©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Desmosomal Cadherin Binding Domains of Plakoglobin (*)

(Received for publication, February 5, 1996; and in revised form, February 27, 1996)

Lora L. Witcher (1) Russell Collins (1) Sailaja Puttagunta (1) Susan E. Mechanic (1) Marylinn Munson (2) Barry Gumbiner (2) Pamela Cowin (1)(§)

From the  (1)Department of Cell Biology, Ronald O. Perelman Department of Dermatology and Kaplan Cancer Center, New York University Medical Center, New York, New York 10016 and the (2)Memorial Sloan Kettering Cancer Center, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Plakoglobin is a major component of both desmosomes and adherens junctions. At these sites it binds to the cytoplasmic domains of cadherin cell-cell adhesion proteins and regulates their adhesive and cytoskeletal binding functions. Plakoglobin also forms distinct cytosolic protein complexes that function in pathways of tumor suppression and cell fate determination. Recent studies in Xenopus suggest that cadherins inhibit the signaling functions of plakoglobin presumably by sequestering this protein at the membrane and depleting its cytosolic pool. To understand the reciprocal regulation between desmosomal cadherins (desmoglein and desmocollin) and plakoglobin, we have sought to identify the binding domains involved in the formation of these protein complexes. Plakoglobin comprises 13 central repeats flanked by amino-terminal and carboxyl-terminal domains. Our results show that repeats 1-4 are involved in binding desmoglein-1. In contrast, the interaction of plakoglobin with desmocollin-1a is sensitive to deletion of either end of the central repeat domain. The binding sites for two adherens junction components, alpha-catenin and classical cadherins, overlap these sites. Competition among these proteins for binding sites on plakoglobin may therefore account for the distinct composition of adherens junctions and desmosomes.


INTRODUCTION

Adherens junctions and desmosomes connect cells together and anchor the cytoskeleton to the plasma membrane. Desmogleins (Dsg) (^1)and desmocollins (Dsc) form the adhesive transmembrane components of desmosomes, whereas classical cadherins perform this function in adherens junctions. Their genes display a highly conserved and unusual genomic structure, suggesting that they arose from a common ancestor(1, 2, 3, 4) . Moreover, their protein products share considerable sequence similarity and a common repetitive organization of their ectodomain (for references see review in (5) ). Recent crystal structure analyses of the amino-terminal extracellular repeat of N-cadherin have shown that the sequences that are highly conserved bind calcium ions and mediate cadherin dimerization in the plane of the membrane(6, 7) . Desmosomal and classical cadherins differ principally in the sequences of their cytoplasmic segment (for references see reviews in (5) and (8) ). Within this domain, however, they share a small region of sequence similarity that interacts with plakoglobin, an 86-kDa cytoplasmic protein(9, 10, 11, 12, 13) .

Plakoglobin is the only component common to both desmosomes and adherens junctions; as such it is uniquely positioned to provide a pivotal point of control in the adhesive mechanism(8, 14) . At adherens junctions, plakoglobin forms a link between classical cadherins and alpha-catenin, which in turn connects the complex to the actin cytoskeleton both directly and through alpha-actinin(15, 16, 17, 18) . The role of plakoglobin at desmosomes is less clear; however, it is essential for desmosome formation and may promote intermediate filament association with the membrane(19, 20, 21) .

Recent studies have revealed a wide range of cellular functions for plakoglobin in addition to its role in adhesive cell-cell junctions. For example, both plakoglobin and its close relative beta-catenin are implicated in pathways controlling cell proliferation, differentiation, and motility by their association in the cytosol with APC, the product of a tumor suppressor gene linked to colon cancer(15, 22, 23, 24, 25) . Furthermore, recent studies suggest their involvement in developmental pathways. Armadillo, the Drosophila homologue of plakoglobin and beta-catenin, participates in a signaling pathway that determines segment polarity via post-transcriptional up-regulation in response to wingless expression(26, 27) . A similar up-regulation of plakoglobin and beta-catenin can be produced in vertebrate cells by expression of Wnt-1, a homologue of wingless(28, 29) . Ectopic expression of plakoglobin, beta-catenin, or Wnt-1 in Xenopus embryos each produce duplication of the embryonic axis, suggesting that they operate in a common conserved signaling pathway(30, 31, 32, 33) . Intriguingly, in the case of plakoglobin, this phenotype is suppressed by co-expression of the Dsg1 cytoplasmic domain, indicating that desmosomal cadherins play a significant role in modulating plakoglobin's signaling functions(33) . As a step toward understanding the reciprocal regulation between these proteins, we have sought to define the regions of plakoglobin involved in their associations. Our results show that the site of Dsg1 interaction is relatively discrete, involving the first three to four repeats of the central domain of plakoglobin. In contrast, the association of Dsc1a with plakoglobin is sensitive to even small deletions at either end of the central repeat domain.


