©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Plakoglobin Domains Required for Association with N-cadherin and -Catenin (*)

(Received for publication, November 21, 1994; and in revised form, March 22, 1995)

Paula A. Sacco Tammy M. McGranahan Margaret J. Wheelock(§)(¶) Keith R. Johnson (§)

From the Department of Biology, University of Toledo, Toledo, Ohio 43606

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cadherins are calcium-dependent, cell surface glycoproteins involved in cell-cell adhesion. To function in cell-cell adhesion, the transmembrane cadherin molecule must be associated with the cytoskeleton via cytoplasmic proteins known as catenins. Three catenins, alpha-catenin, beta-catenin, and -catenin (also known as plakoglobin), have been identified. The domain of the cadherin molecule important for its interaction with the catenins has been mapped to the COOH-terminal 70 amino acids, but less is known about regions of the catenins that allow them to associate with one another or with the cadherin molecule. In this study we have transfected carboxyl-terminal deletions of plakoglobin into the human fibrosarcoma HT-1080 and used immunofluorescence localization and co-immunoprecipitation to map the regions of plakoglobin that allow it to associate with N-cadherin and with alpha-catenin. Plakoglobin is an armadillo family member containing 13 weakly similar internal repeats. These data show that the alpha-catenin-binding region maps within the first repeat and the N-cadherin-binding region maps within repeats 7 and 8.


INTRODUCTION

Plakoglobin is one of the plaque proteins found in both desmosomes and adherens junctions(1) . In addition to membrane-associated plakoglobin, a soluble form has been characterized(2) . Interestingly, only minor differences have been found in the plakoglobin associated with these various cellular compartments(2, 3) .

Desmosomes have two transmembrane components, desmoglein and desmocollin, which are members of the broadly defined cadherin family of molecules. Plakoglobin has been shown to interact with the cytoplasmic tail of desmoglein (4, 5) and desmocollin(6) . The transmembrane component of the adherens junction is a member of the classic cadherin family; these proteins mediate homotypic cell-cell adhesion(7, 8, 9, 10) . In the cytoplasm, classic cadherins associate with three proteins known as alpha-, beta-, and -catenin(11, 12, 13) ; -catenin is identical to plakoglobin(14, 15) . The cytoplasmic components of the desmosome and the adherens junction are important in mediating interactions with the appropriate cytoskeletal elements of the junction, i.e. intermediate filaments and actin, respectively(16, 17, 18) .

The cDNA sequences of plakoglobin from a number of species have revealed close relationships to vertebrate beta-catenins and to Drosophila armadillo, a segment polarity gene(19, 20, 21, 22, 23) . The central region of plakoglobin is composed of 13 weakly similar repeats of approximately 42 amino acids. Developmental studies of armadillo mutants have suggested that the repeats in Armadillo contribute to its function in an additive manner and that the protein is modular(24) . This raises the possibility that plakoglobin likewise functions as a modular protein.

The fact that plakoglobin shares significant regions of homology with beta-catenin (15, 19, 22) is of particular interest in light of recent studies showing that cells express two types of cadherin-catenin complexes: a complex that contains predominantly beta-catenin and one that contains predominantly plakoglobin(25, 26) . The consensus is that plakoglobin (or beta-catenin) binds directly to cadherin and that alpha-catenin is associated with the cadherin through its association with plakoglobin (or beta-catenin). Pulse-chase experiments in Madin-Darby canine kidney cells demonstrated that cadherin-catenin complexes are assembled by first loading plakoglobin (or beta-catenin) onto the newly synthesized cadherin and that alpha-catenin associates with the complex as it arrives at the plasma membrane(25) . Further evidence that beta-catenin associates with cadherin in the absence of alpha-catenin was provided by studies in PC-9 cells, which do not express alpha-catenin but form complexes of beta-catenin and cadherin(27) . These cells do not strongly adhere to one another, but when transfected with alpha-catenin, they adhere normally and form complexes that include alpha-catenin(28) . alpha-Catenin is thought to provide a link between cadherin-beta-catenin and the cytoskeleton.

