Different epithelial intercellular junctions contain distinct complexes incorporating plakoglobin. In adherens junctions, plakoglobin interacts with two molecules, the transmembrane adhesion protein of the cadherin family (e.g. E-cadherin) and alpha-catenin. The latter is thought to anchor the cadherin-plakoglobin complex to the cortical actin cytoskeleton. In desmosomes, plakoglobin forms a complex with desmosomal cadherins, either desmoglein (Dsg) or desmocollin (Dsc), but not with alpha-catenin. To further understand the structure and assembly of the plakoglobin-cadherin complexes we analyzed amino acid residues involved in plakoglobin-Dsg interactions using alanine scanning mutagenesis. Previously, we have shown that plakoglobin interacts with a 72 amino acid-long cytoplasmic domain (C-domain) that is conserved among desmosomal and classic cadherins. In this paper, we show that a row of the large hydrophobic residues located at the C-terminal portion of the Dsg C-domain is indispensable for interaction with plakoglobin. To study a reciprocal site we expressed plakoglobin (MPg) or its mutants tagged by 6 myc epitope in epithelial A-431 cells. Using sucrose gradient centrifugation and subsequent co-immunoprecipitation, MPg was found to be efficiently incorporated into the same type of complexes as endogenous plakoglobin. A major pool of Dsg-plakoglobin complexes sedimented at 8S and exhibited a 1:1 stoichiometry. Using alanine scanning mutagenesis and the co-immunoprecipitation assay we identified nine hydrophobic amino acids within the arm repeats 1–3 of plakoglobin, that are required for binding to Dsg and Dsc. Eight of these amino acids also participate in the interaction with alpha-catenin. No mutations were found to reduce the affinity of plakoglobin binding to E-cadherin. These data provide direct evidence that the same hydrophobic plakoglobin surface is essential for mutually exclusive interaction with distinct proteins such as alpha-catenin and desmosomal cadherins.

Aberle
H.
,
Butz
S.
,
Stappert
J.
,
Weissig
H.
,
Kemler
R.
,
Hoschuetzky
H.
(
1994
).
Assembly of the cadherin-catenin complex in vitro with recombinant proteins.
J. Cell Sci
107
,
3655
3663
Aberle
H.
,
Schwartz
H.
,
Hoschuetzky
H.
,
Kemler
R.
(
1996
).
Single amino acid substitutions in proteins of the Armadillo gene family abolish their binding to-catenin.
J. Biol. Chem
271
,
1520
1526
Bierkamp
C.
,
McLaughlin
K. J.
,
Schwarz
H.
,
Huber
O.
,
Kemler
R.
(
1996
).
Embryonic heart and skin defects in mice lacking plakoglobin.
Dev. Biol
180
,
780
785
Chitaev
N. A.
,
Leube
R. E.
,
Troyanovsky
R. B.
,
Eshkind
L. G.
,
Franke
W. W.
,
Troyanovsky
S. M.
(
1996
).
The binding of plakoglobin to desmosomal cadherins: Patterns of binding sites and topogenic potential.
J. Cell Biol
133
,
359
369
Chitaev
N. A.
,
Troyanovsky
S. M.
(
1997
).
Direct Ca2+-dependent heterophilic interaction between desmosomal cadherins, desmoglein and desmocollin, contributes to cell-cell adhesion.
J. Cell Biol
138
,
193
201
Cowin
P.
,
Kapprell
H.-P.
,
Franke
W. W.
,
Tamkun
J.
,
Hynes
R. O.
(
1986
).
Plakoglobin: A protein common to different kinds of intracellular adhering junctions.
Cell
46
,
1063
1073
Cowin
P.
,
Burke
B.
(
1996
).
Cytoskeleton-membrane interactions.
Curr. Opin. Cell Biol
8
,
56
65
Fagotto
F.
,
Funayama
N.
,
Gluck
U.
,
Gumbiner
B. M.
(
1996
).
Binding to cadherins antagonizes the signalling activity of-catenin during axis formation in Xenopus.
J. Cell Biol
132
,
1105
1114
Finnemann
S.
,
Mitrik
I.
,
Hess
M.
,
Otto
G.
,
Wedlich
D.
(
1997
).
Uncoupling of XB/U-cadherin-catenin complex formation from its function in cell-cell adhesion.
J. Biol. Chem
272
,
11856
11862
Franke
W. W.
,
Goldschmidt
M. D.
,
Zimbelmann
R.
,
Mueller
H. M.
,
Schiller
D. L.
,
Cowin
P.
(
1989
).
Molecular cloning and amino acid sequence of human plakoglobin, the common junctional plaque protein.