EXPERIMENTAL PROCEDURES

cDNA Constructs

The complete bovine Dsg1 cDNA was constructed in the Bluescript SK vector (Stratagene, La Jolla, CA) from three overlapping partial cDNAs(9, 34) . The complete cDNA was fully sequenced and matched that of GenBank data base (GB) accession number X57784. (^2)A 4,571-base pair fragment including the entire coding sequence was subcloned into the HindIII site of the CDM8 mammalian expression vector (Invitrogen, San Diego, CA). DNA sequences encoding the cytoplasmic domain of bovine Dsg1 (amino acid residues 523-994) and bovine Dsg1-DeltaC (amino acid residues 523-689, lacking the region that permits plakoglobin association(9, 21) ) were generated by PCR using the 5` oligonucleotide CCGGAATTCGACTGTGGAGGCGCCCCT and the 3` oligonucleotides CGCGGATCCTTACTTGGTATATTGAACTGT and CGCGGATCCTTACTGGCAGAAGTAACTTTC, respectively. These PCR products were subcloned into the EcoR1/BamHI sites of the pGAD424 vector (Clonetech, Palo Alto, CA) in frame with the activation domain of the GAL4 transcription factor.

Bovine Dsc1a was constructed by replacing the 488-base pair MscI-NheI fragment (bases 2516-2904) of Dsc1b (GB accession number M61750) (35) with an reverse transcription-PCR generated 443-base pair alternatively spliced fragment from the corresponding region of Dsc1a. Sequencing of the PCR-generated segment and flanking sequences confirmed identity to GB accession number X56967. A HindIII-NsiI fragment including the entire Dsc1a coding sequence was subcloned into the HindIII/BstXI site of CDM8.

Human plakoglobin (GB accession number Z68228) (36) was cloned into the EcoR 1 site of the p163/7 mammalian expression vector(37) . The complete coding sequence of human plakoglobin generated by PCR using the oligonucleotides CCGGAATTCATGGAGGTGATGAACCTGATG and CCGGAATTCCTAGGCCAGCATGTGGTC was subcloned into the EcoR1 site of the pGBT9 vector (Clonetech, Palo Alto, CA) in frame with the GAL4 DNA-binding domain. All plakoglobin mutants were constructed by PCR using 5` primers consisting of a three-base spacer followed by an EcoR1 restriction site and six bases conforming to Kozak's rules for the optimal sequence of an initiating methionine (CCGGAATTCGCCGCCATG), followed by 15 nucleotides encoding plakoglobin sequence beginning with the amino acid indicated (Fig. 1). For some mutants, the sequence GAGCAGAAGCTGATCAGCGAGGAGGACCTG, encoding the epitope EQKLISEEDL derived from c-myc was introduced between the initiating ATG codon and the plakoglobin sequence. 3` primers comprised a three-base spacer, an EcoR1 restriction site, a stop codon, and 15 nucleotides encoding the plakoglobin sequence ending with the amino acids indicated (Fig. 1). The deleted forms of human plakoglobin described above (Fig. 1) were subcloned into the EcoR1 site of p163/7 and pGBT9 vectors.


Figure 1: Schematic representation of deletion mutants of human plakoglobin. The diagram at the top represents the amino- and carboxyl-terminal variable domains of plakoglobin flanking the 12 central 42-amino acid arm repeat regions of plakoglobin. The lines below show deletion mutants. The numbers indicate the initial or final amino acids (according to GenBank sequence Z68228) encoded by each construct.



All clones generated by PCR were fully sequenced by the dideoxy chain termination method. All constructs were found to have sequences identical to the parent cDNA clones with the exception of the plakoglobin mutant DeltaC, which contained a single mutation resulting in an alteration in residue 580 from isoleucine to serine. The product of this construct showed no significant difference from that of the original full length cDNA in its ability to associate with Dsg1 or Dsc1a.