To examine further the interactions of plakoglobin with cadherin and alpha-catenin we have identified the regions of plakoglobin that are important in its interaction with the cadherin complex in living cells. We first identified a cell line, the human fibrosarcoma HT-1080(29) , that forms a cadherin-catenin complex but produces only minuscule amounts of plakoglobin. We then transfected HT-1080 cells with full-length and truncated forms of plakoglobin and examined the transfectants for the association of plakoglobin with cadherin and/or alpha-catenin. With this approach we have been able to identify distinct regions of plakoglobin that interact with cadherin and alpha-catenin, supporting the notion that plakoglobin functions as a modular protein.


EXPERIMENTAL PROCEDURES

Cells

The HT-1080 human fibrosarcoma cell line was obtained from American Type Culture Collection (ATCC CCL 121) and maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal calf serum. Transfectants were maintained in the same medium supplemented with 10M dexamethasone (Sigma) and 1 mg/ml G418 (Life Technologies, Inc.).

Molecular Constructions and Transfections

The full-length human plakoglobin clone in pBluescript (21) was a gift from Dr. W. W. Franke, German Cancer Research Center, Heidelberg. The expression vector pLKneo (30) was used for these studies. For expression of full-length plakoglobin, the complete cDNA was inserted into pLKneo. The truncations at the unique SmaI, unique SphI, and most 5` PstI sites were first done in pBluescript, and appropriate restriction fragments were ligated into pLKneo. Truncating the plakoglobin cDNA at these sites resulted in constructions retaining the NH(2)-terminal 572, 233, and 114 amino acids, respectively (Fig. 1). Exonuclease III deletions were made with the Erase-a-Base kit from Promega (Madison, WI) using the full-length pLKneo-plakoglobin construct as the starting plasmid. For all the clones described in the text, the extent of the deletion was determined by nucleotide sequencing. In the deletion clones, translation terminated in the SV-40 sequences downstream of the multiple cloning site in pLKneo. Polymerase chain reactions (PCR) (^1)were used to prepare constructions terminating precisely at several repeat boundaries. The cDNA 3` of the SphI site was replaced with a PCR product extending from the SphI site to the end of repeat 5, 7, or 8. The sequence of each the PCR products agreed with that of plakoglobin(21) . Cell cultures were transfected using calcium phosphate precipitation (31) with materials from Stratagene (La Jolla, CA), and G418-resistant clones were isolated. All data in this manuscript were obtained with stable cell lines.


Figure 1: A map showing functional domains of plakoglobin. Full-length human plakoglobin (745 amino acids) is shown along with the sites of the truncations used in this study. The 13 Armadillo repeats are shown as boxes. The PstI site is located just amino-terminal to the repetitive region, the SphI site is located in the third repeat, and the SmaI site is near the start of the 11th repeat. All of the deletions described in this report are indicated with the number of amino acids remaining in the COOH-terminally truncated plakoglobin. The regions of plakoglobin that allow the truncated protein to retain its association with N-cadherin and alpha-catenin are indicated.



Antibodies and Other Reagents

Mouse monoclonal antibodies to alpha-catenin (1G5) and beta-catenin (12F7 and 9F2) have been described (32) . Mouse monoclonal antibodies 15F11 against plakoglobin and 13A9 against the cytoplasmic domain of N-cadherin were generated as described(32) . The cDNA encoding human N-cadherin (33) was a gift from Dr. John Hemperly (Becton Dickinson and Company Research Center, Research Triangle Park, NC).

Detergent Extractions of Cells

Monolayers of cells were washed with phosphate-buffered saline at room temperature and extracted on ice with 2 ml/75-cm^2 flask 10 mM Tris acetate, pH 8.0, containing 0.5% Nonidet P-40 (BDH Chemicals Ltd., Poole, United Kingdom), 1 mM EDTA, and 2 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 15,000 g for 15 min at 4 °C.

