Molecular organization of the desmoglein-plakoglobin complex.
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.
Chitaev
N A
NA
Department of Dermatology, Washington University Medical School, St Louis, MO 63110, USA.
Averbakh
A Z
AZ
Troyanovsky
R B
RB
Troyanovsky
S M
SM
D000409:Alanine; D000595:Amino Acid Sequence; D015815:Cell Adhesion Molecules / Q000096:biosynthesis / Q000737:chemistry; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000737:chemistry*; D051187:Desmocollins; D051182:Desmogleins; D051180:Desmoplakins; D003896:Desmosomes / Q000502:physiology* / Q000648:ultrastructure; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology; D006801:Humans; D007365:Intercellular Junctions / Q000502:physiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014162:Transfection; D051185:gamma Catenin
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D051187:Desmocollins; D051182:Desmogleins; D051180:Desmoplakins; D011994:Recombinant Proteins; D051185:gamma Catenin; D000409:Alanine