alpha -Catenin Binds Directly to Spectrin and Facilitates Spectrin-Membrane Assembly in Vivo*

Deepti PradhanDagger, Christian R. LombardoDagger§, Susanna RoeDagger, David L. Rimm, and Jon S. Morrow||

From the Department of Pathology, Yale University, New Haven, Connecticut 06510

Received for publication, October 11, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -catenin, a component of the adhesion complex. Spectrin (alpha IIbeta II) and alpha -catenin coprecipitate from extracts of confluent Madin-Darby canine kidney, HT29, and Clone A cells and from solutions of purified spectrin and alpha -catenin in vitro. By surface plasmon resonance and in vitro binding assays, we find that alpha -catenin binds alpha IIbeta II spectrin with an apparent Kd of approx 20-100 nM. By gel-overlay assay, alpha -catenin binds recombinant beta II-spectrin peptides that include the first 313 residues of spectrin but not to peptides that lack this region. Similarly, the binding activity of alpha -catenin is fully accounted for in recombinant peptides encompassing the NH2-terminal 228 amino acid region of alpha -catenin. An in vivo role for the interaction of spectrin with alpha -catenin is suggested by the impaired membrane assembly of spectrin and its enhanced detergent solubility in Clone A cells that harbor a defective alpha -catenin. Transfection of these cells with wild-type alpha -catenin reestablishes alpha -catenin at the plasma membrane and coincidentally recruits spectrin to the membrane. We propose that ankyrin-independent interactions of modest affinity between alpha -catenin and the amino-terminal domain of beta -spectrin augment the interaction between alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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 (approx 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 beta -spectrin, termed membrane association domains 1, 2, and 3 (MAD1,1 MAD2, and MAD3). MAD1 is located in repeat unit 1 of both beta I and beta 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 beta ISigma 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 beta -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.

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 alpha - and beta -catenin (or gamma -catenin) and p120 (for review see Refs. 25 and 27). beta -Catenin (or in some cells gamma -catenin) directly binds the cytoplasmic domain of E-cadherin; alpha -catenin joins the membrane complex via a direct association with beta -catenin or gamma -catenin (28). alpha -Catenin binds and bundles F-actin, an activity that presumably facilitates the attachment of actin filaments to the adhesion complex (29). alpha -Catenin also binds alpha -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 alpha -catenin, and we demonstrate that alpha -catenin is required for spectrin assembly at the plasma membrane in Clone A cells, a human intestinal cell line. These studies thereby identify alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 alpha (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 alpha -catenin constructs used in relation to the full sequence is shown in Fig. 2 and as used previously (29).

Protein Purification-- Recombinant proteins were expressed in the E. coli strains HB101 and DH5alpha . 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 alpha -catenin-GST fusion proteins were utilized as antigens for the generation of polyclonal antibodies that were subsequently affinity purified by absorption to immobilized immunogen. alpha IIbeta 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).

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-alpha -catenin fusion proteins. Binding assays were performed in BSA-coated polypropylene tubes (41). Recombinant alpha -catenin peptides N576 or C447 (1 µg) were incubated with increasing concentrations of 125I-labeled alpha IIbeta 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 gamma -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.

Overlay Binding-- GST was cleaved from recombinant alpha -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 alpha -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 alpha -catenin.

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 alpha IIbeta 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 alpha -catenin or expressed alpha -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 alpha -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.

Transfection Procedure-- Clone A cells (approx 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 alpha 1(E)-catenin (pcDNA3-alpha -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 alpha -catenin expression (46). Alternatively, since stable lines often proved unstable, transiently expressing cells were used for most experiments. The expression of alpha -catenin was verified by immunofluorescence and Western blotting.

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 alpha -catenin (3H4) and 7A11 were used as undiluted culture supernatants; polyclonal antibody (RAFA) to alpha 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.

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 -80 °C until use. alpha IIbeta 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 approx 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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alpha IIbeta II Spectrin and alpha -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 alpha -catenin may mediate the linkage of spectrin to the adhesion complex. In poorly confluent HT29 cells, spectrin is largely cytoplasmic, as is alpha -catenin (Fig. 1, A-C). When these cells become more confluent and establish effective intercellular cadherin-mediated junctions, alpha -catenin is recruited to zones of cell-cell contact (Fig. 1D). Coincident with this recruitment of alpha -catenin, there is recruitment of spectrin from cytoplasmic to membrane pools, in a distribution indistinguishable from alpha -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 alpha -catenin generates a shortened and mutated alpha -catenin transcript (Fig. 3, also see Refs. 46 and 47). Clone A alpha -catenin is unstable and fails to associate with the cadherin-based adhesion complex (39, 46), even though its ability to associate with beta -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 alpha -catenin, Fig. 2B). alpha -Catenin can also be directly demonstrated in immunoprecipitates of alpha IIbeta 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 alpha -catenin and also suggest that these two proteins may associate both in the cytosol and at the membrane.