Electrophoresis and Immunoblotting

Polyacrylamide slab gel electrophoresis in the presence of SDS (SDS-PAGE) was done according to the procedure of Laemmli(34) . Materials were from Bio-Rad. Molecular weight markers were from Sigma. SDS-PAGE-resolved proteins were transferred to nitrocellulose and immunoblotted as described(14) . Quantitative analysis of immunoblots was done using a Molecular Dynamics personal densitometer and Image Quant software.

Immunoprecipitation

A 300-µl aliquot of cell extract was mixed with 300 µl of monoclonal antibody supernatant at 4 °C. After 30 min 50 µl of packed anti-mouse IgG-Sepharose (Organon Teknika-Cappel, Durham, NC) was added, and mixing was continued for 30 min. The Sepharose-bound immune complexes were washed 5 times with 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Tween 20. The pellets were boiled in Laemmli sample buffer and resolved by SDS-PAGE. In some experiments IgG purified from ascites fluid was coupled to CNBr-activated Sepharose 4B (Pharmacia Biotech Inc.) and used as the immunoprecipitating antibody.

Immunofluorescence Microscopy

Cells were grown on glass coverslips, fixed for 30 min in 1% paraformaldehyde buffered with Hanks' balanced salt solution containing 10 mM HEPES pH 7.4, and permeabilized with methanol. The cells were blocked with 10% goat serum and 0.1 M glycine in phosphate-buffered saline for 20 min and exposed to antibodies for 1 h followed by species-specific second antibodies conjugated to fluorescein (Jackson Immunoresearch Laboratories, West Grove, PA). Fluorescence was detected with a Zeiss Axiophot microscope equipped with epifluorescence. All pictures were taken using a 40 objective and Kodak T-MAX 3200 film.


RESULTS

HT-1080 Cells Express an N-cadherin-Catenin Complex but Little Plakoglobin

To study the association of plakoglobin with the cadherin-catenin complex, we sought a cell line that expressed minimal amounts of plakoglobin yet was capable of making normal cell-cell contacts using a cadherin. The HT-1080 human fibrosarcoma fulfilled these criteria. Immunoblot analysis of HT-1080 cells (Fig. 2) showed that they express N-cadherin, beta-catenin, and alpha-catenin at high levels but express almost undetectable levels of plakoglobin (-catenin). Using immunofluorescence microscopy, N-cadherin, alpha-catenin, and beta-catenin were localized to cell-cell borders, while staining with anti-plakoglobin antibodies produced a faint, diffuse signal with no localization to a specific cellular compartment (not shown). When HT-1080 extracts were immunoprecipitated with monoclonal anti-alpha-catenin antibody and the products of the immunoprecipitation reaction immunoblotted with antibodies against N-cadherin and beta-catenin, both N-cadherin and beta-catenin were associated with alpha-catenin (data not shown). Thus, HT-1080 cells synthesize a complex containing N-cadherin, alpha-catenin, and beta-catenin, and such complexes are found at regions of cell-cell contact.


Figure 2: HT-1080 cells express N-cadherin, alpha-catenin, and beta-catenin but very little plakoglobin. A Nonidet P-40 extract of HT-1080 cells was resolved on a 7% SDS-polyacrylamide gel, transblotted to nitrocellulose, and probed with: lane1, 13A9 anti-N-cadherin (N-cad); lane2, 1G5 anti-alpha-catenin (alpha-cat); lane3, 12F7 anti-beta-catenin (beta-cat); lane4, 15F11 anti-plakoglobin (pg). The lines at the left indicate the molecular mass standards at 205, 116, 97, 68, and 45 kDa.