Xenopus alpha-catenin (GB accession number U47624) was cloned by screening a Xenopus stage 17 gt11 library with mouse alpha-E-catenin cDNA. A full-length clone containing the complete open reading frame was subcloned into the pCS2 vector containing a CMV promoter, which was a kind gift of the Hal Weintraub laboratory. The deduced amino acid sequence shows 93% identity to murine alpha-catenin.

Yeast Two-hybrid

Yeast strains SFY526 and HF7c (Clonetech, Palo Alto, CA) were rendered competent by the lithium acetate/polyethylene glycol procedure, cotransformed with 1 µg of each of a pair of DNA constructs and selected by growth on SD plates lacking tryptophan and leucine (SD-trpleu) (38) . HF7c colonies were picked and streaked onto (SD-trpleu) plates and replica plated onto plates also deficient in histidine (SD-trpleuHis) with or without 20 mM 3-aminotriazole and tested for growth. Transformants of both strains were grown in liquid culture (SD-trpleu) at 30 °C to an A of 1.0. The cells were pelleted, lysed, normalized for protein concentration, and assayed using o-nitrophenyl-beta-D-galactoside as a substrate to quantitate beta-galactosidase activity(38) .

Transfection and Immunoprecipitation

Human kidney cells transformed with SV40 T-antigen (293T) were maintained in a 10% CO(2) atmosphere in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 100 units/ml penicillin and streptomycin, and 2 mML-glutamine. Cells grown to 25% confluence in 35-mm dishes were transfected with 5 µg of each DNA construct by the standard calcium phosphate method. 24-48 h later cells were rinsed and cultured for 60 min in 1 ml of methionine/cysteine-free Dulbecco's modified Eagle's medium containing 1% dialyzed fetal calf serum. The cells were then incubated in the same medium with EXPRESS-methionine/cysteine protein labeling mix (DuPont NEN): 75-125 µCi/ml for 3 h to overnight or 1 mCi/ml for 15 min for pulse-chase. Cells were rinsed and chased in Dulbecco's modified Eagle's medium containing 20 mM unlabeled methionine. The cells were lysed and proteins were extracted in 1 ml 25 mM Tris, pH 7.4, 3 mM EDTA, 0.2% Aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5% Triton X-100 (TX-100) for 30 min at 4 °C. Insoluble debris was pelleted for 15 min at 10,000 times g, and the supernatant was precleared for 1 h with 20 µl of 50% protein A-Sepharose beads suspended in phosphate-buffered saline. After removal of these beads, the extract was rotated for 2 h at 4 °C with 5 µl of specific antibody, 20 µl of beads, and, where necessary, 10 µg of rabbit anti-mouse IgG. The beads were pelleted and washed six times in wash buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 M NaCl, 15 mM Tris, pH 7.5, 25 mM KCl, 2 mM EGTA, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.2% aprotinin, 0.1 mM dithiothreitol), then boiled in 40 µl of Laemmli sample buffer, subjected to 8-10% SDS-polyacrylamide gel electrophoresis, and processed for autoradiography. Polyclonal antibodies to Dsg1 and plakoglobin were raised and characterized as described(39) . Anti-myc epitope-tag specific antibodies were a gift of Dr. Harold Varmus (UCSF, San Francisco, CA); PECAM cDNA and Hec7 antibodies were a gift of Dr. Bill Mueller (Rockefeller, New York, NY).


RESULTS

Yeast Two-hybrid Analyses Localize the Desmoglein Binding Site to the First Three Repeats of Plakoglobin

The ability of full-length and deleted forms of plakoglobin to interact with the complete Dsg1 cytoplasmic domain (DSG1-pGAD424) was tested by measuring beta-galactosidase activity (Table 1, second and fourth columns). Controls consisted of yeast expressing these mutants together with the Dsg1 cytoplasmic domain lacking the sequence necessary for plakogobin association (DSG1DeltaC-pGAD424) (Table 1, third and fifth columns)(9, 21) . Yeast expressing full-length plakoglobin and the Dsg1 cytoplasmic domain produced 10-fold higher beta-galactosidase activity than controls. Deletion of the amino-terminal (head) and/or carboxyl-terminal (tail) domains of plakoglobin minimally affected association of these proteins. Yeast expressing mutant plakoglobin lacking the head and first two repeats (DeltaN-2) showed reduced beta-galactosidase activity; no activity was found in those mutants further deleted toward the carboxyl terminus (DeltaN-6, DeltaN-9, DeltaN-13). Similarly, mutants progressively deleted from the carboxyl terminus gave approximately equal levels of beta-galactosidase activity until repeats 1-3 were removed (compare Delta4-C with Delta1-C).