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Fig. 1.   alpha IIbeta II spectrin and alpha -catenin are coincident only along zones of cell-cell contact. The distribution of alpha -catenin (A and D) or alpha IIbeta 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 alpha -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.   alpha IIbeta II spectrin and alpha -catenin associate in vivo. A, alpha IIbeta 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-alpha IIbeta 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 alpha IIbeta 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-alpha -catenin monoclonal antibody 3H4. Note the presence of alpha -catenin in the whole cell lysates (lys) and in the immunoprecipitate with alpha IIbeta II spectrin antibodies but not in the immunoprecipitate using antibodies to alpha Ibeta I spectrin or nonreactive antibodies (nrs). Also apparent is the lower molecular weight of the Clone A alpha -catenin (alpha -cat).



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Fig. 3.   beta II spectrin binds directly to the NH2-terminal 228 amino acids (aa) of alpha -catenin. A, schematic representation of the structure of alpha -catenin, the mutation found in Clone A cells (shaded box (46)), and the recombinant alpha -catenin peptides used in this study. On the right is shown an SDS-PAGE analysis, Coomassie Blue-stained, of the purified alpha IIbeta II spectrin (S) and each of the alpha -catenin peptides (with GST removed). Their Mr divide  1000 is shown. B, surface plasmon resonance analysis of the interaction of GST-alpha -catenin with immobilized alpha IIbeta 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-alpha -catenin, respectively. Similar experiments carried out with different levels of immobilized spectrin, and with recombinant alpha -catenin in which the GST moiety had been removed by thrombin treatment, gave comparable results. Interestingly, analysis of recombinant alpha -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-alpha -catenin peptides at 5.0 µM to alpha IIbeta II spectrin. The alpha -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 alpha IIbeta II spectrin to alpha -catenin peptides. Increasing concentrations of 125I-labeled alpha IIbeta II spectrin were incubated with alpha -catenin peptides N576 (black-square) 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.

Spectrin Binds Directly to the NH2-- terminal 228 Residues of alpha -Catenin---To obtain a more precise analysis of the interaction between alpha -catenin and spectrin, the ability of recombinant alpha -catenin peptides to bind to alpha IIbeta II spectrin was investigated by surface plasmon resonance. A series of recombinant alpha -catenin peptides were prepared as fusions with GST (Fig. 3A). Also prepared was an alpha -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 alpha IIbeta II spectrin was immobilized on the BiacoreTM sensor chip surface, and the changes in resonance units were monitored for different concentrations of alpha -catenin (Fig. 3B) or for different alpha -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 approx 3 of each other for a given peptide. However, no model generated fits with fully random residuals, indicating that the binding of alpha -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 alpha -catenin binding to alpha IIbeta II spectrin is presented in Table I. In this analysis, wild-type alpha -catenin bound spectrin with an apparent KD of 19-80 nM, GST-alpha -catenin bound with an apparent KD of 19-24 nM, and the mutant Clone A alpha -catenin, devoid of GST, bound with an apparent KD of 15-25 nM. The differences in apparent binding affinity between the GST-alpha -catenin versus peptides without GST presumably reflects the propensity of GST to induce homodimerization. Oligomers of alpha -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 alpha -catenin for spectrin versus wild-type alpha -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 alpha -catenin (28).


                              
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Table I
Kinetics and derived dissociation constants as measured by surface plasmon resonance
The values presented represent only an apparent KD, since in neither case (simple bimolecular Langmuir binding or two-exponential fit) were the fitting residuals completely random. Several mechanisms may contribute to this complexity, including surface microheterogeneity, limitations in mass transport to and from the binding surface, and complex conformational dependent binding mechanisms inherent to the proteins themselves. Available data are insufficient to discriminate between these possibilities. The data do demonstrate unequivocal high affinity binding of alpha -catenin to spectrin.

To identify the site in alpha -catenin that interacts with spectrin, additional recombinant peptides derived from the NH2 and COOH termini of alpha -catenin (Fig. 3A) were prepared and assayed qualitatively for their ability to bind directly to alpha IIbeta II spectrin using surface plasmon resonance (Fig. 3C). As before, full-length alpha -catenin (peptide a907) bound avidly. Peptide N576, representing the NH2-terminal half of alpha -catenin, bound in a similar way, achieving approximately half of the RUs of peptide a907. A peptide representing the NH2-terminal 228 residues of alpha -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 alpha -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 alpha -catenin fully accounts for its interaction with spectrin.