HT-1080 Cells Are Capable of Including Plakoglobin in an N-cadherin-Catenin Complex

When full-length plakoglobin was transfected into HT-1080 cells, it was localized at cell-cell borders, similar to alpha-catenin, beta-catenin, and N-cadherin (data not shown). It also co-immunoprecipitated with both alpha-catenin and N-cadherin. Fig. 3shows extracts of transfected and non-transfected cells that were immunoprecipitated with anti-alpha-catenin, anti-plakoglobin, or anti-N-cadherin antibodies and subsequently immunoblotted with anti-plakoglobin antibodies. The above results showed that, although HT-1080 cells formed cadherin-catenin complexes containing very little endogenous plakoglobin, transfectants readily incorporated plakoglobin into the cadherin complex. Thus, HT-1080 cells were shown to be an appropriate cell line in which to begin to map the functional domains of plakoglobin. HT-1080 cells do not synthesize desmosomal components (data not shown), so there are no complicating associations of plakoglobin with desmosomal cadherins.


Figure 3: Transfected plakoglobin is incorporated into the cadherin complex in HT-1080 cells. Extracts of HT-1080 cells transfected with full-length plakoglobin (lanes1, 3, and 5) or mock transfected (lanes2, 4, and 6) were immunoprecipitated with anti-alpha-catenin (alpha-cat, lanes1 and 2), anti-plakoglobin (pg, lanes3 and 4), or anti-N-cadherin (N-cad, lanes5 and 6). The proteins were resolved on a 7% SDS-polyacrylamide gel, transblotted to nitrocellulose, and probed with anti-plakoglobin. Plakoglobin was abundant in all three immunoprecipitation reactions in the transfected cells but barely detectable in the mock transfected cells. The major bands at about 50 kDa are the antibody heavy chains. The lines at the left indicate the molecular mass standards at 116, 97, 68, and 45 kDa.



Mapping Regions of Plakoglobin That Allow It to Associate with N-cadherin and alpha-Catenin

Deletions were generated by cutting the cDNA at selected restriction sites (Fig. 1), resulting in COOH-terminal truncations of plakoglobin that retained 572, 233, and 114 amino acids. Each deleted construct was transfected into HT-1080 cells, and clones were propagated. Cells were extracted, resolved by SDS-PAGE, and immunoblotted with 15F11 anti-plakoglobin antibody (Fig. 4). As described above, non-transfected cells have minimal amounts of plakoglobin, but full-length plakoglobin of about 82 kDa was expressed in transfected HT-1080 cells. Cells transfected with the construct retaining 572 amino acids produced a protein of approximately 60 kDa; cells transfected with the construct retaining 233 amino acids produced a protein of about 30 kDa; and cells transfected with the construct retaining 114 amino acids produced a protein of about 14 kDa. In each case the truncated plakoglobin was stably expressed at high levels. Immunoblots prepared from gels loaded with equal amounts of protein (Fig. 4) were quantitated with densitometry. These data showed that the concentrations of the 572-, 233-, and 114-amino acid constructs were, respectively, 1.9-, 2.7- and 2.6-fold more than that of full-length plakoglobin. All the other truncated plakoglobins used in this study were expressed at levels similar to those presented in Fig. 4.


Figure 4: Quantitative analysis of plakoglobin expression in HT-1080 cells transfected with truncated plakoglobins. Equal amounts of total protein from Nonidet P-40 extracts of transfected HT-1080 cells were resolved on a 10% SDS-polyacrylamide gel, transblotted to nitrocellulose, and probed with anti-plakoglobin (15F11). Lane1, mock transfected cells; lane2, transfected cells expressing full-length plakoglobin; lane3, transfected cells expressing the 572-amino acid construct; lane4, transfected cells expressing the 233-amino acid construct; lane5, transfected cells expressing the 114-amino acid construct. The lines at the left indicate the molecular mass standards at 97, 68, 45, and 29 kDa.



Immunofluorescence localization with anti-plakoglobin antibodies was performed on each cell line. The 572-amino acid construct (Fig. 5B) was found along the cell-cell borders, indistinguishable from the pattern seen with full-length plakoglobin (Fig. 5A). The distributions of the 233-amino acid and 114-amino acid constructs (Fig. 5, C and D, respectively) were distinctly different from those of full-length plakoglobin or the 572-amino acid construct. The two smaller fragments were found diffusely localized in the cytoplasm and not along cell-cell borders.