Proc. Nat. Acad. Sci. USA
86
,
4027
4031
Garrod
D.
,
Chidgey
M.
,
North
A.
(
1996
).
Desmosomes: differentiation, development, dynamics and disease.
Curr. Opin. Cell Biol
8
,
670
678
Gumbiner
G. M.
(
1996
).
Cell adhesion: the molecular basis of tissue architecture and morphogenesis.
Cell
84
,
345
357
Hinck
L.
,
Näthke
I. S.
,
Papkoff
J.
,
Nelson
W. J.
(
1994
).
Dynamics of cadherin/catenin complex formation: novel protein interactions and pathways of complex assembly.
J. Cell Biol
125
,
1327
1340
Horst
M.
,
Hart
N.
,
Hasilik
A.
(
1991
).
Biosynthesis of glycosylated human lysozyme mutants.
J. Biol. Chem
266
,
13914
13919
Huber
O.
,
Bierkamp
C.
,
Kemler
R.
(
1996
).
Cadherins and catenins in development.
Curr. Opin. Cell Biol
8
,
685
691
Huber
O.
,
Krohn
M.
,
Kemler
R.
(
1997
).
A specific domain in-catenin mediates binding to -catenin or plakoglobin.
J. Cell Sci
110
,
1759
1765
Jou
T.-S.
,
Stewart
D. B.
,
Stappert
J.
,
Nelson
W. J.
,
Marrs
J. A.
(
1995
).
Genetic and biochemical dissection of protein linkages in the cadherin-catenin complex.
Proc. Nat. Acad. Sci. USA
92
,
5067
5071
Klymkowsky
M. W.
,
Parr
B.
(
1995
).
A glimpse into the body language of cell: The intimate connection between cell adhesion and gene expression.
Cell
83
,
5
8
Koch
P. J.
,
Goldschmidt
M. D.
,
Walsh
M. J.
,
Zimbelmann
R.
,
Franke
W. W.
(
1991
).
Complete amino acid sequence of the epidermal desmoglein precursor polypeptide and identification of a second type of desmoglein gene.
Eur. J. Cell Biol
55
,
200
208
Koch
P. J.
,
Franke
W. W.
(
1994
).
Desmosomal cadherins: another growing multigene family of adhesion molecules.
Curr. Opin. Cell Biol
6
,
682
687
Knudsen
K. A.
,
Wheelock
M. J.
(
1992
).
Plakoglobin, or an 83-kD homologue distinct from-catenin, interacts with E-cadherin and N-cadherin.
J. Cell Biol
118
,
671
679
Korman
N. J.
,
Eyre
R. W.
,
Klaus-Kovtun
V.
,
Stanley
J. R.
(
1989
).
Demonstration of an adhering-junction molecule (plakoglobin) in the autoantigens of pemphigus foliaceous and pemphigus vulgaris.
New Eng. J. Med
321
,
631
635
Kowalczyk
A. P.
,
Palka
H. L.
,
Luu
H. H.
,
Nilles
L. A.
,
Anderson
J. E.
,
Wheelock
M. J.
,
Green
K. J.
(
1994
).
Posttranslational regulation of plakoglobin expression. Influence of the desmosomal cadherins on plakoglobin metabolic stability.
J. Biol. Chem
49
,
31214
31223
Kowalczyk
A. P.
,
Borgwardt
J. E.
,
Green
K. J.
(
1996
).
Analysis of desmosomal cadherin-adhesive function and stoichiometry of desmosomal cadherin-plakoglobin complexes.
J. Invest. Dermatol
107
,
293
300
Kowalczyk
A. P.
,
Bornslaeger
E. A.
,
Borgwardt
J. E.
,
Palka
H. L.
,
Dhaliwal
A. S.
,
Corcoran
C. M.
,
Denning
M. F.
,
Green
K. J.
(
1997
).
The amino-terminal domain of desmoplakin binds to plakoglobin and clusters desmosomal cadherin-plakoglobin complexes.
J. Cell Biol
139
,
773
784
Marcozzi
C.
,
Burdett
I. D. J.
,
Buxton
R. S.
,
Magee
A. I.
(
1998
).
Coexpression of both types of desmosomal cadherin and plakoglobin confers strong intercellular adhesion.
J. Cell Sci
111
,
495
509
Mathur
M.
,
Goodwin
L.
,
Cowin
P.
(
1994
).
Interactions of the cytoplasmic domain of the desmosomal cadherin Dsg1 with plakoglobin.
J. Biol. Chem
269
,
14075
14080
Miller
J. R.
,
Moon
R. T.
(
1996
).