The ability of these fusion proteins to interact, thereby activating transcription of a HIS3 reporter gene under the control of the GAL1 promoter, was also tested in the HF7c strain by growth on his plates (SD-trpleuhis). All cotransformants, including controls, grew on these plates due to basal levels of HIS3 expression (data not shown). Incorporation of 20 mM 3-aminotriazole, however, reduced this background significantly in all controls except those expressing full-length plakoglobin. Growth in the presence of 20 mM 3-aminotriazole was observed in cells expressing the full-length plakoglobin construct as well as with mutants (Delta4-C, Delta8-C, Delta11-C, DeltaC, DeltaN/DeltaC, DeltaN, DeltaN-2) but not with (Delta1-C, DeltaN-6, DeltaN-9, DeltaN-13) (Fig. 2).


Figure 2: Yeast two-hybrid growth assay of Dsg1 and plakoglobin interactions. Growth of HF7c colonies co-expressing plakoglobin mutants (as indicated) fused to the DNA-binding domain and the desmoglein cytoplasmic domain (DSG) or a truncated form lacking the plakoglobin binding region (DSGDeltaC) fused to the GAL4 activation domain is shown. Growth on SD-trp-leu-his plates is shown on the left and replicas on SD-trp-leu-his+ 20 mM 3-aminotriazole on the right.



Desmoglein and Desmocollin Co-precipitate Different Amounts of Plakoglobin

The interaction of desmosomal cadherins with plakoglobin mutants was also assessed by cotransfection and coimmunoprecipitation. The specificity of the antibodies was first tested by immunoprecipitating Dsg1, Dsc1a, and plakoglobin from 0.5% TX-100 soluble extracts of 293T cells transiently transfected with cDNAs encoding each protein (Fig. 3). All antibodies recognized their cognate antigens and showed no cross-reaction with other overexpressed components (Fig. 3). We next tested that overexpression of two proteins does not, of itself, give rise to spurious complexes by showing that PECAM, a cell adhesion molecule that colocalizes with plakoglobin in the endothelial junction, does not co-immunoprecipitate with plakoglobin (Fig. 4, lanes 3 and 4). In contrast, a complex of Dsg1 and plakoglobin could be coimmunoprecipitated from TX-100 soluble extracts of 293T cells co-expressing both proteins by antibodies specific for either component (Fig. 4, lanes 5 and 9). When such cells were pulse-chased, anti-plakoglobin antibody co-immunoprecipitated Dsg1 precursor and mature Dsg1 (Fig. 4, lanes 6-9). When cells transfected with 5 µg of plakoglobin together with 5 µg of Dsg1 or Dsc1a were immunoprecipitated with the same anti-desmosomal cadherin antibody, a 2-fold difference in expression of the transmembrane components was observed (Fig. 4, lanes 10 and 11). After correction for this fact, 7-fold less plakoglobin was found associated with Dsc1a than with Dsg1, even though equal amounts of plakoglobin were expressed. When the amount of Dsc1a cDNA added to the transfection was titrated (Fig. 4, lanes 11-13) with all other parameters constant, the amount of plakoglobin in the complex increased in proportion to the amount of Dsc1a immunoprecipitated, demonstrating that the relative stiochiometry of the complex remained constant.


Figure 3: Specificity of antibodies. Autoradiogram showing TX-100-soluble extracts of metabolically labeled 293T cells transfected with control CDM8 vector lacking an insert (lanes 1, 5, and 9), bovine Dsc1a (lanes 2, 6, and 10), bovine Dsg1 (lanes 3, 7, and 11), and human plakoglobin (Pg, lanes 4, 8, and 12), which were divided equally into three aliquots and precipitated with the following antibodies: anti-plakoglobin (anti-Pg) (lanes 1-4), anti-desmosomal cadherin (anti-Dsc1a/Dsg1) (lanes 5-8), and anti-Dsg1 (lanes 9-12). Note all antibodies recognize their cognate antigens and show no cross-reaction with other overexpressed proteins.