Figure 5: The 572-amino acid deletion of plakoglobin localizes to sites of cell-cell contact in HT-1080 cells, but the 233-amino acid and 114-amino acid deletions do not. HT-1080 cells transfected with full-length or deleted plakoglobin were grown on glass coverslips and processed for immunofluorescence with anti-plakoglobin 15F11. In cells transfected with the 572-amino acid construct (B), plakoglobin localization looked similar to that seen in cells transfected with full-length plakoglobin (A). Cells transfected with the 233-amino acid construct (C) or with the 114-amino acid construct (D) showed bright, diffuse cytoplasmic staining.



As shown earlier, full-length plakoglobin co-immunoprecipitated with alpha-catenin and with N-cadherin (Fig. 3). Similar experiments were done in order to determine whether or not the truncated plakoglobins associated with these two proteins. It was found that the 572-amino acid construct co-immunoprecipitated with alpha-catenin as well as with N-cadherin (Fig. 6). Thus, the 572-amino acid truncation retained the sequences required for associating with each of these partners.


Figure 6: Mapping of the N-cadherin association domain on plakoglobin. Extracts of HT-1080 cells transfected with truncations of plakoglobin retaining the first 572 amino acids (AA, lanes 1 and 2), 472 amino acids (lanes 3 and 4), 375 amino acids (lanes 5 and 6), or 233 amino acids (lanes7 and 8) of plakoglobin were immunoprecipitated with anti-N-cadherin (N-cad, lanes 1, 3, 5, and 7) or anti-alpha-catenin (alpha-cat, lanes 2, 4, 6, and 8). For these experiments the immunoprecipitating reagents were purified IgG from ascites fluid conjugated to Sepharose. The proteins were resolved on a 12% SDS-polyacrylamide gel, transblotted to nitrocellulose, and probed with monoclonal anti-plakoglobin (15F11). Arrows point out the truncated plakoglobin molecules. The lines at the left indicate the molecular mass standards at 68, 45, and 29 kDa.



When transfectants expressing the 233-amino acid construct were examined, the truncated plakoglobin was found associated with alpha-catenin but not with N-cadherin. Fig. 6, lanes 7 and 8, shows the relevant immunoprecipitates immunoblotted with anti-plakoglobin antibody 15F11. The 233-amino acid construct migrated at about 30 kDa in the same portion of the SDS gel as the antibody light chains. The truncated plakoglobin was apparent in lane 8 when the immunoprecipitating antibody was anti-alpha-catenin. The 233-amino acid construct was not seen when the immunoprecipitating antibody was anti-N-cadherin (lane 7). Thus, the 233-amino acid construct was not able to stably bind N-cadherin, showing that the ability to bind N-cadherin is provided, at least in part, by amino acids 233-572 of plakoglobin. For these and the following experiments we used 1G5 anti-alpha-catenin ascites fluid conjugated to Sepharose as the immunoprecipitating reagent, which resulted in an increased background due to binding of the labeled secondary antibody.

In order to more closely map the sequences necessary for interaction with N-cadherin, we prepared exonuclease III deletions resulting in proteins that retained 233-572 amino acids of plakoglobin. Immunoprecipitations with antibodies against N-cadherin demonstrated that, as the plakoglobin molecule became shorter, the association with N-cadherin became less evident. Fig. 6compares the levels of truncated plakoglobins associated with N-cadherin to the levels associated with alpha-catenin. Comparison of lanes 1, 3, and 5 of Fig. 6revealed a gradual decrease in the ability of the constructs to associate with N-cadherin. The truncation retaining 375 amino acids was just able to stably interact with N-cadherin. Data obtained from clones expressing a 421-amino acid construct supported this gradual decline in association with N-cadherin (not shown). Immunofluorescence localization showed cell border staining in cells expressing the 472-amino acid construct, with some cytoplasmic signal. Cell border staining was not evident in cells expressing the 375-amino acid construct.

