J. S.
(2000)
J. Cell Sci.
113,
2331-2343 |
5. |
Lombardo, C. R.,
Weed, S. A.,
Kennedy, S. P.,
Forget, B. G.,
and Morrow, J. S.
(1994)
J. Biol. Chem.
269,
29212-29219 |
6. |
Davis, L. H.,
and Bennett, V.
(1994)
J. Biol. Chem.
269,
4409-4416 |
7. | Dubreuil, R. R., and Grushko, T. (1999) Biochem. Biophys. Res. Commun. 265, 372-375[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Piepenhagen, P. A.,
and Nelson, W. J.
(1998)
Mol. Biol. Cell
9,
3161-3177 |
9. |
Davis, L. H.,
Otto, E.,
and Bennett, V.
(1991)
J. Biol. Chem.
266,
11163-11169 |
10. |
Willardson, B. M.,
Thevenin, B. J.,
Harrison, M. L.,
Kuster, W. M.,
Benson, M. D.,
and Low, P. S.
(1989)
J. Biol. Chem.
264,
15893-15899 |
11. |
Srinivasan, Y.,
Lewallen, M.,
and Angelides, K. J.
(1992)
J. Biol. Chem.
267,
7483-7489 |
12. |
Li, Z. P.,
Burke, E. P.,
Frank, J. S.,
Bennett, V.,
and Philipson, K. D.
(1993)
J. Biol. Chem.
268,
11489-11491 |
13. |
Zhang, Z.,
Devarajan, P.,
Dorfman, A. L.,
and Morrow, J. S.
(1998)
J. Biol. Chem.
273,
18681-18684 |
14. |
Davis, J. Q.,
and Bennett, V.
(1994)
J. Biol. Chem.
269,
27163-27166 |
15. | Dubreuil, R. R., MacVicar, G., Dissanayake, S., Liu, C., Homer, D., and Hortsch, M. (1996) J. Cell Biol. 133, 647-655[Abstract] |
16. |
Bouley, M.,
Tian, M. Z.,
Paisley, K.,
Shen, Y. C.,
Malhotra, J. D.,
and Hortsch, M.
(2000)
J. Neurosci.
20,
4515-4523 |
17. |
Parra, M.,
Gascard, P.,
Walensky, L. D.,
Gimm, J. A.,
Blackshaw, S.,
Chan, N.,
Takakuwa, Y.,
Berger, T.,
Lee, G.,
Chasis, J. A.,
Snyder, S. H.,
Mohandas, N.,
and Conboy, J. G.
(2000)
J. Biol. Chem.
275,
3247-3255 |
18. |
Devarajan, P.,
Stabach, P. R.,
De Matteis, M. A.,
and Morrow, J. S.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
10711-10716 |
19. | Ladbury, J. E., Lemmon, M. A., Zhou, M., Green, J., Botfield, M. C., and Schlessinger, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3199-3203[Abstract] |
20. | Hyvonen, M., Macias, M. J., Nilges, M., Oschkinat, H., Saraste, M., and Wilmanns, M. (1995) EMBO J. 14, 4676-4685[Abstract] |
21. |
Godi, A.,
Santone, I.,
Pertile, P.,
Devarajan, P.,
Stabach, P. R.,
Morrow, J. S.,
Di Tullio, G.,
Polishchuk, R.,
Petrucci, T. C.,
Luini, A.,
and De Matteis, M. A.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8607-8612 |
22. | Godi, A., Pertile, P., Meyers, R., Marra, P., Di Tullio, G., Iurisci, C., Luini, A., Corda, D., and De Matteis, M. A. (1999) Nat. Cell Biol. 1, 280-287[CrossRef][Medline] [Order article via Infotrieve] |
23. | Donaldson, J. G., and Lippincott-Schwartz, J. (2000) Cell 101, 693-696[Medline] [Order article via Infotrieve] |
24. |
Steiner, J. P.,
Walke, H. T. J.,
and Bennett, V.