Figure 4: Immunoprecipitation of PECAM, Dsg1, Dsc1a, and Plakoglobin Complexes. Autoradiograms of immunoprecipitations of TX-100 soluble extracts of metabolically labeled 293T cells transfected with: PECAM (lanes 1 and 2) and cotransfected with PECAM and plakoglobin (lanes 3 and 4); Dsg1 and plakoglobin (lanes 5-10); and Dsc1a and plakoglobin (lanes 11-13) precipitated with monoclonal antibody: HEC7-specific for PECAM (lanes 1 and 3); and polyclonal antibodies: anti-plakoglobin (alphaPg, lanes 2, 4, and 6-9); anti-Dsg1 (alphaDsg, lane 5) and anti-desmosomal cadherin antibody (anti-Dsg/Dsc, lanes 10-13). Note the lack of interaction between PECAM and plakoglobin (lanes 3 and 4) but complex formation between Dsg 1 and plakoglobin (lane 5). Lanes 6-9 show cells metabolically labeled for 15 min followed by chase periods indicated above the lanes. Anti-plakoglobin antibodies coimmunoprecipitate plakoglobin complexed to the desmoglein precursor (lane 6) and mature Dsg1 (lane 9). Lanes 10-13 show immunoprecipitates with a constant amount of anti-Dsg1/Dsc1a from 293T cells transiently cotransfected with 5 µg of plakoglobin (lanes 10-13) together with 5 µg of Dsg1 (lane 10) or 5, 2.5, and 10 µg, respectively, of Dsc1a indicated above the lanes. Note that equivalent amounts of desmoglein and desmocollin coprecipitate different amounts of plakoglobin and that the stoichiometry of the desmocollin complex remains constant.



Desmoglein Association Requires the Third Repeat of Plakoglobin and Is Further Enhanced by the First and Second Repeats

The plakoglobin deletion mutants previously used in the yeast two-hybrid experiments were subcloned into a mammalian expression vector, co-transfected into 293T cells with the full-length Dsg1 construct, and tested for the ability of their products to co-immunoprecipitate. Association of Dsg1 was observed with full-length (Fig. 5, lanes 1 and 1`) and headless plakoglobin (DeltaN) (Fig. 5, lanes 2 and 2`). Further deletion of repeats 1 and 2 (DeltaN-2), however, led to a significant drop in interaction (Fig. 5, lanes 3 and 3`), and mutants lacking the first six repeats or more showed no detectable association (Fig. 5, lanes 4`, 4, 5`, and 5). Additional deletion mutants were created to further delineate the binding site. Deletion of the first four repeats (DeltaN-4) resulted in loss of association (Fig. 5, lanes 8 and 8`). Sequential deletion of the head (Fig. 5, compare lanes 1` and 2`), head and first repeat (DeltaN-1), and head and first two repeats (DeltaN-2) (Fig. 5, compare lanes 6 and 7 and lanes 6` and 7`) progressively impairs Dsg1 binding. All mutants sequentially deleted from the carboxyl terminus showed significant association with Dsg1 (Fig. 6, lanes 1-5, 9, and 9`) up to the point of removal of the third repeat (Fig. 6, lanes 6-8 and 6`-8`). In summary, these data show that the head and first three repeats are sufficient for desmoglein association, that the third repeat is essential, and that the head and repeats 1 and 2 enhance this interaction. In the absence of the head and first four repeats the rest of the protein is unable to bind Dsg 1.


Figure 5: Co-immunoprecipitation of Dsg1 with full-length and mutant forms of plakoglobin sequentially deleted from the amino terminus. Autoradiogram of immunoprecipitations with the anti-plakoglobin (Pg) antibody (lanes 1-8) or anti-Dsg1 antibody (lanes 1`-8`) of TX-100-soluble fractions of metabolically labeled 293T cells transiently cotransfected with Dsg1 and the plakoglobin constructs indicated above the lanes.




Figure 6: Co-immunoprecipitation of Dsg1 with full-length and mutant forms of plakoglobin sequentially deleted from the carboxyl terminus. Autoradiogram of immunoprecipitates with anti-Dsg1 antibody (lanes 1-5 and 6`-9`) and anti-myc antibody (lanes 6-9 and 12-14) of TX-100 soluble fractions of metabolically labeled 293T cells transiently cotransfected with the plakoglobin constructs indicated above the lanes and either Dsg1 (lanes 1-9 and 6`-9`) or alpha-catenin (lanes 10-14).