To address the question of whether or not the borders of the Armadillo repeats delineated functional domains, we used PCR to generate truncations terminating precisely at the ends of repeats 5, 7, and 8 (amino acids 334, 414, and 458, respectively). Table 1presents the immunofluorescence and immunoprecipitation data generated by all of our constructs. The ability of these shortened molecules to compete with endogenous beta-catenin for binding to N-cadherin suggests that the amino acids from 376 to 414 (comprising repeats 7 and 8) are particularly important for interaction with N-cadherin.



When cells expressing the 114-amino acid construct were extracted and immunoprecipitated, neither the anti-N-cadherin (not shown) nor the anti-alpha-catenin immunoprecipitate included the 114-amino acid construct (Fig. 7), although the protein was present in the cell extract (lane 6, indicated by an arrow). Thus at least one region of plakoglobin required for interaction with alpha-catenin resides between amino acids 114 and 233. Fig. 7also shows that when plakoglobin was truncated at 161 amino acids it readily associated with alpha-catenin, but, when truncated at 133 amino acids, the association was barely detected. These data suggest that the 47 residues from amino acids 115 to 161 play a major role in association with alpha-catenin with the 19 amino acids from 115 to 133 allowing weak association. All of the deletions used in these experiments as well as the regions of plakoglobin that likely interact with alpha-catenin and N-cadherin are presented in Table 1and shown diagrammatically in Fig. 1.


Figure 7: Mapping of the alpha-catenin association domain on plakoglobin. Extracts of HT-1080 cells transfected with truncations retaining 161 amino acids (AA, lane1), 133 amino acids (lane3), or 114 amino acids (lane5) of plakoglobin were immunoprecipitated (IP) with anti-alpha-catenin. Cell extract (ext) was run for comparison. The proteins were resolved on a 12% SDS-polyacrylamide gel, transblotted to nitrocellulose, and probed with anti-plakoglobin. The 161-amino acid construct co-immunoprecipitated with alpha-catenin while the 133-amino acid construct barely associated with alpha-catenin and the 114-amino acid construct no longer associated with alpha-catenin.




DISCUSSION

To function in cell-cell adhesion, the transmembrane cadherin molecule must be associated with the cytoskeleton via the cytoplasmic catenins. The COOH-terminal 70 amino acids of classical cadherins have been shown to be necessary for association with the catenins(12, 35, 36) . Assembly of the cadherin-catenin complex has been studied(13, 26, 37, 38, 39) , but less is known of the domains of the catenins that allow them to associate with the cadherin molecule or with one another. In this study we show that distinct regions of plakoglobin allow its association with N-cadherin and with alpha-catenin.

The localization of N-cadherin, alpha-catenin, and beta-catenin to cell-cell borders implies that HT-1080 cells interact with one another via their cadherins. However, the cells form an adhesion complex that contains only minute amounts of plakoglobin. Since the endogenous plakoglobin does not localize to cell-cell borders, HT-1080 cells apparently do not need plakoglobin in the cadherin-catenin complex in order to form cell-cell contacts. In AtT20 cells (25) and in Madin-Darby canine kidney cells(26) , data have been presented showing that cadherin-catenin complexes contain either beta-catenin or plakoglobin but not both. The sequence similarity between beta-catenin and plakoglobin suggests they may be able to substitute for one another and perhaps perform similar functions in the cadherin-catenin complex. The two molecules are not interchangeable, however, since plakoglobin, but not beta-catenin, associates with desmoglein and desmocollin. Perhaps because HT-1080 cells do not form desmosomes, beta-catenin alone is sufficient to provide the necessary functions for cell-cell adhesion. Interestingly, we could not detect obvious morphological or behavioral differences in cells transfected with plakoglobin. Thus, complexes containing plakoglobin appear not to confer a distinct phenotype in HT-1080 cells.