(1989)
J. Biol. Chem.
264,
2783-2791 |
25. | Peifer, M., McCrea, P. D., Green, K. J., Wieschaus, E., and Gumbiner, B. M. (1992) J. Cell Biol. 118, 681-691[Abstract] |
26. | McNeill, H., Ryan, T. A., Smith, S. J., and Nelson, W. J. (1993) J. Cell Biol. 120, 1217-1226[Abstract] |
27. | Provost, E., and Rimm, D. L. (1999) Curr. Opin. Cell Biol. 11, 567-572[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Koslov, E., R.,
Maupin, P.,
Pradhan, D.,
Morrow, J. S.,
and Rimm, D. L.
(1997)
J. Biol. Chem.
272,
27301-37306 |
29. | Rimm, D. L., Koslov, E. R., Kebriaei, P., Cianci, C. D., and Morrow, J. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8813-8817[Abstract] |
30. |
Nieset, J. E.,
Redfield, A. R.,
Jin, F.,
Knudsen, K. A.,
Johnson, K. R.,
and Wheelock, M. J.
(1997)
J. Cell Sci.
110,
1013-1022 |
31. | Gumbiner, B. M., and McCrea, P. D. (1993) J. Cell Sci. 17 (suppl.), 155-158[Abstract] |
32. | Hulsken, J., Birchmeier, W., and Behrens, J. (1994) J. Cell Biol. 127, 2061-2069[Abstract] |
33. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
34. | Kennedy, S. P., Warren, S. L., Forget, B. G., and Morrow, J. S. (1991) J. Cell Biol. 115, 267-277[Abstract] |
35. | Rimm, D. L., Kebriaei, P., and Morrow, J. S. (1994) Biochem. Biophys. Res. Commun. 203, 1691-1699[CrossRef][Medline] [Order article via Infotrieve] |
36. | Smith, D. B., and Johnson, K. S. (1988) Gene (Amst.) 67, 31-40[Medline] [Order article via Infotrieve] |
37. | Bennett, V., Baines, A. J., and Davis, J. (1986) Methods Enzymol. 134, 55-69[Medline] [Order article via Infotrieve] |
38. | Devarajan, P., Stabach, P. R., Mann, A. S., Ardito, T., Kashgarian, M., and Morrow, J. S. (1996) J. Cell Biol. 133, 819-830[Abstract] |
39. | Breen, E., Clarke, A., Steele, G., Jr., and Mercurio, A. M. (1993) Cell Adhes. Commun. 1, 239-250[Medline] [Order article via Infotrieve] |
40. | Devarajan, P., Scaramuzzino, D. A., and Morrow, J. S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2965-2969[Abstract] |
41. |
Lombardo, C. R.,
Willardson, B. M.,
and Low, P. S.
(1992)
J. Biol. Chem.
267,
9540-9546 |
42. | Canziani, G., Zhang, W., Cines, D., Rux, A., Willis, S., Cohen, G., Eisenberg, R., and Chaiken, I. (1999) Methods 19, 253-269[CrossRef][Medline] [Order article via Infotrieve] |
43. | Panayotou, G. (1998) Methods Mol. Biol. 88, 1-10[Medline] [Order article via Infotrieve] |
44. | Zhou, M., Felder, S., Rubinstein, M., Hurwitz, D. R., Ullrich, A., Lax, I., and Schlessinger, J. (1993) Biochemistry 32, 8193-8198[Medline] [Order article via Infotrieve] |
45. | Karlsson, R., Michaelsson, A., and Mattsson, L. (1991) J. Immunol. Methods 145, 229-240[Medline] [Order article via Infotrieve] |
46. | Roe, S., Koslov, E. R., and Rimm, D. L. (1998) Cell Adhes. Commun. 5, 283-296[Medline] [Order article via Infotrieve] |
47. | Furukawa, Y., Nakatsuru, S., Nagafuchi, A., Tsukita, S., Muto, T., Nakamura, Y., and Horii, A. (1994) Cytogenet. Cell Genet. 65, 74-78[Medline] [Order article via Infotrieve] |