Biochemical characterisation of the actin-binding properties of utrophin

Utrophin is a large ubiquitously expressed cytoskeletal protein that is imp ortant for maturation of vertebrate neuromuscular junctions. It is highly h omologous to dystrophin, the protein defective in Duchenne and Becker muscu lar dystrophies. Utrophin binds to the actin cytoskeleton via an N-terminal actin-binding domain, which is related to the actin-binding domains of mem bers of the spectrin superfamily of proteins. We have determined the actin- binding properties of this utrophin domain and investigated its binding sit e on F-actin. An F-actin cosedimentation assay confirmed that the domain bi nds more tightly to beta-F-actin than to alpha-F-actin and that the full-le ngth utrophin domain binds more tightly to both actin isoforms than a trunc ated construct, lacking a characteristic utrophin N-terminal extension. Bot h domain constructs exist in solution as compact monomers and bind to actin as 1:1 complexes. Analysis of the products of partial proteolysis of the d omain in the presence of F-actin showed that the N-terminal extension was p rotected by binding to actin. The actin isoform dependence of utrophin bind ing could reflect differences at the N-termini of the actin isoforms, thus localising the utrophin-binding site on actin. The involvement of the actin N-terminus in utrophin binding was also supported by competition binding a ssays using myosin subfragment S1, which also binds F-actin near its N-term inus. Cross-linking studies suggested that utrophin contacts two actin mono mers in the actin filament as does myosin S1. These biochemical approaches complement our structural studies and facilitate characterisation of the ac tin-binding properties of the utrophin actin-binding domain. (C) 2000 Wiley -Liss, Inc.