As mentioned earlier, plakoglobin is a member of the armadillo family of proteins(40) . The effects of carboxyl-terminal truncations of Armadillo on the development of Drosophila larvae were studied by Peifer and Wieschaus(24) . When the severity of each mutation was scored using time of embryonic lethality as a parameter, the severity correlated with the extent of the truncation suggesting a modular structure of the Armadillo protein. It was proposed that modules contributed in an additive fashion to the function of Armadillo. Our experiments with plakoglobin parallel those done with Armadillo; in both studies, the effects of carboxyl-terminal truncations were examined. Our data support a modular structure for plakoglobin since, as the extent of the deletion increased, association with N-cadherin was lost first and then association with alpha-catenin. If our data and those of Peifer and Wieschaus (24) are taken together, one interpretation is that, as the truncations of Armadillo became longer, the shortened protein first lost the ability to interact with cadherin and then lost the ability to productively associate with alpha-catenin. The developmental data would then suggest that preventing the association of an armadillo family member with both alpha-catenin and cadherin has more serious consequences than preventing the association with only the cadherin. This in turn suggests that cadherin-less complexes containing alpha-catenin and Armadillo may have functions during development.

It is also interesting to note that some of the mutants studied by Peifer and Wieschaus (24) resulted in Armadillo being truncated at positions corresponding to the carboxyl-terminal region of plakoglobin. Although embryos with these mutations survived a relatively long time and synthesized enough of the truncated Armadillo for it to be readily detected in immunoblots, the mutations were lethal. This underscores the importance of the carboxyl terminus of armadillo family members, although its function is unknown.

The diagram shown in Fig. 1assigns functions to some regions of plakoglobin. A domain of plakoglobin needed for association with alpha-catenin was mapped to amino acids 115-161, roughly corresponding to the first repeat. Since the truncated plakoglobins are probably competing with alpha-catenin's other partners (such as beta-catenin), these data suggest that sequences near repeat 1 play a major role in the recognition of alpha-catenin.

In contrast to the small region identified in the alpha-catenin studies, our data suggest a gradual decrease in the ability of plakoglobin and N-cadherin to associate as plakoglobin is shortened from the end of repeat 8 (amino acid 458) to the end of repeat 6 (amino acid 375). Such a large region of association suggests multiple protein-protein interactions are required for plakoglobin to interact with N-cadherin. Immunofluorescence showed that constructs retaining at least 458 amino acids localized to cell borders, which suggests they can successfully compete with beta-catenin for binding to N-cadherin. However, we have preliminary data, based on washing immunoprecipitations more stringently, that suggests COOH-terminally truncated plakoglobins are not as stably associated with N-cadherin as are full-length plakoglobin or beta-catenin. Studies are in progress to more completely characterize the stability of the interaction of truncated plakoglobins with N-cadherin.

As mentioned above, there are regions of plakoglobin whose function was not revealed in our assays. A number of proteins have been localized to the plaques of adherens junctions (for a review see (41) ). Perhaps beta-catenin and plakoglobin have roles in recruiting other junctional components. It would not be surprising if plakoglobin and beta-catenin have different affinities for these other molecules, a feature that could play a role in signaling events. Conditions must be developed to preserve the interactions of these other adherens junction components in order to study their interactions with the cadherin-catenin complex; such studies are ongoing in our laboratory.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM51188, by grants from the Ohio Chapters of the American Cancer Society and the American Heart Association, and by the Ohio Board of Regents. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These authors contributed equally to this study.

To whom correspondence should be addressed: Dept. of Biology, 2801 W. Bancroft St., University of Toledo, Toledo, OH 43606. Tel.: 419-537-4918; Fax: 419-537-7737.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Drs. Werner W. Franke, John Hemperly, and Nicolas Fasel for reagents; Drs. Pamela J. Jensen and Karen A. Knudsen for helpful comments; Dr. Karen A. Knudsen for help with densitometry; and James K. Wahl III for excellent technical help.


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