The Plakoglobin Binding Sites for Desmoglein and alpha-Catenin Partially Overlap

To determine the proximity of this site to those variously reported for alpha-catenin, we show that plakoglobin mutants lacking the head or first repeat (DeltaN, Delta1-C) do not bind alpha-catenin (Fig. 6, lanes 11 and 12) confirming the work of others(15, 40) . We extend these results to show that the head and first repeat together are sufficient for association with alpha-catenin (compare Delta1-C with Delta2-C, Fig. 6, lanes 12 and 13). Thus, the sites for desmoglein and alpha-catenin lie in close proximity and head and repeat 1 contribute toward two distinct interactions of plakoglobin, providing the binding site for alpha-catenin and enhancing the interaction of Dsg1.

Desmocollin Association with Plakoglobin Requires Both Ends of the Plakoglobin Repeat Region

The plakoglobin mutants were co-transfected together with Dsc1a and immunoprecipitated with the anti-desmosomal cadherin antibody (Fig. 7). Deletion of the head (DeltaN) and/or tail (DeltaN/DeltaC, DeltaC) or head and first repeat (DeltaN-1) produced little effect on Dsc1a interaction (Fig. 7, lanes 2, 3, 7, and 8). However, further amino-terminal and all carboxyl-terminal deletions of the repeat region led to loss of interaction, implicating both ends of the repeats in Dsc1a association (Fig. 7, lanes 4-6 and 9-12).


Figure 7: Co-immunoprecipitation of Dsc1a with full-length and mutant forms of plakoglobin from transiently transfected 293T cells. Autoradiogram of immunoprecipitates with anti-desmosomal cadherin antibody (anti-Dsg/Dsc) of TX-100 soluble fractions of metabolically labeled 293T cells transiently cotransfected with the plakoglobin constructs indicated above the lanes and Dsc1a. Arrowheads indicate where the plakoglobin (Pg) mutants would migrate as judged from sister gels (data not shown).




DISCUSSION

Plakoglobin belongs to a family of functionally diverse proteins (41, 42) that contain varying numbers of 42-amino acid sequences termed ``arm'' repeats after Armadillo, the protein in which they were first observed(26, 43) . Five of these proteins (plakoglobin, beta-catenin, Armadillo, desmosomal band six protein and p120) associate with cadherins, but plakoglobin is the only one known to associate with both classical cadherins at adherens junctions and desmosomal cadherins at desmosomes(9, 10, 11, 12, 13, 44, 45, 46, 47, 48) . Definition of its interactions are therefore crucial to our understanding of the structure and function of the two major vertebrate cell-cell adhesive junctions.

Plakoglobin is also found in the cytoplasm, where it forms a homodimer of unknown function (14, 49) (for alternative view, see (24) ); like beta-catenin, plakoglobin can form cytosolic complexes with the APC tumor suppressor gene product, implicating it in growth control(15, 22, 23, 24, 25) . Studies in Xenopus suggest that cytosolic pools of plakoglobin participate in the transmission of developmental patterning signals that, intriguingly, are suppressed by Dsg1 expression(33) . From this it has been inferred that desmosomal cadherins influence signaling pathways governing patterning by sequestering plakoglobin at the membrane, and it is reasonable to surmise that they may also affect plakoglobin's cytosolic role in growth control. Thus, definition of cadherin-plakoglobin interactions is important for our understanding of the connections between these processes and cell adhesion.

Our results show that plakoglobin binds to the precursor and mature forms of Dsg1, analogous to the early association reported to occur between beta-catenin and E-cadherin(50) . The plakoglobin binding site for Dsg1 involves a relatively discrete region of amino-terminal repeats. Specifically, our results indicate that the head and first three repeats are sufficient for Dsg1 interaction. Amino-terminally deleted mutants show that although removal of the head domain lessens binding slightly, progressive removal of the first and second repeats severely compromises Dsg1 association, and further deletion of the third and fourth repeats eliminates interaction. Taken together, these data suggest that the head and repeats 1 and 2 cooperate to enhance Dsg1 association with the third and possibly fourth repeats. The first and third repeats of plakoglobin are very similar to those of beta-catenin and Armadillo, so it is rather surprising to find them engaged in an association considered unique to plakoglobin. The second repeat, however, is less conserved and may therefore impart specificity to the complex.

