Concerted mutation of Phe residues belonging to the β-dystroglycan ectodomain strongly inhibits the interaction with α-dystroglycan in vitro

Abstract

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. © 2006 The Authors.