Signal transduction through-catenin and specification of cell fate during embryogenesis.
Genes Dev
10
,
2527
2539
Ozawa
M.
,
Kemler
R.
(
1992
).
Molecular organization of the uvomorulin-catenin complex.
J. Cell Biol
116
,
989
996
Pai
L.-M.
,
Kirkpatrick
C.
,
Blanton
J.
,
Oda
H.
,
Takeichi
M.
,
Peifer
M.
(
1996
).
Drosophila-catenin and E-cadherin bind to distinct regions of Drosophila armadillo.
J. Cell Biochem
271
,
32411
32420
Pasdar
M.
,
Krzeminski
K. A.
,
Nelson
W. J.
(
1991
).
Regulation of desmosome assembly in MDCK epithelial cells: coordination of membrane core and cytoplasmic plaque domain assembly at the plasma membrane.
J. Cell Biol
113
,
645
655
Peifer
M.
,
Berg
S.
,
Reynolds
A. B.
(
1994
).
A repeating amino acid motif shared by proteins with diverse cellular roles.
Cell
76
,
789
791
Roh
J.-Y.
,
Stanley
J. R.
(
1995
).
Plakoglobin binding by human Dsg3 (pemphigus vulgaris antigen) in keratinocytes requires the cadherin-like intracytoplasmic segment.
J. Invest. Dermatol
104
,
720
724
Ruiz
P.
,
Brinkmann
V.
,
Ledermann
B.
,
Behrend
M.
,
Grund
C.
,
Thalhammer
C.
,
Vogel
F.
,
Birchmeier
C.
,
Gunthert
U.
,
Franke
W. W.
,
Birchmeier
W.
(
1996
).
Targeted mutation of plakoglobin in micereveals essential functions of desmosomes in the embryonic heart.
J. Cell Biol
135
,
215
225
Sacco
P. A.
,
McGranahan
T. M.
,
Wheelock
M. J.
,
Johnson
K. R.
(
1995
).
Identification of plakoglobin domains required for association with N-cadherin with-catenin.
J. Biol. Chem
270
,
20201
20206
Schwarz
M. A.
,
Owaribe
K.
,
Kartenbeck
J.
,
Franke
W. W.
(
1990
).
Desmosomes and hemidesmosomes; Constitutive molecular components.
Annu. Rev. Cell Biol
6
,
461
491
Stappert
J.
,
Kemler
R.
(
1994
).
A short core region of E-cadherin is essential for catenin binding and is highly phosphorylated.
Cell Adhes. Commun
2
,
319
327
Troyanovsky
S. M.
,
Eshkind
L. G.
,
Troyanovsky
R. B.
,
Leube
R. E.
,
Franke
W. W.
(
1993
).
Contributions of cytoplasmic domains of desmosomal cadherins to desmosome assembly and intermediate filament anchorage.
Cell
72
,
561
574
Troyanovsky
S. M.
,
Troyanovsky
R. B.
,
Eshkind
L. G.
,
Krutovskikh
V. A.
,
Leube
R. E.
,
Franke
W. W.
(
1994
).
Identification of theplakoglobin-binding domain in desmoglein and its role in plaque assembly and intermediate filament anchorage.
J. Cell Biol
127
,
151
160
Troyanovsky
S. M.
,
Troyanovsky
R. B.
,
Eshkind
L. G.
,
Leube
R. E.
,
Franke
W. W.
(
1994
).
Identification of amino acid sequence motifs in the desmosomal glycoprotein, desmocollin, that are required for plakoglobin binding and plaque formation.
Proc. Nat. Acad. Sci. USA
91
,
10790
10794
Troyanovsky
R. B.
,
Chitaev
N. A.
,
Troyanovsky
S. M.
(
1996
).
Cadherin binding sites of plakoglobin: localization, specificity and role in targeting to adhering junctions.
J. Cell Sci
109
,
3069
3078
Wahl
J. K.
,
Sacco
P. A.
,
McGranahan-Sadler
T. M.
,
Sauppe
L. M.
,
Wheelock
M. J.
,
Johnson
K. R.
(
1996
).
Plakoglobin domains that define its association with the desmosomal cadherins and the classical cadherins: identification of unique and shared domains.
J. Cell Sci
109
,
1143
1154
Witcher
L.
,
Collins
R.
,
Puttagunta
S.
,
Mechanic
S. E.
,
Munson
M.
,
Gumbiner
B.
,
Cowin
P.
(
1996
).
Desmosomal cadherin binding domains of plakoglobin.
J. Biol. Chem
271
,
10904
10909
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