In contrast to the localized region of plakoglobin sufficient to bind Dsg1, Dsc1a association is impaired by deletions of either the carboxyl-terminal repeats or repeat 2. These results are compatible with two possibilities. In the first model, Dsc1a association would require simultaneous binding to both ends of the repeat domain. Alternatively, the interaction may occur over a region between repeats 2-13 in a manner that is conformationally sensitive to deletions at either end. Interestingly, this region overlaps binding sites for E- and N-cadherin(15, 40) . Likewise, the Dsg1 binding site lies next to and may involve that for alpha catenin(15, 40) . It is therefore likely that binding of desmosomal cadherins will sterically hinder the association of other partners, accounting for the distinct complexes formed by plakoglobin with alpha-catenin and classical cadherins at adherens junctions and with Dsg1 and Dsc-1a at desmosomes.

Considerably more plakoglobin binds to Dsg1 than to Dsc1a. Furthermore, overexpression of the Dsg1 cytoplasmic domain produces a dominant negative effect on desmosome formation, whereas expression of the analogous region of Dsc1a does not(21) . Given the capacity of Dsg1 for binding plakoglobin and the significance of this association for both the formation of desmosomes and suppression of cytosolic roles of plakoglobin, naturally occurring mutations in the interactive domains of Dsg1 and plakoglobin are likely to have marked consequences not only for cell adhesion but also for proliferation and differentiation.

The interactions of plakoglobin with its partners are similar in several respects to those described for another arm protein, the regulatory A subunit of phosphatase 2A (PP2A-A)(51) . For example, the catalytic C subunit binds to a discrete site at one end of PP2A-A, as do Dsg1 and alpha-catenin on plakoglobin. There is clear evidence for competition and cooperation among some of the binding partners of PP2A, whereas others bind independently(51) . Our results show that Dsg1 and Dsc1a can interact with plakoglobin separately. Whether they can bind simultaneously to a single plakoglobin molecule awaits the development of Dsc-1-specific antibodies. This question relates to an important potential role for plakoglobin in organizing cadherins into an adhesive zipper.

A model has been proposed for PP2A-A in which each repeat forms two amphipathic helices that interact along their hydrophobic faces(51) . Consecutive helix pairs stack to form a concertina-like rod with interrepeat and intrarepeat loops projecting from opposite surfaces of the rod. A similar arrangement of secondary structures may exist for plakoglobin, because residues required to form the putative amphipathic helices are conserved in all arm proteins(42) . Despite the potential similarities between PP2A-A and plakoglobin in their secondary structures and topology of their intermolecular associations, biochemical analyses suggest that they differ in their tertiary structure; PP2A-A is a rod-like molecule, whereas plakoglobin behaves as a globular protein(49, 51) . As Dsc1a association requires both ends of the repeat domain of plakoglobin, one can envisage a horseshoe, rather than a rod-like shape for plakoglobin. Dsc1a would bind directly to both ends or to a central conformationally dependent binding pocket created by intramolecular association of the ends. Clearly these models are highly speculative, and crystal structure analyses to resolve the structure of plakoglobin are currently underway.

The emerging model of arm proteins is one in which individual repeats provide the principal binding sites for particular partners and neighboring repeats contribute to the specificity and stability of these associations. Partners recognize partially overlapping regions and may compete or cooperate for binding. Thus distinct tripartite complexes with different functions are formed, integrating cellular processes as diverse as adhesion, proliferation, and differentiation. Definition of the binding sites on plakoglobin now permits the development of minimally altered mutants deficient in cadherin binding functions to test the outcome of uncoupling membrane and cytosolic functions of this protein.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM47429 (to P. C.) and GM37432 (to B. G.). 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.

§
To whom correspondence should be addressed: Dept. of Cell Biology, Ronald O. Perelman Dept. of Dermatology and Kaplan Cancer Center, New York University Medical Center, 550 First Ave., New York, NY 10016. Tel.: 212-263-8715/6915; Fax: 212-263-8139.

(^1)
The abbreviations used are: Dsg, desmoglein; Dsc, desmocollin; GB, GenBank; PCR, polymerase chain reaction; PECAM, platelet-endothelial cell adhesion molecule; PP2A, protein phosphatase 2A; TX-100, Triton X-100; SD, synthetic media + 2% dextrose.

(^2)
The cDNA sequences referred to in the text are: bovine desmoglein 1 GB, X57784; bovine desmocollin 1a GB, X56967; bovine desmocollin 1b GB, M61750; Human plakoglobin GB, Z68228; Xenopus alpha catenin GB, U47624.


ACKNOWLEDGEMENTS

We thank Erik Levy and Dr. Alan Frey for critically reading the manuscript, Dr. Gert Kreibich for insightful comments, and Dr. Warren Jelinek for advice with yeast two-hybrid experiments.


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