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Concerted mutation of Phe residues belonging to the β‐dystroglycan ectodomain strongly inhibits the interaction with α‐dystroglycan in vitro
Author(s) -
Bozzi Manuela,
Sciandra Francesca,
Ferri Lorenzo,
Torreri Paola,
Pavoni Ernesto,
Petrucci Tamara C.,
Giardina Bruno,
Brancaccio Andrea
Publication year - 2006
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2006.05492.x
Subject(s) - ectodomain , dystroglycan , in vitro , chemistry , mutation , microbiology and biotechnology , genetics , biology , biochemistry , laminin , gene , receptor , cell
The dystroglycan adhesion complex consists of two noncovalently interacting proteins: α‐dystroglycan, a peripheral extracellular subunit that is extensively glycosylated, and the transmembrane β‐dystroglycan, whose cytosolic tail interacts with dystrophin, thus linking the F‐actin cytoskeleton to the extracellular matrix. Dystroglycan is thought to play a crucial role in the stability of the plasmalemma, and forms strong contacts between the extracellular matrix and the cytoskeleton in a wide variety of tissues. Abnormal membrane targeting of dystroglycan subunits and/or their aberrant post‐translational modification are often associated with several pathologic conditions, ranging from neuromuscular disorders to carcinomas. A putative functional hotspot of dystroglycan is represented by its intersubunit surface, which is contributed by two amino acid stretches: approximately 30 amino acids of β‐dystroglycan (691–719), and approximately 15 amino acids of α‐dystroglycan (550–565). Exploiting alanine scanning, we have produced a panel of site‐directed mutants of our two consolidated recombinant peptides β‐dystroglycan (654–750), corresponding to the ectodomain of β‐dystroglycan, and α‐dystroglycan (485–630), spanning the C‐terminal domain of α‐dystroglycan. By solid‐phase binding assays and surface plasmon resonance, we have determined the binding affinities of mutated peptides in comparison to those of wild‐type α‐dystroglycan and β‐dystroglycan, and shown the crucial role of two β‐dystroglycan phenylalanines, namely Phe692 and Phe718, for the α–β interaction. Substitution of the α‐dystroglycan residues Trp551, Phe554 and Asn555 by Ala does not affect the interaction between dystroglycan subunits in vitro . As a preliminary analysis of the possible effects of the aforementioned mutations in vivo , detection through immunofluorescence and western blot of the two dystroglycan subunits was pursued in dystroglycan‐transfected 293‐Ebna cells.