Finally, it was of interest to determine whether alpha -catenin would also bind alpha IIbeta II spectrin in vitro in solution (versus immobilized spectrin on the Biacore sensor surface). Increasing concentrations of purified 125I-labeled alpha IIbeta 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 gamma -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 alpha IIbeta II spectrin and the NH2-terminal 228 residues of alpha -catenin. Consistent with this binding locus, no differences in spectrin binding by Clone A alpha -catenin were detected. Clone A alpha -catenin deletes residues 197-354 of the native protein, suggesting that the actual interaction site in alpha -catenin for spectrin is proximal to residue 197.

alpha -Catenin Binds to the First 313 Residues of beta II Spectrin-- The site to which alpha -catenin binds in beta II spectrin was identified by gel-overlay assay (Fig. 4). Recombinant GST fusion peptides representing all regions of human beta II spectrin were transferred to PVDF membranes and overlaid with alpha -catenin (from which GST had been removed) (Fig. 4). Of the peptides examined, only those (beta IIN-1, beta IIN-4, and beta IIN-6) that included the NH2-terminal region of beta II spectrin bound alpha -catenin (Fig. 4B, center). To assess further the relative affinities of alpha -catenin for this region of beta II spectrin, overlay experiments were designed using a range of peptide concentrations (Fig. 4B, right). Regardless of concentration, alpha -catenin did not bind to GST alone or to beta II9-C. Conversely, strong binding was detected at every concentration of the beta IIN-1 peptide. This active peptide, the smallest one examined in these experiments, spans residues 1-313 of beta II spectrin and places the alpha -catenin-binding site within this region.



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Fig. 4.   The first 313 residues of beta II spectrin effect alpha -catenin binding. A, summary of the recombinant beta II spectrin peptides used in relation to the overall functional domain structure of beta II spectrin. The major known ligand-binding regions in beta 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 alpha -catenin binding to beta 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 alpha -catenin and developed with the 3H4 antibody to alpha -catenin. Note the strong binding of alpha -catenin to beta IIN-6, beta IIN-4, and beta 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 beta II9-C, beta IIN-1, or GST alone at three loadings (1.0, 0.5, or 0.1 µg) overlaid with alpha -catenin (GST-free). Note the strong binding to beta IIN-1 with no detectable binding to beta II9-C or GST at any concentration.

alpha -Catenin Facilitates Spectrin Membrane Assembly in Vivo-- Clone A cells are defective in cell-cell adhesion and harbor an internal deletion in the expressed alpha -catenin (39, 46). This mutation leads to the loss of alpha -catenin associated with the plasma membrane, reduced cell-cell adhesion, and coincidentally, reduced assembly of alpha IIbeta 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 beta -catenin staining (46). To test whether the failure of spectrin assembly in these cells was due to the defect in alpha -catenin, Clone A cells were transiently transfected with wild-type alpha -catenin, and the assembly of alpha -catenin and alpha IIbeta II spectrin at the membrane was monitored (Fig. 5). The transfected wild-type alpha -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, alpha IIbeta II spectrin was restored to its plasma membrane location (Fig. 5, B and D). Thus, wild-type alpha -catenin is fully competent to rescue the impaired membrane assembly of spectrin in Clone A cells.



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Fig. 5.   Wild-type alpha -catenin restores spectrin assembly at the membrane in Clone A cells. Clone A cells were transiently transfected with wild-type alpha -catenin and stained with the monoclonal antibody 7A11 which only recognizes wild-type alpha -catenin (46) (A and C) or with RAF-A, an antibody to alpha IIbeta II spectrin (B and D). Note the cluster of transfected cells that express alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

These findings establish that alpha -catenin can bind directly to spectrin, that these proteins are associated in cultured epithelial cells in vivo, and that alpha -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 alpha -catenin and the first 313 residues of beta II spectrin. (ii) Spectrin and alpha -catenin co-localize and co-precipitate in confluent monolayers of Clone A, HT29, and MDCK cells. (iii) Spectrin and alpha -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 alpha -catenin. Collectively, these findings suggest that in addition to its other roles, alpha -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).

The demonstration of a direct interaction between alpha -catenin and spectrin is reminiscent of the binding of alpha -catenin to actinin (30). A member of the spectrin gene superfamily, alpha -actinin shares the repeat structure of spectrin and binds F-actin. However, unlike for spectrin, a sequence in the two central alpha -actinin repeat units appears to interact with a region in alpha -catenin that is downstream of the spectrin-binding site identified here. This result is a bit surprising given the similarity of alpha -actinin to spectrin and suggests that spectrin and alpha -actinin, despite their similarities, play distinct roles in the physiology of the adhesion complex. The presence of distinct binding sites for both alpha -actinin and spectrin (and actin, Ref. 29) in alpha -catenin, as well as independent binding sites for actin in both spectrin and alpha -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 alpha -catenin with spectrin, F-actin, and alpha -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 alpha -catenin from Clone A cells binds spectrin normally (as it does F-actin and beta -catenin (46)), the deletion in this catenin (residues 197-354) overlaps a region previously demonstrated to bind alpha -actinin (residues 325-394 (30)). Perhaps a loss of alpha -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.


    FOOTNOTES

* 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.

Dagger 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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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